
John G Hildebrand
- Regents Professor Emeritus
- (520) 621-6626
- Gould-Simpson, Rm. 621
- Tucson, AZ 85721
- jhildebr@arizona.edu
Biography
John G. Hildebrand is Foreign Secretary of the U.S. National Academy of Sciences and Regents Professor of Neuroscience and Professor of Chemistry & Biochemistry, Ecology & Evolutionary Biology, Entomology, and Molecular & Cellular Biology at the University of Arizona in Tucson. He was founding head of the University’s Division of Neurobiology (1985-2009) and of the Department of Neuroscience (2009-2013) after the Division became a Department. He earned his baccalaureate degree (biology) at Harvard University and Ph.D. (biochemistry) at the Rockefeller University and moved to Arizona in 1985 after 16 years of faculty service at Harvard and Columbia Universities. His research fields are insect neurobiology and behavior, physiology and functions of the olfactory system, chemical ecology, and the biology of arthropod vectors of pathogens. A past president of the Association for Chemoreception Sciences, International Society of Chemical Ecology, and International Society for Neuroethology, he is a member of the U.S. National Academy of Sciences, American Academy of Arts and Sciences, American Philosophical Society, German National Academy of Sciences 'Leopoldina’, Norwegian Academy of Science and Letters, Royal Norwegian Society of Sciences and Letters, and Brazilian Academy of Sciences; an Honorary Fellow of the Royal Entomological Society (UK); and a Fellow of the American Association for the Advancement of Science (AAAS), the Entomological Society of America, and the International Society for Neuroethology.
Degrees
- Ph.D. Biochemistry
- Rockefeller University, New York, New York, United States
- Succinyl phosphate and the succinyl Coenzyme A synthetase reaction
- B.A. Biology
- Harvard University, Cambridge, Massachusetts, United States
Work Experience
- Columbia University, New York, New York (1980 - 1985)
- Harvard Medical School, Boston, Massachusetts (1969 - 1980)
Awards
- Elected Foreign Member
- Norwegian Academy of Sciences and Letters, Spring 1999
- Elected Member
- German National Academy of Sciences 'Leopoldina', Fall 1998
- American Philosophical Society, Spring 2014
- National Academy of Sciences, Spring 2007
- Elected Corresponding Member
- Brazilian Academy of Sciences, Fall 2019
- Wigglesworth Award
- Royal Entomological Society (UK), Fall 2016
- Galileo Circle Fellow
- College of Science, University of Arizona, Spring 2014
- Sigma Xi Distinguished Lecturer
- Spring 2014
- Honored Faculty Award
- UofA Graduate Interdisciplinary Programs, Spring 2013
- 50-Year Member Recognition
- American Chemical Society, Spring 2012
- AChemS Max Mozell Award for Outstanding Achievement in the Chemical Senses
- Spring 2012
- Elected Fellow
- International Society for Neuroethology, Spring 2012
- American Academy of Arts and Sciences, Spring 2001
- Elected Honorary Fellow
- Royal Entomological Society (UK), Spring 2012
- Einstein Professorship
- Chinese Academy of Sciences, P.R.C., Beijing, Fall 2008
- Laurea honoris causa
- University of Cagliari, Italy, Spring 2000
Interests
No activities entered.
Courses
2024-25 Courses
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Biomedical Ethics
HNRS 195B (Fall 2024)
2023-24 Courses
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Biomedical Ethics
HNRS 195B (Fall 2023)
2022-23 Courses
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Biomedical Ethics
HNRS 195B (Fall 2022)
2021-22 Courses
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Special Topics in Science
HNRS 195I (Fall 2021)
2020-21 Courses
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Special Topics in Science
HNRS 195I (Fall 2020)
2019-20 Courses
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Special Topics in Science
HNRS 195I (Fall 2019)
2018-19 Courses
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Special Topics in Science
HNRS 195I (Fall 2018)
2017-18 Courses
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Special Topics in Science
HNRS 195I (Fall 2017)
2016-17 Courses
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Special Topics in Science
HNRS 195I (Fall 2016)
Scholarly Contributions
Chapters
- Lei, H., Oland, L. A., Riffell, J. A., Beyerlein, A., & Hildebrand, J. G. (2016). Implications from microcircuits of a moth antennal lobe for olfactory information processing.. In Handbook of Brain Microcircuits.
- Riffell, J. A., & Hildebrand, J. G. (2016). Adaptive processing in the insect olfactory system. In The Ecology of Animal Senses(pp 3-24). Springer.
Journals/Publications
- Hildebrand, J. G., Davidowitz, G., Potter, K. A., Tseng, A. S., & Wilson, K. (2017). The effects of the alkaloid scopolamine on the performance and behavior of two caterpillar species. Arthropod-Plant Interactions, 12, 21-29.
- Mitchell, R. F., Hall, L. P., Reagel, P. F., McKenna, D. D., Baker, T. C., & Hildebrand, J. G. (2016). Odorant receptors and antennal lobe morphology offer a new approach to understanding olfaction in the Asian longhorned beetle.. Journal of Comparative Physiology A.
- Wilson, J. K., Tseng, A. S., Potter, K. A., Davidowitz, G., & Hildebrand, J. G. (2016). The effects of the alkaloid scopolamine on the performance and behavior of two caterpillar species. Arthropod-Plant Interactions.
- Goldman-Huertas, B., Mitchell, R. F., Lapoint, R. T., Faucher, C. P., Hildebrand, J. G., & Whitreman, N. K. (2015). Evolution of herbivory in Drosophilidae linked to loss of behaviors, antennal responses, odorant receptors and ancestral diet.. Proc Natl Acad Sci USA, 112, 3026 - 3031.
- Lei, H., Chiu, H., & Hildebrand, J. G. (2013). Responses of protocerebral neurons in male Manduca sexta to sex-pheromone mixtures. Journal of comparative physiology. A, Neuroethology, sensory, neural, and behavioral physiology, 199(11).More infoMale Manduca sexta moths are attracted to a mixture of two components of the female's sex pheromone at the natural concentration ratio. Deviation from this ratio results in reduced attraction. Projection neurons innervating prominent male-specific glomeruli in the male's antennal lobe produce maximal synchronized spiking activity in response to synthetic mixtures of the two components centering around the natural ratio, suggesting that behaviorally effective mixture ratios are encoded by synchronous neuronal activity. We investigated the physiological activity and morphology of downstream protocerebral neurons that responded to antennal stimulation with single pheromone components and their mixtures at various concentration ratios. Among the tested neurons, only a few gave stronger responses to the mixture at the natural ratio whereas most did not distinguish among the mixtures that were tested. We also found that the population response distinguished among the two pheromone components and their mixtures, prior to the peak population response. This observation is consistent with our previous finding that synchronous firing of antennal-lobe projection neurons reaches its maximum before the firing rate reaches its peak. Moreover, the response patterns of protocerebral neurons are diverse, suggesting that the representation of olfactory stimuli at the level of protocerebrum is complex.
- Martin, J. P., Lei, H., Riffell, J. A., & Hildebrand, J. G. (2013). Synchronous firing of antennal-lobe projection neurons encodes the behaviorally effective ratio of sex-pheromone components in male Manduca sexta. Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology, 199(11), 963-979.More infoPMID: 24002682;PMCID: PMC3840155;Abstract: Olfactory stimuli that are essential to an animal's survival and reproduction are often complex mixtures of volatile organic compounds in characteristic proportions. Here, we investigated how these proportions are encoded in the primary olfactory processing center, the antennal lobe, of male Manduca sexta moths. Two key components of the female's sex pheromone, present in an approximately 2:1 ratio, are processed in each of two neighboring glomeruli in the macroglomerular complex (MGC) of males of this species. In wind-tunnel flight experiments, males exhibited behavioral selectivity for ratios approximating the ratio released by conspecific females. The ratio between components was poorly represented, however, in the firing-rate output of uniglomerular MGC projection neurons (PNs). PN firing rate was mostly insensitive to the ratio between components, and individual PNs did not exhibit a preference for a particular ratio. Recording simultaneously from pairs of PNs in the same glomerulus, we found that the natural ratio between components elicited the most synchronous spikes, and altering the proportion of either component decreased the proportion of synchronous spikes. The degree of synchronous firing between PNs in the same glomerulus thus selectively encodes the natural ratio that most effectively evokes the natural behavioral response to pheromone. © 2013 Springer-Verlag Berlin Heidelberg.
- Riffell, J. A., Lei, H., Abrell, L., & Hildebrand, J. G. (2013). Neural basis of a pollinator's buffet: Olfactory specialization and learning in Manduca sexta moth. Science, 339(6116), 200-204.More infoPMID: 23223454;Abstract: Pollinators exhibit a range of innate and learned behaviors that mediate interactions with flowers, but the olfactory bases of these responses in a naturalistic context remain poorly understood. The hawkmoth Manduca sexta is an important pollinator for many night-blooming flowers but can learn - through olfactory conditioning - to visit other nectar resources. Analysis of the flowers that are innately attractive to moths shows that the scents all have converged on a similar chemical profile that, in turn, is uniquely represented in the moth's antennal (olfactory) lobe. Flexibility in visitation to nonattractive flowers, however, is mediated by octopamine-associated modulation of antennal-lobe neurons during learning. Furthermore, this flexibility does not extinguish the innate preferences. Such processing of stimuli through two olfactory channels, one involving an innate bias and the other a learned association, allows the moths to exist within a dynamic floral environment while maintaining specialized associations.
- Hinterwirth, A. J., Medina, B., Lockey, J., Otten, D., Voldman, J., Lang, J. H., Hildebrand, J. G., & Daniel, T. L. (2012). Wireless stimulation of antennal muscles in freely flying hawkmoths leads to flight path Changes. PLoS ONE, 7(12).More infoPMID: 23300751;PMCID: PMC3530516;Abstract: Insect antennae are sensory organs involved in a variety of behaviors, sensing many different stimulus modalities. As mechanosensors, they are crucial for flight control in the hawkmoth Manduca sexta. One of their roles is to mediate compensatory reflexes of the abdomen in response to rotations of the body in the pitch axis. Abdominal motions, in turn, are a component of the steering mechanism for flying insects. Using a radio controlled, programmable, miniature stimulator, we show that ultra-low-current electrical stimulation of antennal muscles in freely-flying hawkmoths leads to repeatable, transient changes in the animals' pitch angle, as well as less predictable changes in flight speed and flight altitude. We postulate that by deflecting the antennae we indirectly stimulate mechanoreceptors at the base, which drive compensatory reflexes leading to changes in pitch attitude. © 2012 HInterwirth et al.
- Reisenman, C. E., Savary, W., Cowles, J., Gregory, T. L., & Hildebrand, J. G. (2012). The distribution and abundance of triatomine insects, potential vectors of Chagas Disease, in a metropolitan area in southern Arizona, United States. Journal of medical entomology, 49(6).More infoTriatomine insects are a problem for human health in southwestern United States because of the moderate-to-severe allergic reactions their bites can cause and because they are potential vectors of Chagas Disease. Although both infected insects and wild mammalian reservoirs are plentiful in southern U.S., only seven cases of autochthonous transmission (plus 16 new presumed cases) of this disease have been reported to date. Therefore, the purpose of this study was to investigate triatomine distribution and abundance in a metropolitan area in southern Arizona. Species, life-stage, locality, and date of collection were recorded for 1,878 triatomine insects collected during 4 yr inside and around houses. For both sexes of the most abundant species, Triatoma rubida (Uhler) (>95% of triatomines collected), dispersal followed a typical year-to-year pattern: dispersal started at the beginning of May and peaked during the first-second week of June. T. rubida was found widely distributed in suburban areas. Triatomines of the two less abundant species, T. recurva (Stal) and T. protracta (Uhler), were collected in all suburban areas throughout the 4-yr survey. All of these population characteristics were observed both at a large (i.e., all collection sites pooled) and a small (i.e., single collection sites) scale. In total, approximately 55-60% of the triatomines were collected inside houses, and 30-35% of those were found in or near beds; thus, it is likely that they fed on humans. To our knowledge, this study is the first comprehensive multi-year analysis of triatomine distribution and abundance in the U.S., providing data that allow inferences about risks to human health.
- Tsang, W. M., Stone, A. L., Otten, D., Aldworth, Z. N., Daniel, T. L., Hildebrand, J. G., Levine, R. B., & Voldman, J. (2012). Insect-machine interface: A carbon nanotube-enhanced flexible neural probe. Journal of Neuroscience Methods, 204(2), 355-365.More infoPMID: 22155384;Abstract: We developed microfabricated flexible neural probes (FNPs) to provide a bi-directional electrical link to the moth Manduca sexta. These FNPs can deliver electrical stimuli to, and capture neural activity from, the insect's central nervous system. They are comprised of two layers of polyimide with gold sandwiched in between in a split-ring geometry that incorporates the bi-cylindrical anatomical structure of the insect's ventral nerve cord. The FNPs provide consistent left and right abdominal stimulation both across animals and within an individual animal. The features of the stimulation (direction, threshold charge) are aligned with anatomical features of the moth. We also have used these FNPs to record neuronal activity in the ventral nerve cord of the moth. Finally, by integrating carbon nanotube (CNT)-Au nanocomposites into the FNPs we have reduced the interfacial impedance between the probe and the neural tissue, thus reducing the magnitude of stimulation voltage. This in turn allows use of the FNPs with a wireless stimulator, enabling stimulation and flight biasing of freely flying moths. Together, these FNPs present a potent new platform for manipulating and measuring the neural circuitry of insects, and for other nerves in humans and other animals with similar dimensions as the ventral nerve cord of the moth. © 2011 Elsevier B.V.
- Lei, H., Reisenman, C. E., Wilson, C. H., Gabbur, P., & Hildebrand, J. G. (2011). Spiking patterns and their functional implications in the antennal lobe of the tobacco hornworm Manduca sexta. PLoS ONE, 6(8).More infoPMID: 21897842;PMCID: PMC3163580;Abstract: Bursting as well as tonic firing patterns have been described in various sensory systems. In the olfactory system, spontaneous bursts have been observed in neurons distributed across several synaptic levels, from the periphery, to the olfactory bulb (OB) and to the olfactory cortex. Several in vitro studies indicate that spontaneous firing patterns may be viewed as "fingerprints" of different types of neurons that exhibit distinct functions in the OB. It is still not known, however, if and how neuronal burstiness is correlated with the coding of natural olfactory stimuli. We thus conducted an in vivo study to probe this question in the OB equivalent structure of insects, the antennal lobe (AL) of the tobacco hornworm Manduca sexta. We found that in the moth's AL, both projection (output) neurons (PNs) and local interneurons (LNs) are spontaneously active, but PNs tend to produce spike bursts while LNs fire more regularly. In addition, we found that the burstiness of PNs is correlated with the strength of their responses to odor stimulation - the more bursting the stronger their responses to odors. Moreover, the burstiness of PNs was also positively correlated with the spontaneous firing rate of these neurons, and pharmacological reduction of bursting resulted in a decrease of the neurons' responsiveness. These results suggest that neuronal burstiness reflects a physiological state of these neurons that is directly linked to their response characteristics. © 2011 Lei et al.
- Martin, J. P., Beyerlein, A., Dacks, A. M., Reisenman, C. E., Riffell, J. A., Lei, H., & Hildebrand, J. G. (2011). The neurobiology of insect olfaction: Sensory processing in a comparative context. Progress in Neurobiology, 95(3), 427-447.More infoPMID: 21963552;Abstract: The simplicity and accessibility of the olfactory systems of insects underlie a body of research essential to understanding not only olfactory function but also general principles of sensory processing. As insect olfactory neurobiology takes advantage of a variety of species separated by millions of years of evolution, the field naturally has yielded some conflicting results. Far from impeding progress, the varieties of insect olfactory systems reflect the various natural histories, adaptations to specific environments, and the roles olfaction plays in the life of the species studied. We review current findings in insect olfactory neurobiology, with special attention to differences among species. We begin by describing the olfactory environments and olfactory-based behaviors of insects, as these form the context in which neurobiological findings are interpreted. Next, we review recent work describing changes in olfactory systems as adaptations to new environments or behaviors promoting speciation. We proceed to discuss variations on the basic anatomy of the antennal (olfactory) lobe of the brain and higher-order olfactory centers. Finally, we describe features of olfactory information processing including gain control, transformation between input and output by operations such as broadening and sharpening of tuning curves, the role of spiking synchrony in the antennal lobe, and the encoding of temporal features of encounters with an odor plume. In each section, we draw connections between particular features of the olfactory neurobiology of a species and the animal's life history. We propose that this perspective is beneficial for insect olfactory neurobiology in particular and sensory neurobiology in general. © 2011 Elsevier Ltd.
- Meinwald, J., & Hildebrand, J. G. (2011). Teaching science appreciation. Science, 331(6020), 1010-1011.More infoPMID: 21350147;
- Reisenman, C. E., Dacks, A. M., & Hildebrand, J. G. (2011). Local interneuron diversity in the primary olfactory center of the moth Manduca sexta. Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology, 197(6), 653-665.More infoPMID: 21286727;Abstract: Local interneurons (LNs) play important roles in shaping and modulating the activity of output neurons in primary olfactory centers. Here, we studied the morphological characteristics, odor responses, and neurotransmitter content of LNs in the antennal lobe (AL, the insect primary olfactory center) of the moth Manduca sexta. We found that most LNs are broadly tuned, with all LNs responding to at least one odorant. 70% of the odorants evoked a response, and 22% of the neurons responded to all the odorants tested. Some LNs showed excitatory (35%) or inhibitory (33%) responses only, while 33% of the neurons showed both excitatory and inhibitory responses, depending on the odorant. LNs that only showed inhibitory responses were the most responsive, with 78% of the odorants evoking a response. Neurons were morphologically diverse, with most LNs innervating almost all glomeruli and others innervating restricted portions of the AL. 61 and 39% of LNs were identified as GABA-immunoreactive (GABA-ir) and non-GABA-ir, respectively. We found no correlations between odor responses and GABA-ir, neither between morphology and GABA-ir. These results show that, as observed in other insects, LNs are diverse, which likely determines the complexity of the inhibitory network that regulates AL output. © 2011 Springer-Verlag.
- Reisenman, C. E., Gregory, T., Guerenstein, P. G., & Hildebrand, J. G. (2011). Feeding and defecation behavior of Triatoma rubida (Uhler, 1894) (Hemiptera: Reduviidae) under laboratory conditions, and its potential role as a vector of chagas disease in Arizona, USA. American Journal of Tropical Medicine and Hygiene, 85(4), 648-656.More infoPMID: 21976567;PMCID: PMC3183772;Abstract: Chagas disease is caused by the parasite Trypanosoma cruzi, which is transmitted to humans by blood-sucking triatomine insects. This disease is endemic throughout Mexico and Central and South America, but only a few autochthonous cases have been reported in the United States, despite the fact that infected insects readily invade houses and feed on humans. Competent vectors defecate during or shortly after feeding so that infective feces contact the host. We thus studied the feeding and defecation behaviors of the prevalent species in southern Arizona, Triatoma rubida. We found that whereas defecation during feeding was frequent in females (93%), it was very rare in immature stages (3%), and absent in males. Furthermore, more than half of the immature insects that exhibited multiple feeding bouts (62%) defecated during interruptions of feeding, i.e., while likely on or near the host. These results indicate that T. rubida potentially could transmit T. cruzi to humans. Copyright © 2011 by The American Society of Tropical Medicine and Hygiene.
- Alarcon, R., Riffell, J. A., Davidowitz, G. -., Hildebrand, J. G., & Bronstein, J. -. (2010). Sex-dependent variation in the floral preferences of the hawkmoth Manduca sexta. Animal Behavior, 80, 289-296.
- Alarcón, R., Riffell, J. A., Davidowitz, G., Hildebrand, J. G., & Bronstein, J. L. (2010). Sex-dependent variation in the floral preferences of the hawkmoth Manduca sexta. Animal Behaviour, 80(2), 289-296.More infoAbstract: Studies of plant-pollinator interactions have often documented species differences in preferences for floral advertisements and rewards. However, the contribution of intraspecific variation in behaviours, especially between sexes, remains less understood. We explored resource preference and resource use by male and female Manduca sexta hawkmoths, relative to two important nectar resources in southern Arizona, U.S.A. Manduca sexta is the major pollinator of one of these species (Datura wrightii, Solanaceae). Because females must also seek out D. wrightii as an oviposition resource, females were predicted to feed upon it more than would males, which should be free to choose the best nectar resource. Using naïve laboratory-reared moths in flight arena experiments, we found that both sexes preferred Datura wrightii over Agave palmeri (Agavaceae). Exposure to only one species and an odourless paper control, however, revealed sex-specific differences in foraging behaviour, with females feeding longer from A. palmeri and males feeding longer from D. wrightii, leading us to reject our hypothesis. Differences in feeding preferences directly translated into differences in energy intake. Females gained significantly more energy than did males by feeding from A. palmeri. We also examined whether behavioural preferences of moths in the laboratory translated into foraging behaviour in the field. Pollen load analysis of moths caught in 2004 showed that females carried significantly more A. palmeri pollen than did males, whereas males carried more D. wrightii pollen than did females. Whereas most studies examine pollination associations at the species level, our results highlight the potential importance of between-sex variation in floral visits. © 2010.
- Dacks, A. M., Guerenstein, P. G., Reisenman, C. E., Martin, J. P., Lei, H., & Hildebrand, J. G. (2010). Olfaction in Invertebrates: Manduca. Encyclopedia of Neuroscience, 49-57.More infoAbstract: Current knowledge of the olfactory system of. Manduca sexta is discussed within the context of the natural history of this model organism. The anatomy of the olfactory system is described progressing from the antennas to the antennal lobes and then to higher centers of olfactory processing in the brain. The principles of olfactory information processing revealed from studies of this organism are discussed with respect to its ability to identify odors and evaluate their concentration and spatiotemporal dynamics. The cellular substrate underlying multiple functional roles of antennal lobe circuitry is also described. © 2009 Elsevier Ltd All rights reserved.
- Daly, D. C., Mercier, P. P., Bhardwaj, M., Stone, A. L., Aldworth, Z. N., Daniel, T. L., Voldman, J., Hildebrand, J. G., & Chandrakasan, A. P. (2010). A pulsed UWB receiver SoC for insect motion control. IEEE Journal of Solid-State Circuits, 45(1), 153-166.More infoAbstract: A 2.5 mW wireless flight control system for cyborg moths is presented, consisting of a 3-to-5 GHz non-coherent pulsed ultra-wideband receiver system-on-chip with an integrated 4-channel pulse-width modulation stimulator mounted on a 1.5 cm by 2.6 cm printed circuit board. The highly duty cycled, energy detection receiver requires 0.5-to-1.4 nJ/bit and achieves a sensitivity of 76 dBm at a data rate of 16 Mb/s (10-3 BER). A multi-stage inverter-based RF front end with resonant load and differential signal chain allow for robust, low energy operation. Digital calibration is used in the baseband amplifier, ADC and DLL to cancel voltage and timing offsets. Through the use of a flexible PCB and 3-D die stacking, the total weight of the electronics is kept to 1 g, within the carrying capacity of an adult Manduca sexta moth. Preliminary wireless flight control of a moth in a wind tunnel is demonstrated. © 2009 IEEE.
- Hildebrand, J. G. (2010). Profile of John G. Hildebrand. Interview by Bijal P. Trivedi.. Proceedings of the National Academy of Sciences of the United States of America, 107(32), 13981-13983.More infoPMID: 20660735;PMCID: PMC2922587;
- Kalberer, N. M., Reisenman, C. E., & Hildebrand, J. G. (2010). Male moths bearing transplanted female antennae express characteristically female behaviour and central neural activity. Journal of Experimental Biology, 213(8), 1272-1280.More infoPMID: 20348339;Abstract: The primary olfactory centres of the sphinx moth Manduca sexta, the antennal lobes, contain a small number of sexually dimorphic glomeruli: the male-specific macroglomerular complex and the large female glomeruli. These glomeruli play important roles in sex-specific behaviours, such as the location of conspecific females and the selection of appropriate host plants for oviposition. The development of sexually dimorphic glomeruli depends strictly on the ingrowth of sex-specific olfactory receptor cell afférents. In the present study we tested the role of female-specific olfactory receptor cells (ORCs) in mediating femalespecific host plant approach behaviour and in determining the response of downstream antennal lobe neurons. We generated male gynandromorphs by excising one imaginai disc from a male larva and replacing it with the antennal imaginai disc from a female donor. Most male gynandromorphs had an apparently normal female antenna and a feminised antennal lobe. These gynandromorphs were tested for flight responses in a wind tunnel towards tomato plants, a preferred host plant for oviposition in M. sexta. Male gynandromorphs landed on host plants as often as normal females, demonstrating that the presence of the induced female-specific glomeruli was necessary and sufficient to produce female-like, odour-oriented behaviour, i.e. orientation towards host plants. We also characterised the physiological and morphological properties of antennal lobe neurons of male gynandromorphs. We found that projection neurons with arborisations in the induced female-specific glomeruli showed physiological responses akin to those of female-specific projection neurons in normal females. These results therefore indicate that ORCs confer specific odour tuning to their glomerular targets and, furthermore, instruct odour-specific behaviour. © 2010. Published by The Company of Biologists Ltd.
- Martin, J. P., & Hildebrand, J. G. (2010). Innate recognition of pheromone and food odors in moths: A common mechanism in the antennal lobe?. Frontiers in Behavioral Neuroscience, 4(SEP).More infoAbstract: The survival of an animal often depends on an innate response to a particular sensory stimulus. For an adult male moth, two categories of odors are innately attractive: pheromone released by conspecific females, and the floral scents of certain, often co-evolved, plants. These odors consist of multiple volatiles in characteristic mixtures. Here, we review evidence that both categories of odors are processed as sensory objects, and we suggest a mechanism in the primary olfactory center, the antennal lobe (AL), that encodes the configuration of these mixtures and may underlie recognition of innately attractive odors. In the pheromone system, mixtures of two or three volatiles elicit upwind flight. Peripheral changes are associated with behavioral changes in speciation, and suggest the existence of a pattern recognition mechanism for pheromone mixtures in the AL. Moths are similarly innately attracted to certain floral scents. Though floral scents consist of multiple volatiles that activate a broad array of receptor neurons, only a smaller subset, numerically comparable to pheromone mixtures, is necessary and sufficient to elicit behavior. Both pheromone and floral scent mixtures that produce attraction to the odor source elicit synchronous action potentials in particular populations of output (projection) neurons (PNs) in the AL. We propose a model in which the synchronous output of a population of PNs encodes the configuration of an innately attractive mixture, and thus comprises an innate mechanism for releasing odor-tracking behavior. The particular example of olfaction in moths may inform the general question of how sensory objects trigger innate responses. © 2010 Martin and Hildebrand.
- Reisenman, C. E., Lawrence, G., Guerenstein, P. G., Gregory, T., Dotson, E., & Hildebrand, J. G. (2010). Infection of kissing bugs with Trypanosoma cruzi, Tucson, Arizona, USA. Emerging Infectious Diseases, 16(3), 400-405.More infoPMID: 20202413;PMCID: PMC3322010;Abstract: Triatomine insects (Hemiptera: Reduviidae), commonly known as kissing bugs, are a potential health problem in the southwestern United States as possible vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Although this disease has been traditionally restricted to Latin America, a small number of vector-transmitted autochthonous US cases have been reported. Because triatomine bugs and infected mammalian reservoirs are plentiful in southern Arizona, we collected triatomines inside or around human houses in Tucson and analyzed the insects using molecular techniques to determine whether they were infected with T. cruzi. We found that 41.5% of collected bugs (n = 164) were infected with T. cruzi, and that 63% of the collection sites (n = 22) yielded ≥1 infected specimens. Although many factors may contribute to the lack of reported cases in Arizona, these results indicate that the risk for infection in this region may be higher than previously thought.
- Reisenman, C. E., Riffell, J. A., Bernays, E. A., & Hildebrand, J. G. (2010). Antagonistic effects of floral scent in an insect-plant interaction. Proceedings of the Royal Society B: Biological Sciences, 277(1692), 2371-2379.More infoPMID: 20335210;PMCID: PMC2894902;Abstract: In southwestern USA, the jimsonweed Datura wrightii and the nocturnal moth Manduca sexta form a pollinator-plant and herbivore-plant association. Because the floral scent is probably important in mediating this interaction, we investigated the floral volatiles that might attract M. sexta for feeding and oviposition. We found that flower volatiles increase oviposition and include small amounts of both enantiomers of linalool, a common component of the scent of hawkmoth-pollinated flowers. Because (+)-linalool is processed in a female-specific glomerulus in the primary olfactory centre of M. sexta, we hypothesized that the enantiomers of linalool differentially modulate feeding and oviposition. Using a synthetic mixture that mimics the D. wrightii floral scent, we found that the presence of linalool was not necessary to evoke feeding and that mixtures containing (+)-and/or (-)-linalool were equally effective in mediating this behaviour. By contrast, females oviposited more on plants emitting (+)-linalool (alone or in mixtures) over control plants, while plants emitting (-)-linalool (alone or in mixtures) were less preferred than control plants. Together with our previous investigations, these results show that linalool has differential effects in feeding and oviposition through two neural pathways: one that is sexually isomorphic and non-enantioselective, and another that is female-specific and enantioselective. This journal is © 2010 The Royal Society.
- Riffell, J. A., Lei, H., Abrell, L., Hildebrand, J. G., Abrell, L. M., Lei, H., & Hildebrand, J. G. (2013). Neural basis of a pollinator's buffet: olfactory specialization and learning in Manduca sexta. Science (New York, N.Y.), 339(6116).More infoPollinators exhibit a range of innate and learned behaviors that mediate interactions with flowers, but the olfactory bases of these responses in a naturalistic context remain poorly understood. The hawkmoth Manduca sexta is an important pollinator for many night-blooming flowers but can learn--through olfactory conditioning--to visit other nectar resources. Analysis of the flowers that are innately attractive to moths shows that the scents all have converged on a similar chemical profile that, in turn, is uniquely represented in the moth's antennal (olfactory) lobe. Flexibility in visitation to nonattractive flowers, however, is mediated by octopamine-associated modulation of antennal-lobe neurons during learning. Furthermore, this flexibility does not extinguish the innate preferences. Such processing of stimuli through two olfactory channels, one involving an innate bias and the other a learned association, allows the moths to exist within a dynamic floral environment while maintaining specialized associations.
- Tsang, W. M., Stone, A. L., Aldworth, Z. N., Hildebrand, J. G., Daniel, T. L., Akinwande, A. I., & Voldman, J. (2010). Flexible split-ring electrode for insect flight biasing using multisite neural stimulation. IEEE Transactions on Biomedical Engineering, 57(7), 1757-1764.More infoPMID: 20176539;Abstract: We describe a flexible multisite microelectrode for insect flight biasing using neural stimulation. The electrode is made of two layers of polyimide (PI) with gold sandwiched in between in a split-ring geometry. The split-ring design in conjunction with the flexibility of the PI allows for a simple insertion process and provides good attachment between the electrode and ventral nerve cord of the insect. Stimulation sites are located at the ends of protruding tips that are circularly distributed inside the split-ring structure. These protruding tips penetrate into the connective tissue surrounding the nerve cord. We have been able to insert the electrode into pupae of the giant sphinx moth Manduca sexta as early as seven days before the adult moth emerges, and we are able to use the multisite electrode to deliver electrical stimuli that evoke multidirectional, graded abdominal motions in both pupae and adult moths. Finally, in loosely tethered flight, we have used stimulation through the flexible microelectrodes to alter the abdominal angle, thus causing the flying moth to deviate to the left or right of its intended path. © 2006 IEEE.
- Tsang, W. M., Stone, A., Aldworth, Z., Otten, D., Akinwande, A. I., Daniel, T., Hildebrand, J. G., Levine, R. B., & Voldman, J. (2010). Remote control of a cyborg moth using carbon nanotube-enhanced flexible neuroprosthetic probe. Proceedings of the IEEE International Conference on Micro Electro Mechanical Systems (MEMS), 39-42.More infoAbstract: We report the first remote flight control of an insect using microfabricated flexible neuroprosthetic probes (FNPs) that directly interface with the animal's central nervous system. The FNPs have a novel split-ring design that incorporates the anatomical bi-cylinder structure of the nerve cord and allows for an efficient surgical process for implantation (Figure 1a). Additionally, we have integrated carbon nanotube (CNT)-Au nanocomposites into the FNPs to enhance the charge injection capability of the probe. The FNPs integrated with a wireless system are able to evoke multi-directional, graded abdominal motions in the moths thus altering their flight path. ©2010 IEEE.
- Daly, D. C., Mercier, P. P., Bhardwaj, M., Stone, A. L., Voldman, J., Levine, R. B., Hildebrand, J. G., & Chandrakasan, A. P. (2009). A pulsed UWB receiver SoC for insect motion control. Digest of Technical Papers - IEEE International Solid-State Circuits Conference.
- Lei, H., Riffell, J. A., Gage, S. L., & Hildebrand, J. G. (2009). Contrast enhancement of stimulus intermittency in a primary olfactory network and its behavioral significance. Journal of Biology, 8(2).More infoPMID: 19232128;PMCID: PMC2687775;Abstract: Background. An animal navigating to an unseen odor source must accurately resolve the spatiotemporal distribution of that stimulus in order to express appropriate upwind flight behavior. Intermittency of natural odor plumes, caused by air turbulence, is critically important for many insects, including the hawkmoth, Manduca sexta, for odor-modulated search behavior to an odor source. When a moth's antennae receive intermittent odor stimulation, the projection neurons (PNs) in the primary olfactory centers (the antennal lobes), which are analogous to the olfactory bulbs of vertebrates, generate discrete bursts of action potentials separated by periods of inhibition, suggesting that the PNs may use the binary burst/non-burst neural patterns to resolve and enhance the intermittency of the stimulus encountered in the odor plume. Results. We tested this hypothesis first by establishing that bicuculline methiodide reliably and reversibly disrupted the ability of PNs to produce bursting response patterns. Behavioral studies, in turn, demonstrated that after injecting this drug into the antennal lobe at the effective concentration used in the physiological experiments animals could no longer efficiently locate the odor source, even though they had detected the odor signal. Conclusions. Our results establish a direct link between the bursting response pattern of PNs and the odor-tracking behavior of the moth, demonstrating the behavioral significance of resolving the dynamics of a natural odor stimulus in antennal lobe circuits. © 2009 Lei et al.; licensee BioMed Central Ltd.
- Reisenman, C. E., Riffell, J. A., & Hildebrand, J. G. (2009). Neuroethology of oviposition behavior in the moth manduca sexta. Annals of the New York Academy of Sciences, 1170, 462-467.More infoPMID: 19686178;PMCID: PMC2766020;Abstract: Olfactory cues play decisive roles in the lives of most insect species, providing information about biologically relevant resources, such as food, mates, and oviposition sites. The nocturnal moth Manduca sexta feeds on floral nectar from a variety of plants (and thus serves as a pollinator), but females oviposit almost exclusively on solanaceous plants, which they recognize on the basis of olfactory cues. Plants, however, respond to herbivory by releasing blends of volatiles that attract natural enemies of herbivores. Thus, oviposition behavior probably results from the sensory evaluation not only of attractive host plant volatiles but also of repellent volatiles that indicate the acceptability or inappropriateness, respectively, of host plants for the females' offspring. Here we describe results from chemical-ecological, neurophysiological, and behavioral experiments aimed at understanding the neural mechanisms that control oviposition behavior in M. sexta. © 2009 New York Academy of Sciences.
- Riffell, J. A., Lei, H., & Hildebrand, J. G. (2009). Neural correlates of behavior in the moth Manduca sexta in response to complex odors. Proceedings of the National Academy of Sciences of the United States of America, 106(46), 19219-19226.More infoPMID: 19907000;PMCID: PMC2780752;Abstract: With Manduca sexta as a model system, we analyzed how natural odor mixtures that are most effective in eliciting flight and foraging behaviors are encoded in the primary olfactory center in the brain, the antennal lobe. We used gas chromatography coupled with multiunit neural-ensemble recording to identify key odorants from flowers of two important nectar resources, the desert plants Datura wrightii and Agave palmeri, that elicited responses from individual antennal-lobe neurons. Neural-ensemble responses to the A. palmeri floral scent, comprising >60 odorants, could be reproduced by stimulation with a mixture of six of its constituents that had behavioral effectiveness equivalent to that of the complete scent. Likewise, a mixture of three floral volatiles from D. wrightii elicited normal flight and feeding behaviors. By recording responses of neural ensembles to mixtures of varying behavioral effectiveness, we analyzed the coding of behaviorally "meaningful" odors. We considered four possible ensemble-coding mechanisms - mean firing rate, mean instantaneous firing rate, pattern of synchronous ensemble firing, and total net synchrony of firing - and found that mean firing rate and the pattern of ensemble synchrony were best correlated with behavior (R = 41% and 43%, respectively). Stepwise regression analysis showed that net synchrony and mean instantaneous firing rate contributed little to the variation in the behavioral results. We conclude that a combination of mean-rate coding and synchrony of firing of antennal-lobe neurons underlies generalization among related, behaviorally effective floral mixtures while maintaining sufficient contrast for discrimination of distinct scents.
- Riffell, J. A., Lei, H., Christensen, T. A., & Hildebrand, J. G. (2009). Characterization and Coding of Behaviorally Significant Odor Mixtures. Current Biology, 19(4), 335-340.More infoPMID: 19230669;PMCID: PMC2677194;Abstract: For animals to execute odor-driven behaviors, the olfactory system must process complex odor signals and maintain stimulus identity in the face of constantly changing odor intensities [1-5]. Surprisingly, how the olfactory system maintains identity of complex odors is unclear [6-10]. We took advantage of the plant-pollinator relationship between the Sacred Datura (Datura wrightii) and the moth Manduca sexta [11, 12] to determine how olfactory networks in this insect's brain represent odor mixtures. We combined gas chromatography and neural-ensemble recording in the moth's antennal lobe to examine population codes for the floral mixture and its fractionated components. Although the floral scent of D. wrightii comprises at least 60 compounds, only nine of those elicited robust neural responses. Behavioral experiments confirmed that these nine odorants mediate flower-foraging behaviors, but only as a mixture. Moreover, the mixture evoked equivalent foraging behaviors over a 1000-fold range in dilution, suggesting a singular percept across this concentration range. Furthermore, neural-ensemble recordings in the moth's antennal lobe revealed that reliable encoding of the floral mixture is organized through synchronized activity distributed across a population of glomerular coding units, and this timing mechanism may bind the features of a complex stimulus into a coherent odor percept. © 2009 Elsevier Ltd. All rights reserved.
- Dacks, A. M., Christensen, T. A., & Hildebrand, J. G. (2008). Modulation of olfactory information processing in the antennal lobe of Manduca sexta by serotonin. Journal of Neurophysiology, 99(5), 2077-2085.More infoPMID: 18322001;Abstract: The nervous system copes with variability in the external and internal environment by using neuromodulators to adjust the efficacy of neural circuits. The role of serotonin (5HT) as a neuromodulator of olfactory information processing in the antennal lobe (AL) of Manduca sexta was examined using multichannel extracellular electrodes to record the responses of ensembles of AL neurons to olfactory stimuli. In one experiment, the effects of 5HT on the concentration-response functions for two essential plant oils across a range of stimulus intensities were examined. In a second experiment, the effect of 5HT on the ability of ensembles to discriminate odorants from different chemical classes was examined. Bath application of 5HT enhanced AL unit responses by increasing response duration and firing rate, which in turn increased the amount of spike time cross-correlation and -covariance between pairs of units. 5HT had the greatest effect on overall ensemble activation at higher odorant concentrations, resulting in an increase in the gain of the dose-response function of individual units. Additionally, response thresholds shifted to lower odorant concentrations for some units, suggesting that 5HT increased their sensitivity. Serotonin enhanced ensemble discrimination of different concentrations of individual odorants as well as discrimination of structurally dissimilar odors at the same concentration. Given the known circadian fluctuations of 5HT in the AL of this species, these findings support the hypothesis that 5HT periodically enhances sensitivity and responsiveness in the AL of Manduca to maximize efficiency when the requirement for olfactory acuity is the greatest. Copyright © 2008 The American Physiological Society.
- Guerenstein, P. G., & Hildebrand, J. G. (2008). Roles and effects of environmental carbon dioxide in insect life. Annual Review of Entomology, 53, 161-178.More infoPMID: 17803457;Abstract: Carbon dioxide (CO2) is a ubiquitous sensory cue that plays multiple roles in insect behavior. In recent years understanding of the well-known role of CO2 in foraging by hematophagous insects (e.g., mosquitoes) has grown, and research on the roles of CO2 cues in the foraging and oviposition behavior of phytophagous insects and in behavior of social insects has stimulated interest in this area of insect sensory biology. This review considers those advances, as well as some of the mechanistic bases of the modulation of behavior by CO2 and important progress in our understanding of the detection and CNS processing of CO2 information in insects. Finally, this review briefly addresses how the ongoing increase in atmospheric CO2 levels may affect insect life. Copyright © 2008 by Annual Reviews. All rights reserved.
- Hildebrand, J. G., & Riffell, J. A. (2008). Journal of Chemical Ecology: Preface. Journal of Chemical Ecology, 34(7), 820-821.More infoPMID: 18581176;
- Reisenman, C. E., Heinbockel, T., & Hildebrand, J. G. (2008). Inhibitory interactions among olfactory glomeruli do not necessarily reflect spatial proximity. Journal of Neurophysiology, 100(2), 554-564.More infoPMID: 18417626;PMCID: PMC2525721;Abstract: Inhibitory interactions shape the activity of output neurons in primary olfactory centers and promote contrast enhancement of odor representations. Patterns of interglomerular connectivity, however, are largely unknown. To test whether the proximity of glomeruli to one another is correlated with interglomerular inhibitory interactions, we used intracellular recording and staining methods to record the responses of projection (output) neurons (PNs) associated with glomeruli of known olfactory tuning in the primary olfactory center of the moth Manduca sexta. We focused on Toroid I, a glomerulus in the male-specific macroglomerular complex (MGC) specialized to one of the two key components of the conspecific females' sex pheromone, and the adjacent, sexually isomorphic glomerulus 35, which is highly sensitive to Z-3-hexenyl acetate (Z3-6:OAc). We used the two odorants to activate these reference glomeruli and tested the effects of olfactory activation in other glomeruli. We found that Toroid-I PNs were not inhibited by input to G35, whereas G35 PNs were inhibited by input to Toroid-I PNs. We also recorded the responses of PNs arborizing in other sexually isomorphic glomeruli to stimulation with the sex pheromone and Z3-6:OAc. We found that inhibitory responses were not related to proximity to the MGC and G35: both distant and adjacent PNs were inhibited by stimulation with the sex pheromone, some others were affected by only one odorant, and yet others by neither. Similar results were obtained in female PNs recorded in proximity to female-specific glomeruli. Our findings indicate that inhibitory interactions among glomeruli are widespread and independent of their spatial proximity. Copyright © 2008 The American Physiological Society.
- Riffell, J. A., Abrell, L., & Hildebrand, J. G. (2008). Physical processes and real-time chemical measurement of the insect olfactory environment. Journal of Chemical Ecology, 34(7), 837-853.More infoPMID: 18548311;PMCID: PMC2778261;Abstract: Odor-mediated insect navigation in airborne chemical plumes is vital to many ecological interactions, including mate finding, flower nectaring, and host locating (where disease transmission or herbivory may begin). After emission, volatile chemicals become rapidly mixed and diluted through physical processes that create a dynamic olfactory environment. This review examines those physical processes and some of the analytical technologies available to characterize those behavior-inducing chemical signals at temporal scales equivalent to the olfactory processing in insects. In particular, we focus on two areas of research that together may further our understanding of olfactory signal dynamics and its processing and perception by insects. First, measurement of physical atmospheric processes in the field can provide insight into the spatiotemporal dynamics of the odor signal available to insects. Field measurements in turn permit aspects of the physical environment to be simulated in the laboratory, thereby allowing careful investigation into the links between odor signal dynamics and insect behavior. Second, emerging analytical technologies with high recording frequencies and field-friendly inlet systems may offer new opportunities to characterize natural odors at spatiotemporal scales relevant to insect perception and behavior. Characterization of the chemical signal environment allows the determination of when and where olfactory-mediated behaviors may control ecological interactions. Finally, we argue that coupling of these two research areas will foster increased understanding of the physicochemical environment and enable researchers to determine how olfactory environments shape insect behaviors and sensory systems. © 2008 Springer Science+Business Media, LLC.
- Riffell, J. A., Alarcón, R., Abrell, L., Davidowitz, G., Bronstein, J. L., & Hildebrand, J. G. (2008). Behavioral consequences of innate preferences and olfactory learning in hawkmoth-flower interactions. Proceedings of the National Academy of Sciences of the United States of America, 105(9), 3404-3409.More infoPMID: 18305169;PMCID: PMC2265144;Abstract: Spatiotemporal variability in floral resources can have ecological and evolutionary consequences for both plants and the pollinators on which they depend. Seldom, however, can patterns of flower abundance and visitation in the field be linked with the behavioral mechanisms that allow floral visitors to persist when a preferred resource is scarce. To explore these mechanisms better, we examined factors controlling floral preference in the hawk-moth Manduca sexta in the semiarid grassland of Arizona. Here, hawkmoths forage primarily on flowers of the bat-adapted agave, Agave palmeri, but shift to the moth-adapted flowers of their larval host plant, Datura wrightii, when these become abundant. Both plants emit similar concentrations of floral odor, but scent composition, nectar, and flower reflectance are distinct between the two species, and A. palmeri flowers provide six times as much chemical energy as flowers of D. wrightii. Behavioral experiments with both naïve and experienced moths revealed that hawkmoths learn to feed from agave flowers through olfactory conditioning but readily switch to D. wrightii flowers, for which they are the primary pollinator, based on an innate odor preference. Behavioral flexibility and the olfactory contrast between flowers permit the hawkmoths to persist within a dynamic environment, while at the same time to function as the major pollinator of one plant species. © 2008 by The National Academy of Sciences of the USA.
- Dacks, A. M., Christensen, T. A., & Hildebrand, J. G. (2006). Phylogeny of a serotonin-immunoreactive neuron in the primary olfactory center of the insect brain. Journal of Comparative Neurology, 498(6), 727-746.More infoPMID: 16927264;Abstract: Serotonin (5-hydroxytryptamine; 5HT) functions in insects as a neurotransmitter, neuromodulator, and neurohormone. In the sphinx moth Manduca sexta, each of the paired antennal lobes (ALs; the primary olfactory centers in the insect brain) has one 5HT-immunoreactive (5HT-ir) neuron that projects into the protocerebrum, crosses the posterior midline, and innervates the contralateral AL; this is referred to as the contralaterally projecting, serotonin-immunoreactive deutocerebral (CSD) neuron. These neurons are thought to function as centrifugal modulators of olfactory sensitivity. To examine the phylogenetic distribution of 5HT-ir neurons apparently homologous to the CSD neuron, we imaged 5HT-like immunoreactivity in the brains of 40 species of insects belonging to 38 families in nine orders. CSD neurons were found in other Lepidoptera, Trichoptera, Diptera, Coleoptera, and Neuroptera but not in the Hymenoptera. In the paraneopteran and polyneopteran species (insects that undergo incomplete metamorphosis) examined, AL 5HT neurons innervate the ispsilateral AL and project to the protocerebrum. Our findings suggest that the characteristic morphology of the CSD neurons originated in the holometabolous insects (those that undergo complete metamorphosis) and were lost in the Hymenoptera. In a subset of the Diptera, the CSD neurons branch within the contralateral AL and project back to the ipsilateral AL via the antennal commissure. The evolution of AL 5HT neurons is discussed in the context of the physiological actions of 5HT observed in the lepidopteran AL. © 2006 Wiley-Liss, Inc.
- Davis, N. T., & Hildebrand, J. G. (2006). Neuroanatomy of the sucking pump of the moth, Manduca sexta (Sphingidae, Lepidoptera). Arthropod Structure and Development, 35(1), 15-33.More infoPMID: 18089055;Abstract: Knowledge of the neuroanatomy of the sucking pump of Manduca sexta (Sphingidae) is valuable for studies of olfactory learning, pattern generators, and postembryonic modification of motor circuitry. The pump comprises a cibarial valve, a buccal pump, and an esophageal sphincter valve. Cibarial opener and closer muscles control the cibarial valve. Six pairs of dilator muscles and a compressor muscle operate the buccal pump. The cibarial opener and one pair of buccal dilator muscles are innervated by paired neurons in the tritocerebrum, and the cibarial opener has double, bilateral innervation. Their tritocerebral innervation indicates that these muscles evolved from labro-clypeal muscles. The remaining paired buccal dilator muscles each are innervated by an unpaired motor neuron in the frontal ganglion. These motor neurons project bilaterally through the frontal connectives to dendritic arborizations in the tritocerebrum. These projections also have a series of dendritic-like arborizations in the connectives. The cibarial closer and buccal compressor muscles are also innervated by motor neurons in the frontal ganglion, but only the closer muscle neuron projects bilaterally to the tritocerebrum. The innervation of the pump muscles indicates that they are associated with the stomodaeum, and, therefore, the buccal pump evolved from the anterior stomodaeum rather than from the cibarium. © 2005 Elsevier Ltd. All rights reserved.
- Abrell, L., Guerenstein, P. G., Mechaber, W. L., Stange, G., Christensen, T. A., Nakanishi, K., & Hildebrand, J. G. (2005). Effect of elevated atmospheric CO2 on oviposition behavior in Manduca sexta moths. Global Change Biology, 11(8), 1272-1282.More infoAbstract: Moths can detect changes in environmental carbon dioxide (CO2) with extremely high sensitivity, but the role of CO2 in the biology of these and other insects is not well understood. Although CO2 has been demonstrated to influence egg-laying (oviposition) behavior of the pyralid moth Cactoblastis cactorum and nectar foraging of the sphingid moth Manduca sexta, information about the generalized role of CO2 in the behavioral biology of these species is lacking. Comparative data are necessary to properly assess how the behaviors of different species may be modified by steadily rising levels of greenhouse gases in the environment. Experiments carried out in Biosphere 2 addressed whether changes in ambient CO2 levels play a role in the oviposition behaviors of M. sexta moths. In the first series of experiments, oviposition was measured inside a flight cage with different levels of nearly ambient or elevated CO2 (400, 800 or 1200 ppm). For each concentration, hostplants used as oviposition sites were grown from seed at a CO2 level that matched the environment inside the flight cage. Under homogenous levels of CO2, we observed no significant difference in oviposition behavior at the concentrations tested. In a second series of experiments, two groups of hostplants, each surrounded by a mini free-air CO2 enrichment (FACE) ring, were assembled inside a flight cage. In this choice test, a dynamic plume of artificially high CO2 was generated around one group of test plants, while ambient CO2 was released around the second (control) group. After eggs were counted on both plant groups, M. sexta females showed a small preference for ovipositing on the control plants. Therefore, in contrast to C. cactorum females tested under similar dynamic flow conditions, M. sexta female oviposition was not strongly inhibited by elevated CO2. To investigate this phenomenon further, we used electrophysiological recording and found that the CO2 receptor cells in M. sexta, unlike those in C. cactorum, are not readily affected by elevated levels of ambient CO2. These findings therefore suggest that elevated background levels of CO2 affect the physiology of the CO2 detection system of M. sexta to a lesser extent than that of C. cactorum, and this correlates well with the observed differences in oviposition behavior between the two species under elevated levels of environmental CO2. Hostplants of C. cactorum are crassulacean acid metabolism plants that generate nocturnal CO2 sinks on the cladode surfaces, whereas, M. sexta hostplants are nocturnal sources of respiratory CO2. We hypothesize that the abrupt and continuing increase in global ambient CO2 levels will differentially alter the behavior and physiology of moths that use CO2 sinks and sources as sensory cues to find hostplants. © 2005 Blackwell Publishing Ltd.
- Dacks, A. M., Christensen, T. A., Agricola, H., Wollweber, L., & Hildebrand, J. G. (2005). Octopamine-immunoreactive neurons in the brain and subesophageal ganglion of the hawkmoth Manduca sexta. Journal of Comparative Neurology, 488(3), 255-268.More infoPMID: 15952164;PMCID: PMC1363738;Abstract: Octopamine is a neuroactive monoamine that functions as a neurohormone, a neuromodulator, and a neurotransmitter in many invertebrate nervous systems, but little is known about the distribution of octopamine in the brain. We therefore used a monoclonal antibody to study the distribution of octopamine-like immunoreactivity in the brain of the hawkmoth Manduca sexta. Immunoreactive processes were observed in many regions of the brain, with the distinct exception of the upper division of the central body. We focused our analysis on nine ventral unpaired median (VUM) neurons with cell bodies in the labial neuromere of the subesophageal ganglion. Seven of these neurons projected caudally through the ventral nerve cord. Two neurons projected rostrally into the brain (supraesophageal ganglion), and one of these was a bilateral neuron that sent projections to the γ-lobe of the mushroom body and the lateral protocerebrum. Octopamine-immunoreactive processes from one or more cells originating in the subesophageal ganglion also form direct connections between the antennal lobes and the calyces of the mushroom bodies. © 2005 Wiley-Liss, Inc.
- Mercer, A. R., Kloppenburg, P., & Hildebrand, J. G. (2005). Plateau potentials in developing antennal-lobe neurons of the moth, Manduca sexta. Journal of Neurophysiology, 93(4), 1949-1958.More infoPMID: 15548619;Abstract: Using whole cell recordings from antennal-lobe (AL) neurons in vitro and in situ, in semi-intact brain preparations, we examined membrane properties that contribute to electrical activity exhibited by developing neurons in primary olfactory centers of the brain of the sphinx moth, Manduca sexta. This activity is characterized by prolonged periods of membrane depolarization that resemble plateau potentials. The presence of plateau potential-generating mechanisms was confirmed using a series of tests established earlier. Brief depolarizing current pulses could be used to trigger a plateau state. Once triggered, plateau potentials could be terminated by brief pulses of hyperpolarizing current. Both triggering and terminating of firing states were threshold phenomena, and both conditions resulted in all-or-none responses. Rebound excitation from prolonged hyperpolarizing pulses could also be used to generate plateau potentials in some cells. These neurons were found to express a hyperpolarization-activated inward current. Neither the generation nor the maintenance of plateau potentials was affected by removal of Na+ ions from the extracellular medium or by blockade of Na+ currents with TTX. However, blocking of Ca 2+ currents with Cd2+ (5 × 10-4 M) inhibited the generation of plateau potentials, indicating that, in Manduca AL neurons, plateau potentials depend on Ca2+. Examining Ca2+ currents in isolation revealed that activation of these currents occurs in the absence of experimentally applied depolarizing stimuli. Our results suggest that this activity underlies the generation of plateau potentials and characteristic bursts of electrical activity in developing AL neurons of M. sexta. Copyright © 2005 The American Physiological Society.
- Reisenman, C. E., Christensen, T. A., & Hildebrand, J. G. (2005). Chemosensory selectivity of output neurons innervating an identified, sexually isomorphic olfactory glomerulus. Journal of Neuroscience, 25(35), 8017-8026.More infoPMID: 16135759;PMCID: PMC1351300;Abstract: The antennal lobe (AL) of insects, like the olfactory bulb of vertebrates, is characterized by discrete modules of synaptic neuropil called glomeruli. In some insects (e.g., moths and cockroaches), a few glomeruli are sexually dimorphic and function in labeled lines for processing of sensory information about sex pheromones. Controversy still exists, however, about whether projection (output) neurons (PNs) of glomeruli in the main AL are also narrowly tuned. We examined this critical issue in the AL of the moth Manduca sexta. We used intracellular recording and staining techniques to investigate the chemosensory tuning of PNs innervating an identifiable, sexually isomorphic glomerulus, G35, in the main AL. We found that the morphological features and chemosensory tuning of G35-PNs were nearly identical in females and males. G35-PNs responded to low concentrations of the plant-derived volatile compound cis-3-hexenyl acetate (c3HA), but the sensitivity threshold of female PNs was lower than that of male PNs. The propionate andbutyrate homologs of c3HA could evoke excitatory responses but only at moderate-to-high concentrations. Other plant volatiles did not evoke responses from G35-PNs. Moreover, PNs innervating glomeruli near G35 (in females) showed little or no response to c3HA. Female G35-PNs were hyperpolarized by (±)linalool, a compound that excites PNs in an adjacent glomerulus, thus providing evidence for lateral-inhibitory interactions between glomeruli. Our results show that PNs arborizing in an identified glomerulus in the main olfactory pathway are morphologically and physiologically equivalent in both sexes and have characteristic, limited molecular receptive ranges that are highly conserved across individuals. Copyright © 2005 Society for Neuroscience.
- Daly, K. C., Christensen, T. A., Lei, H., Smith, B. H., & Hildebrand, J. G. (2004). Learning modulates the ensemble representations for odors in primary olfactory networks. Proceedings of the National Academy of Sciences of the United States of America, 101(28), 10476-10481.More infoPMID: 15232007;PMCID: PMC478594;Abstract: Recent evidence suggests that odor-driven responses in the insect antennal lobe (AL) can be modified by associative and nonassociative processes, as has been shown in the vertebrate olfactory bulb. However, the specific network changes that occur in response to olfactory learning remain unknown. To characterize changes in AL network activity during learning, we developed an in vivo protocol in Manduca sexta that allows continuous monitoring of neural ensembles and feeding behavior over the course of olfactory conditioning. Here, we show that Pavlovian conditioning produced a net recruitment of responsive neural units across the AL that persisted after conditioning. Recruitment only occurred when odor reliably predicted food. Conversely, when odor did not predict food, a net loss of responsive units occurred. Simultaneous measures of feeding responses indicated that the treatment-specific patterns of neural recruitment were positively correlated with changes in the insect's behavioral response to odor. In addition to recruitment, conditioning also produced consistent and profound shifts in the temporal responses of 16% of recorded units. These results show that odor representations in the AL are dynamic and related to olfactory memory consolidation. We furthermore provide evidence that the basis of the learning-dependent changes in the AL is not simply an increase in activity in the neural network representing an odorant. Rather, learning produces a restructuring of spatial and temporal components of network responses to odor in the AL.
- Gibson, N. J., Hildebrand, J. G., & Tolbert, L. P. (2004). Glycosylation patterns are sexually dimorphic throughout development of the olfactory system in Manduca sexta. Journal of Comparative Neurology, 476(1), 1-18.More infoPMID: 15236463;Abstract: In the moth Manduca sexta, development of the adult olfactory system depends on complex interactions between olfactory receptor neurons in the antenna, antennal-lobe neurons in the brain, and several classes of glial cells. As one approach to characterizing molecules that may play roles in these interactions, we used lectins to screen antennae and antennal lobes at different stages of adult development. We find that each of the major neural cell types has a distinct pattern of labeling by lectins. Effects of enzymatic and other treatments on lectin labeling lead us to conclude that the predominant lectin ligands are: glycosphingolipids and an O-linked, fucose-containing glycoprotein on axons of olfactory receptor neurons, O-linked glycoproteins on antennal-lobe neurons, and N-linked glycoproteins on all classes of glial cells in the primary olfactory pathway. Wheat germ agglutinin labels all olfactory axons uniformly during much of development, but labeling becomes restricted to the pheromone-responsive olfactory receptor neurons in the adult male. Succinylated WGA reveals differences in these axon classes earlier, as glomerului develop from protoglomeruli. The adult female displays a less pronounced difference in labeling of axons targeting ordinary and sexually dimorphic glomeruli. Differences in labeling of receptor axons targeted to ordinary and sexually dimorphic glomeruli may be correlated with differences in function or connectivity in different regions of the antennal lobe. © 2004 Wiley-Liss, Inc.
- Guerenstein, P. G., Christensen, T. A., & Hildebrand, J. G. (2004). Sensory processing of ambient CO2 information in the brain of the moth Manduca sexta. Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology, 190(9), 707-725.More infoPMID: 15235811;Abstract: Insects use information about CO2 to perform vital tasks such as locating food sources. In certain moths, CO2 is involved in oviposition behavior. The labial palps of adult moths that feed as adults have a pit organ containing sensory receptor cells that project into the antennal lobes, the sites of primary processing of olfactory information in the brain. In the moth Manduca sexta and certain other species of Lepidoptera, these receptor cells in the labial-palp pit organ have been shown to be tuned to CO 2, and their axons project to a single, identified glomerulus in the antennal lobe, the labial-palp pit organ glomerulus. At present, however, nothing is known about the function of this glomerulus or how CO2 information is processed centrally. We used intracellular recording and staining to reveal projection (output) neurons in the antennal lobes that respond to CO2 and innervate the labial-palp pit organ glomerulus. Our results demonstrate that this glomerulus is the site of first-order processing of sensory information about ambient CO2. We found three functional types of CO2-responsive neurons (with their cell bodies in the antennal lobe or the protocerebrum) that provide output from the antennal lobe to higher centers in the brain. Some physiological characteristics of those neurons are described. © Springer-Verlag 2004.
- Heinbockel, T., Christensen, T. A., & Hildebrand, J. G. (2004). Representation of binary pheromone blends by glomerulus-specific olfactory projection neurons. Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology, 190(12), 1023-1037.More infoPMID: 15378331;Abstract: An outstanding challenge in olfactory neurobiology is to explain how glomerular networks encode information about stimulus mixtures, which are typical of natural olfactory stimuli. In the moth Manduca sexta, a species-specific blend of two sex-pheromone components is required for reproductive signaling. Each component stimulates a different population of olfactory receptor cells that in turn target two identified glomeruli in the macroglomerular complex of the male's antennal lobe. Using intracellular recording and staining, we examined how responses of projection neurons innervating these glomeruli are modulated by changes in the level and ratio of the two essential components in stimulus blends. Compared to projection neurons specific for one component, projection neurons that integrated information about the blend (received excitatory input from one component and inhibitory input from the other) showed enhanced ability to track a train of stimulus pulses. The precision of stimulus-pulse tracking was furthermore optimized at a synthetic blend ratio that mimics the physiological response to an extract of the female's pheromone gland. Optimal responsiveness of a projection neuron to repetitive stimulus pulses therefore appears to depend not only on stimulus intensity but also on the relative strength of the two opposing synaptic inputs that are integrated by macroglomerular complex projection neurons. © Springer-Verlag 2004.
- Hildebrand, J., Guerenstein, P. G., A Yepez, E., Van Haren, J., Williams, D. G., & Hildebrand, J. G. (2004). Floral CO(2) emission may indicate food abundance to nectar-feeding moths. Die Naturwissenschaften, 91(7).More infoAs part of a study of the roles of the sensory subsystem devoted to CO(2) in the nectar-feeding moth Manduca sexta, we investigated CO(2) release and nectar secretion by flowers of Datura wrightii, a preferred hostplant of Manduca. Datura flowers open at dusk and wilt by the following noon. During the first hours after dusk, when Manduca feeds, the flowers produce considerable amounts of nectar and emit levels of CO(2) that should be detectable by moths nearby. By midnight, however, both nectar secretion and CO(2) release decrease significantly. Because nectar production requires high metabolic activity, high floral CO(2) emission may indicate food abundance to the moths. We suggest that hovering moths could use the florally emitted CO(2) to help them assess the nectar content before attempting to feed in order to improve their foraging efficiency.
- Lei, H., Christensen, T. A., & Hildebrand, J. G. (2004). Spatial and temporal organization of ensemble representations for different odor classes in the moth antennal lobe. Journal of Neuroscience, 24(49), 11108-11119.More infoPMID: 15590927;Abstract: In the insect antennal lobe, odor discrimination depends on the ability of the brain to read neural activity patterns across arrays of uniquely identifiable olfactory glomeruli. Less is understood about the complex temporal dynamics and interglomerular interactions that underlie these spatial patterns. Using neural-ensemble recording, we show that the evoked firing patterns within and between groups of glomeruli are odor dependent and organized in both space and time. Simultaneous recordings from up to 15 units per ensemble were obtained from four zones of glomerular neuropil in response to four classes of odorants: pheromones, monoterpenoids, aromatics, and aliphatics. Each odor class evoked a different pattern of excitation and inhibition across recording zones. The excitatory response field for each class was spatially defined, but inhibitory activity was spread across the antennal lobe, reflecting a center-surround organization. Some chemically related odorants were not easily distinguished by their spatial patterns, but each odorant evoked transient synchronous firing across a uniquely different subset of ensemble units. Examination of 535 cell pairs revealed a strong relationship between their recording positions, temporal correlations, and similarity of odor response profiles. These findings provide the first definitive support for a nested architecture in the insect olfactory system that uses both spatial and temporal coordination of firing to encode chemosensory signals. The spatial extent of the representation is defined by a stereotyped focus of glomerular activity for each odorant class, whereas the transient temporal correlations embedded within the ensemble provide a second coding dimension that can facilitate discrimination between chemically similar volatiles.
- Reisenman, C. E., Christensen, T. A., Francke, W., & Hildebrand, J. G. (2004). Enantioselectivity of Projection Neurons Innervating Identified Olfactory Glomeruli. Journal of Neuroscience, 24(11), 2602-2611.More infoPMID: 15028752;Abstract: Projection neurons (PNs) with arborizations in the sexually dimorphic "lateral large female glomerulus" (latLFG) in the antennal lobe (AL) of the moth Manduca sexta previously were shown to respond preferentially to antennal stimulation with (±)linalool, a volatile compound commonly emitted by plants. In the present study, using intracellular recording and staining techniques, we examined the responsiveness of latLFG-PNs to the enantiomers, (+)linalool and (-)linalool and found that (1) latLFG-PNs are more responsive to antennal stimulation with (+)linalool than with (-)linalool, (2) PNs with arborizations in a glomerulus adjacent to the latLFG are preferentially responsive to (-)linalool, and (3) PNs with arborizations confined to other glomeruli near the latLFG are equally responsive to both enantiomers of linalool. Structure-activity studies showed that the hydroxyl group in this tertiary terpene alcohol is the key feature of the molecule determining the response of enantioselective PNs to linalool. In contrast, the responses of non-enantioselective PNs are less dependent on the alcoholic functionality of linalool. Our findings show that PNs innervating a uniquely identifiable glomerulus respond preferentially to a particular enantiomer of an odor substance. Moreover, PNs with arborizations in a glomerulus adjacent to the latLFG, although less sensitive than latLFG-PNs to linalool, respond preferentially to the opposite enantiomer, demonstrating that information about stimulus-absolute configuration can be encoded in different olfactory glomeruli.
- Thom, C., Guerenstein, P. G., Mechaber, W. L., & Hildebrand, J. G. (2004). Erratum: Floral Co2 reveals flower profitability to moths. Journal of Chemical Ecology, 30(7), 1477-.
- Thom, C., Guerenstein, P. G., Mechaber, W. L., & Hildebrand, J. G. (2004). Floral CO 2 reveals flower profitability to moths. Journal of Chemical Ecology, 30(6), 1285-1288.More infoPMID: 15303329;Abstract: The hawkmoth Manduca sexta (Lepidoptera: Sphingidae), an experimentally favorable Lepidopteran that is highly sensitive to carbon dioxide (CO 2), feeds on the nectar of a range of flowering plants, such as Datura wrightii (Solanaceae). Newly opened Datura flowers give off dramatically elevated levels of CO 2 and offer ample nectar. Thus, floral CO 2 emission could indicate food-source profitability. This study documents that foraging Manduca moths prefer surrogate flowers that emit high levels of CO 2, characteristic of newly opened Datura flowers. We show for the first time that CO 2 may play an important role in the foraging behavior of nectar-feeding insects.
- Christensen, T. A., Lei, H., & Hildebrand, J. G. (2003). Coordination of central odor representations through transient, non-oscillatory synchronization of glomerular output neurons. Proceedings of the National Academy of Sciences of the United States of America, 100(19), 11076-11081.More infoPMID: 12960372;PMCID: PMC196929;Abstract: At the first stage of processing in the olfactory pathway, the patterns of glomerular activity evoked by different scents are both temporally and spatially dynamic. In the antennal lobe (AL) of some insects, coherent firing of AL projection neurons (PNs) can be phase-locked to network oscillations, and it has been proposed that oscillatory synchronization of PN activity may encode the chemical identity of the olfactory stimulus. It remains unclear, however, how the brain uses this time-constrained mechanism to encode chemical identity when the stimulus itself is unpredictably dynamic. In the olfactory pathway of the moth Manduca sexta, we find that different odorants evoke gamma-band oscillations in the AL and the mushroom body (a higher-order network that receives input from the AL), but oscillations within or between these two processing stages are not temporally coherent. Moreover, the timing of action potential firing in PNs is not phase-locked to oscillations in either the AL or mushroom body, and the correlation between PN synchrony and field oscillations remains low before, during, and after olfactory stimulation. These results demonstrate that olfactory circuits in the moth are specialized to preserve time-varying signals in the insect's olfactory space, and that stimulus dynamics rather than intrinsic oscillations modulate the uniquely coordinated pattern of PN synchronization evoked by each olfactory stimulus. We propose that non-oscillatory synchronization provides an adaptive mechanism by which PN ensembles can encode stimulus identity while concurrently monitoring the unpredictable dynamics in the olfactory signal that typically occur under natural stimulus conditions.
- Davis, N. T., Blackburn, M. B., Golubeva, E. G., & Hildebrand, J. G. (2003). Localization of myoinhibitory peptide immunoreactivity in Manduca sexta and Bombyx mori, with indications that the peptide has a role in molting and ecdysis. Journal of Experimental Biology, 206(9), 1449-1460.More infoPMID: 12654884;Abstract: For normal development of Manduca sexta larvae, the ecdysteroid titer must drop following its sudden rise at the start of the molting cycle; this sudden decline in titer may be due to myoinhibitory peptide I (MIP I), which has an inhibitory effect on the release of ecdysone by the prothoracic glands of Bombyx mori in vitro. Using an antiserum to MIP, we have demonstrated secretion of an MIP-like peptide by the epiproctodeal glands of Manduca sexta, which are located on each proctodeal nerve, just anterior to the rectum. These MIP-immunoreactive glands are also present in B. mori. In fourth-instar larvae of M. sexta, the epiproctodeal glands show a distinct cycle of synthesis and sudden release of MIP that coincides with the time of the rapid decline in ecdysteroid titer. The function of the epiproctodeal glands appears to be the timely release of MIP during the molting cycle, so as to inhibit the prothoracic glands and thus to facilitate the sudden decline in ecdysteroid titer. In addition, we have found that MIP immunoreactivity is co-localized with that of crustacean cardioactive peptide (CCAP) in the 704 interneurons; these peptides appear to be co-released at the time of ecdysis. It is known that CCAP can initiate the ecdysis motor program; our results suggest that MIP may also be involved in activating ecdysis behavior.
- Fraser, A. M., Mechaber, W. L., & Hildebrand, J. G. (2003). Electroantennographic and behavioral responses of the sphinx moth Manduca sexta to host plant headspace volatiles. Journal of Chemical Ecology, 29(8), 1813-1833.More infoPMID: 12956509;Abstract: Coupled gas chromatography with electroantennographic detection (GC-EAD) using antennae of adult female Manduca sexta was employed to screen for olfactory stimulants present in headspace collections from four species of larval host plants belonging to two families: Solanaceae - Lycopersicon esculentum (tomato), Capiscum annuum (bell pepper), and Datura wrightii; and Martyniaceae - Proboscidea parviflora. Headspace volatiles were collected from undamaged foliage of potted, living plants. GC-EAD revealed 23 EAD-active compounds, of which 15 were identified by GC-mass spectrometry. Identified compounds included aliphatic, aromatic, and terpenoid compounds bearing a range of functional groups. Nine EAD-active compounds were common to all four host plant species: (Z)-3-hexenyl acetate, nonanal, decanal, phenylacetaldehyde, methyl salicylate, benzyl alcohol, geranyl acetone, (E)-nerolidol, and one unidentified compound. Behavioral responses of female moths to an eight-component synthetic blend of selected tomato headspace volatiles were tested in a laboratory wind tunnel. Females were attracted to the blend. A comparison of responses from antennae of males and females to bell pepper headspace volatiles revealed that males responded to the same suite of volatiles as females, except for (Z)-3-hexenyl benzoate. EAD responses of males also were lower for (Z)- and (E)-nerolidol and one unidentified compound. Electroantennogram EAG dose-response curves for the 15 identified EAD-active volatiles were recorded. At the higher test doses (10-100 μg), female antennae yielded larger EAG responses to terpenoids and to aliphatic and aromatic esters. Male antennae did respond to the higher doses of (Z)-3-hexenyl benzoate, indicating that they can detect this compound. On the basis of ubiquity of the EAD-active volatiles identified to date in host plant headspace collections, we suggest that M. sexta uses a suite of volatiles to locate and identify appropriate host plants.
- Shiga, S., Davis, N. T., & Hildebrand, J. G. (2003). Role of neurosecretory cells in the photoperiodic induction of pupal diapause of the tobacco hornworm Manduca sexta. Journal of Comparative Neurology, 462(3), 275-285.More infoPMID: 12794732;Abstract: In the tobacco hornworm, Manduca sexta, pupal diapause can be induced by exposure of fifth-instar larvae to a short-day photoperiod. We studied the effect of surgical ablation of tissues containing the neurosecretory cells of the brain of fifth-instar larvae on the photoperiodic induction of pupal diapause. At the end of the experiments, we immunostained the neurosecretory cells to determine the success of the ablations. Under long-day conditions (LD 16:8 at 22°C), all intact larvae, most of the sham-operated larvae, and control-operated larvae developed into nondiapausing pupae. Under short-day conditions (LD 10:14 at 22°C), most intact, sham-operated, and control-operated larvae developed into diapausing pupae. Removal of type-II cells did not interfere with the photoperiodic response. Under long-day conditions, elimination of type-Ia1 cells did not affect the incidence of nondiapausing pupae. When type-Ia1 cells were removed under short-day conditions, however, the incidence of nondiapausing pupae was higher (51%, n = 41) than that of the intact (16%, n = 75), sham-operated (24%, n = 88), control-operated larvae (5%, n = 40), and larvae with type-II cells removed (11%, n = 27). Thus, removal of type-Ia1 cells can impede induction of diapause. These results indicate that the type-Ia1 neurosecretory cells have an important role in the induction of pupal diapause. © 2003 Wiley-Liss, Inc.
- Christensen, T. A., & Hildebrand, J. G. (2002). Pheromonal and host-odor processing in the insect antennal lobe: How different?. Current Opinion in Neurobiology, 12(4), 393-399.More infoPMID: 12139986;Abstract: In the olfactory bulb of vertebrates and the antennal lobe of insects, precise connections between sensory receptor cells and olfactory glomeruli form the basis of a highly organized chemotopic map at the first stage of central processing in the brain. Beyond this basic level of organization, the olfactory system is typically separated into two subsystems: a 'main' olfactory pathway that detects and processes information about most environmental odorants, and an 'accessory' olfactory pathway that is devoted to information about social signals such as sex pheromones. A growing number of studies show, however, that it is not always possible to draw clear functional distinctions between the two subsystems. These findings have led some to speculate that the organizational principles by which olfactory stimuli are represented across glomeruli may be more similar in these two olfactory subsystems than previously thought.
- Lei, H., Christensen, T. A., & Hildebrand, J. G. (2002). Local inhibition modulates odor-evoked synchronization of glomerulus-specific output neurons. Nature Neuroscience, 5(6), 557-565.More infoPMID: 12006983;Abstract: At the first stage of olfactory processing in the brain, synchronous firing across glomeruli may help to temporally bind multiple and spatially distributed input streams activated by a given odor. This hypothesis, however, has never been tested in an organism in which the odor-tuning properties of several spatially identifiable glomeruli are known. Using the sphinx moth, an insect that meets these specific criteria, we recorded odor-evoked responses simultaneously from pairs of projection neurons (PNs) innervating the same or different glomeruli in the macroglomerular complex (MGC), which is involved in processing pheromonal information. PNs that branched in the same glomerulus and were activated by the same pheromone component also showed the strongest coincident responses to each odor pulse. Glomerulus-specific PN pairs were also inhibited by the pheromone component that selectively activated PNs in the neighboring glomerulus, and about 70% of all intraglomerular pairs showed increased synchronization when stimulated with a mixture of the two odorants. Thus, when two adjacent glomeruli receive their inputs simultaneously, the temporal tuning of output from each glomerulus is enhanced by reciprocal and inhibitory interglomerular interactions.
- Mechaber, W. L., Capaldo, C. T., & Hildebrand, J. G. (2002). Behavioral responses of adult female tobacco hornworms, Manduca sexta, to hostplant volatiles change with age and mating status.. Journal of insect science (Online), 2, 5-.More infoPMID: 15455039;PMCID: PMC355905;Abstract: We present evidence for two behaviors influenced by intact, vegetative plant odor - upwind flight and abdomen curling - in female Manduca sexta and demonstrate the influence of the age and mating status of the moths on these behaviors. We compared the behavioral responses of laboratory-reared M. sexta. of discrete ages and physiological states (2,3, and 4 day old for virgin; 2 and 3 day old for mated) as individual moths flew upwind in a flight tunnel to a source of hostplant volatiles. We monitored odor-modulated flight and abdomen curling in the presence of volatiles released by potted hostplants. Mated 3 day old females exhibited the highest incidence of odor-modulated flight and abdomen curling. Similarly, as virgin moths aged, a greater percentage of the individuals displayed odor-modulated flight patterns and abdomen curling. In contrast, younger virgin moths exhibited high levels of abdomen curling only after contact with the plant.
- Mercer, A. R., & Hildebrand, J. G. (2002). Developmental changes in the density of ionic currents in antennal-lobe neurons of the sphinx moth, Manduca sexta. Journal of Neurophysiology, 87(6), 2664-2675.More infoPMID: 12037169;Abstract: Early in metamorphic adult development, action potentials elicited from Manduca sexta antennal lobe neurons are small in amplitude, long in duration, and calcium dependent. As development proceeds, the action potential waveform becomes larger in amplitude, shorter in duration, and increasingly sodium dependent. Whole cell voltage-clamp analysis of Manduca antennal-lobe neurons in vitro has been used to identify voltage-activated currents that contribute to developmental changes in the electrical excitability of these cells. Proximal Branching neurons [putative projection (output) neurons] and Rick Rack neurons (putative local antennal-lobe interneurons) are examined in detail early (pupal stage 5) and late (pupal stage 14) in adult metamorphosis. In both cell types, four voltage-gated and two calcium-dependent ionic currents have been identified. Cell-type-specific changes in the density of sodium, calcium, and potassium currents correlate temporally with changes in cell excitability and spike waveform. Developmental changes in ionic current profiles are accompanied also by the emergence of cell-type-specific response characteristics in the cells. Together with the accompanying paper, this study provides an important foundation for examining the impact of developmental changes in electrical excitability on the growth, electrical properties and connectivity of neurons in central olfactory pathways of the moth.
- Mercer, A. R., & Hildebrand, J. G. (2002). Developmental changes in the electrophysiological properties and response characteristics of Manduca antennal-lobe neurons. Journal of Neurophysiology, 87(6), 2650-2663.More infoPMID: 12037168;Abstract: Using whole cell patch-clamp recordings, we have examined changes in the electrophysiological properties and response characteristics of antennal lobe (AL) neurons associated with the metamorphic adult development of the sphinx moth, Manduca sexta. Whole cell current profiles and electrical excitability were examined in dispersed AL neurons in vitro, and in medial-group AL neurons in situ in semi-intact brain preparations. Around stages 2-4 of the 18 stages of metamorphic adult development, whole cell current profiles were dominated by large outward (K+) currents. Calcium-dependent action potentials could be elicited at this stage, but only a small percentage of cells exhibited sodium spikes. From stages 3 to 10, there was a rapid increase in the proportion of AL neurons exhibiting rapidly activating, transient sodium currents, and many cells in vitro exhibited spontaneous bursts of spike activity at this time. As development progressed, action-potential waveforms became shorter in duration and larger in amplitude. Cell-type-specific differences in the prevalence of spontaneous activity, and in the electrophysiological properties and response characteristics of AL neurons, were most apparent late in metamorphosis. While removal of antennal sensory input to the ALs early (stage 1-2) in metamorphosis had no detectable effect on the development of cell excitability, a significantly higher percentage of neurons in vitro from stage 4 pupae exhibited sodium-based action potentials following the addition of serotonin to the culture medium. Characteristic forms of electrical excitability in developing Manduca AL neurons, and their modulation by serotonin, seem likely to play a central role in the functional development of the ALs.
- Nighorn, A., & Hildebrand, J. G. (2002). Dissecting the molecular mechanisms of olfaction in a malaria-vector mosquito. Proceedings of the National Academy of Sciences of the United States of America, 99(3), 1113-1114.More infoPMID: 11830653;PMCID: PMC122153;
- Christensen, T. A., D'Alessandro, G., Lega, J., & Hildebrand, J. G. (2001). Morphometric modeling of olfactory circuits in the insect antennal lobe: I. Simulations of spiking local interneurons. BioSystems, 61(2-3), 143-153.More infoPMID: 11716974;PMCID: PMC2773206;Abstract: Inhibitory local interneurons (LNs) play a critical role in shaping the output of olfactory glomeruli in both the olfactory bulb of vertebrates and the antennal lobe of insects and other invertebrates. In order to examine how the complex geometry of LNs may affect signaling in the antennal lobe, we constructed detailed multi-compartmental models of single LNs from the sphinx moth, Manduca sexta, using morphometric data from confocal-microscopic images. Simulations clearly revealed a directionality in LNs that impeded the propagation of injected currents from the sub-micron-diameter glomerular dendrites toward the much larger-diameter integrating segment (IS) in the coarse neuropil. Furthermore, the addition of randomly-firing synapses distributed across the LN dendrites (simulating the noisy baseline activity of afferent input recorded from LNs in the odor-free state) led to a significant depolarization of the LN. Thus the background activity typically recorded from LNs in vivo could influence synaptic integration and spike transformation in LNs through voltage-dependent mechanisms. Other model manipulations showed that active currents inserted into the IS can help synchronize the activation of inhibitory synapses in glomeruli across the antennal lobe. These data, therefore, support experimental findings suggesting that spiking inhibitory LNs can operate as multifunctional units under different ambient odor conditions. At low odor intensities, (i.e. subthreshold for IS spiking), they participate in local, mostly intra-glomerular processing. When activated by elevated odor concentrations, however, the same neurons will fire overshooting action potentials, resulting in the spread of inhibition more globally across the antennal lobe. Modulation of the passive and active properties of LNs may, therefore, be a deciding factor in defining the multi-glomerular representations of odors in the brain. Copyright © 2001 Elsevier Science Ireland Ltd.
- Davis, N. T., Dulcis, D., & Hildebrand, J. G. (2001). Innervation of the heart and aorta of Manduca sexta. Journal of Comparative Neurology, 440(3), 245-260.More infoPMID: 11745621;Abstract: Innervation of the heart and aorta of Manduca sexta was studied by using anatomic, neuronal tracing and immunocytochemical techniques. The study was undertaken to provide a foundation for investigating the neural mechanisms controlling cardiac reversal in adults. Lateral cardiac nerves were not found in the larval or adult heart. The larval heart and aorta seem to lack innervation, but a neurohemal system for the release of a cardioactive peptide is associated with the larval alary muscles. At adult metamorphosis, this neurohemal system regresses, and, at the same time, processes grow onto the anterior aorta. These processes seem to be neurohemal and originate from two pairs of neurosecretory cells located in the subesophageal ganglion. This system is immunoreactive to cardioactive peptides and may function, therefore, in hormonal modulation of the activity of the adult heart. Also during metamorphosis, synaptic innervation develops on the terminal heart chamber, and this innervation is from axons extending through the seventh and eighth dorsal nerves of the terminal abdominal ganglion. These axons originate from cells that have been identified as serial homologs of motor neuron-1 of other abdominal ganglia. These neurons are immunoreactive to a cardioactive peptide, and this peptide probably modulates the synaptic innervation of the terminal heart chamber. During metamorphosis, the target of the motor neurons-1 of the seventh and eighth segments becomes respecified from larval skeletal muscles to the terminal chamber of the adult heart. © 2001 Wiley-Liss, Inc.
- Dulcis, D., Davis, N. T., & Hildebrand, J. G. (2001). Neuronal control of heart reversal in the hawkmoth Manduca sexta. Journal of Comparative Physiology - A Sensory, Neural, and Behavioral Physiology, 187(10), 837-849.More infoPMID: 11800040;Abstract: Cardiograms demonstrate that heart activity of Manduca sexta changes from larva, to pupa, to adult. The larval heart has only anterograde contractions. During metamorphosis, heart activity becomes a cyclic alternation of anterograde and retrograde contractions. Thus, the adult heart has both an anterograde and a retrograde pacemaker. External stimuli also can initiate cardiac reversal. Cardiac reversal is blocked by tetrodotoxin, indicating that reversal is under neuronal control. A branch of each dorsal nerve 8 innervates the posterior chamber of the heart, the location of the anterograde pacemaker. Only retrograde contractions occur when dorsal nerves 8 are cut. Stimulation of ml-1 8 initiates anterograde contractions; when stimulation ceases, the heart reverses to retrograde contractions. These experiments indicate that the anterograde pacemaker receives neural input that makes it the dominant pacemaker. In the absence of neural input this pacemaker is inactive, and the retrograde pacemaker becomes active. Application of crustacean cardioactive peptide accelerates the heart but does not eliminate cardiac reversal. The terminal chamber of the heart is also innervated by a branch of each dorsal nerve 7; stimulation of this nerve increases the strength of contraction of the terminal chamber but has no effect on contractions of the remainder of the heart or on cardiac reversal.
- Gibson, N. J., Roessler, W. R., Nighorn, A. J., Oland, L. A., Hildebrand, J. G., & Tolbert, L. P. (2001). Neuron-glia communication via nitric oxide is essential in establishing antennal-lobe structure in Manduca sexta. Developmental Biology, 240, 326-39.
- Gibson, N. J., Rössler, W., Nighorn, A. J., Oland, L. A., Hildebrand, J. G., & Tolbert, L. P. (2001). Neuron-glia communication via nitric oxide is essential in establishing antennal-lobe structure in Manduca sexta. Developmental Biology, 240(2), 326-339.More infoPMID: 11784067;Abstract: Nitric oxide synthase recently has been shown to be present in olfactory receptor cells throughout development of the adult antennal (olfactory) lobe of the brain of the moth Manduca sexta. Here, we investigate the possible involvement of nitric oxide (NO) in antennal-lobe morphogenesis. Inhibition of NO signaling with a NO synthase inhibitor or a NO scavenger early in development results in abnormal antennal lobes in which neuropil-associated glia fail to migrate. A more subtle effect is seen in the arborization of dendrites of a serotonin-immunoreactive neuron, which grow beyond their normal range. The effects of NO signaling in these types of cells do not appear to be mediated by activation of soluble guanylyl cyclase to produce cGMP, as these cells do not exhibit cGMP immunoreactivity following NO stimulation and are not affected by infusion of a soluble guanylyl cyclase inhibitor. Treatment with Novobiocin, which blocks ADP-ribosylation of proteins, results in a phenotype similar to those seen with blockade of NO signaling. Thus, axons of olfactory receptor cells appear to trigger glial cell migration and limit arborization of serotonin-immunoreactive neurons via NO signaling. The NO effect may be mediated in part by ADP-ribosylation of target cell proteins. © 2001 Elsevier Science.
- Shields, V. D., & Hildebrand, J. G. (2001). Recent advances in insect olfaction, specifically regarding the morphology and sensory physiology of antennal sensilla of the female sphinx moth Manduca sexta. Microscopy Research and Technique, 55(5), 307-329.More infoPMID: 11754510;PMCID: PMC2386875;Abstract: The antennal flagellum of female Manduca sexta bears eight sensillum types: two trichoid, two basiconic, one auriculate, two coeloconic, and one styliform complex sensilla. The first type of trichoid sensillum averages 34 μm in length and is innervated by two sensory cells. The second type averages 26 μm in length and is innervated by either one or three sensory cells. The first type of basiconic sensillum averages 22 μm in length, while the second type averages 15 μm in length. Both types are innervated by three bipolar sensory cells. The auriculate sensillum averages 4 μm in length and is innervated by two bipolar sensory cells. The coeloconic type-A and type-B both average 2 μm in length. The former type is innervated by five bipolar sensory cells, while the latter type, by three bipolar sensory cells. The styliform complex sensillum occurs singly on each annulus and averages 38-40 μm in length. It is formed by several contiguous sensilla. Each unit is innervated by three bipolar sensory cells. A total of 2,216 sensilla were found on a single annulus (annulus 21) of the flagellum. Electrophysiological responses from type-A trichoid sensilla to a large panel of volatile odorants revealed three different subsets of olfactory receptor cells (ORCs). Two subsets responded strongly to only a narrow range of odorants, while the third responded strongly to a broad range of odorants. Anterograde labeling of ORCs from type-A trichoid sensilla revealed that their axons projected mainly to two large female glomeruli of the antennal lobe. © 2001 Wiley-Liss, Inc.
- Vickers, N. J., Christensen, T. A., Baker, T. C., & Hildebrand, J. G. (2001). Odour-plume dynamics influence file brain's olfactory code. Nature, 410(6827), 466-470.More infoPMID: 11260713;Abstract: The neural computations used to represent olfactory information in the brain have long been investigated. Recent studies in the insect antennal lobe suggest that precise temporal and/or spatial patterns of activity underlie the recognition and discrimination of different odours, and that these patterns may be strengthened by associative learning. It remains unknown, however, whether these activity patterns persist when odour intensity varies rapidly and unpredictably, as often occurs in nature. Here we show that with naturally intermittent odour stimulation, spike patterns recorded from moth antennal-lobe output neurons varied predictably with the fine-scale temporal dynamics and intensity of the odour. These data support the hypothesis that olfactory circuits compensate for contextual variations in the stimulus pattern with high temporal precision. The timing of output neuron activity is constantly modulated to reflect ongoing changes in stimulus intensity and dynamics that occur on a millisecond timescale.
- Christensen, T. A., Pawlowski, V. M., Lei, H., & Hildebrand, J. G. (2000). Multi-unit recordings reveal context-dependent modulation of synchrony in odor-specific neural ensembles. Nature Neuroscience, 3(9), 927-931.More infoPMID: 10966624;Abstract: We used neural ensemble recording to examine odor-evoked ensemble patterns in the moth antennal (olfactory) lobe. Different odors are thought to evoke unique spatiotemporal patterns of glomerular activity, but little is known about the population dynamics underlying formation of these patterns. Using a silicon multielectrode array, we observed dynamic network interactions within and between glomeruli. Whereas brief odor pulses repeatedly triggered activity in the same coding ensemble, the temporal pattern of synchronous activity superimposed on the ensemble was neither oscillatory nor odor specific. Rather, synchrony strongly depended on contextual variables such as odor intensity and intermittency. Also, because of emergent inhibitory circuit interactions, odor blends evoked temporal ensemble patterns that could not be predicted from the responses to the individual odorants. Thus even at this early stage of information processing, the timing of odor-evoked neural representations is modulated by key stimulus factors unrelated to the molecular identity of the odor.
- King, J. R., Christensen, T. A., & Hildebrand, J. G. (2000). Response characteristics of an identified, sexually dimorphic olfactory glomerulus. Journal of Neuroscience, 20(6), 2391-2399.More infoPMID: 10704513;Abstract: Partitioning of synaptic neuropil into glomeruli is a common feature of primary olfactory centers in most animal species. The functional significance of glomeruli, however, is not yet well understood. The present study is part of our effort to test the hypothesis that each glomerulus is a functional unit dedicated to processing information about a particular odorant or attribute of odor molecules and that the glomerular array constitutes a map of 'odor space.' We investigated the physiological and morphological features of uniglomerular projection neurons (PNs) associated with an identified glomerulus in each antennal lobe of the female sphinx moth, Manduca sexta. This 'lateral large female glomerulus' (latLFG) is sexually dimorphic and therefore may play a female-specific role, such as processing of information about one or more odorants important for orientation of a female to host plants for oviposition. Together with the medial LFG (medLFG), the latLFG resides outside the array of spheroidal ordinary glomeruli, near the entrance of the antennal (olfactory) nerve. Each LFG is innervated by four to five PNs. Using intracellular recording and staining, we examined the responses of latLFG-PNs to odorants that represent major classes of volatiles released by host plants of M. sexta. All latLFG-PNs were excited when the ipsilateral antenna was stimulated with low concentrations of the monoterpenoid linalool. Dose-response analysis showed that neither other monoterpenoids nor representatives of other classes of host plant volatiles were similarly stimulatory to latLFG-PNs. These findings are consistent with the idea that each glomerulus has a characteristic, limited molecular receptive range.
- Lehman, H. K., Murgiuc, C. M., & Hildebrand, J. G. (2000). Characterization and developmental regulation of tyramine-β-hydroxylase in the CNS of the moth, Manduca sexta. Insect Biochemistry and Molecular Biology, 30(5), 377-386.More infoPMID: 10745161;Abstract: Octopamine (OA) is present in insect nervous tissue, but little is known about its biosynthesis. In the CNS of Manduca sexta, OA levels increase markedly during postembryonic adult development. To study this increase, we developed an assay for tyramine-β-hydroxylase, the putatively rate-limiting enzyme for OA biosynthesis. Tyramine-β-hydroxylase activity in extracts of M. sexta CNS tissue: (1) was time- and protein-dependent, and with protein concentrations up to 2 μg/μl, was linear for 20 min; (2) had a pH optimum of 7.0 for conversion of tyramine to OA; (3) required ascorbate, copper, and catalase; and (4) had an apparent K(M, tyramine) of 0.22±0.04 mM. These characteristics resemble those of the mammalian enzyme dopamine-β- hydroxylase, suggesting that these two enzymes are functionally related. During adult development, tyramine-β-hydroxylase activity increased 11-fold in the brain and 9-fold in the abdominal ganglia, paralleling increases in OA levels in those CNS structures during metamorphosis. The apparent kinetic constants of tyramine-β-hydroxylase suggested that the amount of this enzyme present in the tissues increases. The increase in OA levels during adult development thus appears to be due to an increase in the level of enzyme available for OA synthesis and may reflect an increase in the number of octopaminergic neurons. (C) 2000 Published by Elsevier Science Ltd.
- Mechaber, W. L., & Hildebrand, J. G. (2000). Novel, non-solanaceous hostplant record for Manduca sexta (Lepidoptera: Sphingidae) in the Southwestern United States. Annals of the Entomological Society of America, 93(3), 447-451.More infoAbstract: Field observations in the Tucson, AZ, basin reveal that Manduca sexta (L.) has been incorrectly described in the past as a specialist herbivore that oviposits and feeds exclusively on plants in the family Solanaceae. We present evidence that previously unreported, non-solanaceous host-plants are routinely used by M. sexta females for oviposition. These plants permit successful larval development, through to emergence of the next generation of adults. The novel hostplants. 2 species of the genus Proboscidea, belong to the Martyniaceae, a family taxonomically distant from the Solanaceae. Our observations on oviposition and larval feeding were conducted during 2 consecutive field seasons. During the 2nd field season, we counted M. sexta eggs on native hostplants, both solanaceous (Datura wrightii, Regel) and non-solanaceous (2 Proboscidea species), and found higher abundance of eggs on the Proboscidea spp., in approximately a 3:1 ratio. Our results challenge the commonly held view that M. sexta is a specialist exclusively on solanaceous plants.
- Rospars, J. P., & Hildebrand, J. G. (2000). Sexually dimorphic and isomorphic glomeruli in the antennal lobes of the sphinx moth manduca sexta. Chemical Senses, 25(2), 119-129.More infoPMID: 10781018;Abstract: Antennal robes of adult male and female Manduca sexta were compared in order to investigate the nature and extent of sexual dimorphism of the primary olfactory center of this lepidopteran species. Complete identification of the glomeruli led to the conclusion that all female glomeruli have homologous male counterparts. Thus, there is no sex-specific glomerulus present in one sex and absent in the other. Sexual dimorphism (i.e. glomeruli present but morphologically different in males and females), however, does occur in the three glomeruli composing the male macroglomerular complex. The female homologs of this complex consist of two previously identified 'large female glomeruli' and one newly identified normal-sized glomerulus. The lateral and medial large female glomeruli are interpreted to be homologous to the first two macroglomerular-complex glomeruli - the cumulus and toroid 1. The third male component, the toroid 2, was tentatively identified with a normal-sized spheroidal glomerulus of the female, called here the 'small female glomerulus'. The 60 'ordinary' glomeruli that make up the rest of the glomerular neuropil were found to be homologous in males and females, with the exception of two anomalous (or uncertain) glomeruli. Some variations in relative position and size observed among those glomeruli suggest a diffuse, quantitative kind of sexual dimorphism.
- Rössler, W., Tolbert, L. P., & Hildebrand, J. G. (2000). Importance of timing of olfactory receptor-axon outgrowth for glomerulus development in Manduca sexta. Journal of Comparative Neurology, 425(2), 233-243.More infoPMID: 10954842;Abstract: In the moth Manduca sexta, development of glomeruli in the antennal (olfactory) lobes (ALs) follows a precise timetable and involves interactions of olfactory receptor cell (ORC) axons with AL glial cells and neurons. To study the importance of timing for these intercellular interactions, we experimentally desynchronized the development of the ALs and the ORCs by altering the temperature of the developing antenna and brain for defined periods of time during development. Selective cooling of the antenna relative to the body resulted in a delay of ORC-axon outgrowth, and slightly warming the antenna while cooling the body caused precocious ingrowth of axons into the AL. Whereas cooling of the antenna for 24 hours caused only a delay in the formation of glomeruli, cooling for 48 hours led to significant disruption of glomerular development. Glial cells did not form normal glomerular borders, and glomeruli were shaped abnormally. Axons of pheromone-specific ORCs projected to their correct target, but terminal branches within the macroglomerular complex (MGC) were not clearly segregated. The results suggest that proper formation of glial glomerular borders requires interaction of ORC axons and glial cells within a sensitive period, whereas targeting of ORC axons appears to be effective over extended periods in development. Precocious ingrowth of ORC axons after warming the antenna and cooling the body for 48 hours resulted in enlarged protoglomeruli. Glial borders formed normally, but a subpopulation of MGC-specific ORC axons grew past the MGC. The decreased accuracy of targeting in these cases suggests that targeting mechanisms are not fully developed before the time when ORC axons normally would enter the brain. (C) 2000 Wiley-Liss, Inc.
- Shields, V. D., & Hildebrand, J. G. (2000). Responses of a population of antennal olfactory receptor cells in the female moth Manduca sexta to plant-associated volatile organic compounds. Journal of Comparative Physiology - A Sensory, Neural, and Behavioral Physiology, 186(12), 1135-1151.More infoPMID: 11288825;Abstract: Extracellular electrophysiological recordings were made from individual type-A trichoid sensilla on the antenna of the female sphinx moth Manduca sexta. A single annulus of the antenna bears about 1,100 of these sensilla, and each is innervated by two olfactory receptor cells. We tested the responses of these receptor cells to a panel of 102 volatile compounds, as well as three plant-derived odor mixtures, and could discern three different functional types of type-A trichoid sensilla. One subset of receptor cells exhibited an apparently narrow molecular receptive range, responding strongly to only one or two terpenoid odorants. The second subset was activated exclusively by aromatics and responded strongly to two to seven odorants. The third subset had a broad molecular receptive range and responded strongly to odorants belonging to several chemical classes. We also found receptor cells that did not respond to any of the odorants tested but were spontaneously active. Certain odorants elicited excitatory responses in some sensilla but inhibitory responses in others, and some receptor cells were strongly excited by certain odorants but inhibited by others. Impregnation of groups of receptor cells in type-A trichoid sensilla with rhodamine-dextran demonstrated that their axons project mainly to the large female glomeruli of the antennal lobe.
- E., P., Davis, N. T., Meredith, J. A., Christensen, T. A., & Hildebrand, J. G. (1999). Role of the ventral nerve cord and terminal abdominal ganglion in the regulation of sex pheromone production in the tobacco budworm (Lepidoptera: Noctuidae). Annals of the Entomological Society of America, 92(6), 891-901.More infoAbstract: We sought to clarify the role of the nervous system, and its relationship to hormonal regulation, in controlling sex-pheromone biosynthesis in the female tobacco budworm moth, Heliothis virescens (F.). Isolation of the terminal abdominal ganglion (TAG) from the rest of the ventral nerve cord (VNC), or transection of the terminal nerves that extend from the TAG to the periphery, resulted in severe reduction in the production of sex pheromone that normally occurs during the scotophase. The amount of pheromone extracted from the sex-pheromone glands of these surgically manipulated moths, however, was greater than that extracted from glands of normal females during the photophase, a period when pheromone levels are low or undetectable. Injection of an extract of female TAGs into virgin females stimulated production of sex pheromone. Fractionation of extracts of the TAG and of the brain/subesophageal ganglion (Brain-SEG) complex by solid-phase extraction, ion exchange, and reverse-phase liquid chromatography revealed that the TAG contains pheromonotropic peptides with retention characteristics very similar to those of the Brain-SEG. Immunochemical studies showed that the TAG-derived pheromonotropic peptides have some degree of homology, at their C-termini, to the pheromone-biosynthesis-activating neuropeptide of the corn earworm, Helicoverpa zea (Hez-PBAN). Discrepancies between chromatographic properties of Hez-PBAN and the TAG pheromonotropic peptides, however, indicated that the peptides from H. virescens are not identical to Hez-PBAN. Immunocytochemical studies revealed that axons of PBAN-immunoreactive neurons in the SEG project through the VNC and terminate in the neuropil of the TAG. Because the immunoreactivity was not found in nerves or neurohemal organs of the TAG, it is likely that the pheromonotropic peptides in the TAG function within the TAG rather than as neurohormones or peripheral neurotransmitters. It is not yet clear whether these PBAN-like peptides are involved in control of the pheromone gland. Nevertheless, our findings suggest that in H. virescens, optimal production of sex pheromone depends on both neural and hormonal regulation.
- Heinbockel, T., Christensen, T. A., & Hildebrand, J. G. (1999). Temporal tuning of odor responses in pheromone-responsive projection neurons in the brain of the sphinx moth Manduca sexta. Journal of Comparative Neurology, 409(1), 1-12.More infoPMID: 10363707;Abstract: By means of intracellular recording and staining, we studied the ability of several distinct classes of projection (output) neurons, which innervate the sexually dimorphic macroglomerular complex (MGC-PNs) in the antennal lobe of the male moth Manduca sexta, to encode naturally intermittent sex pheromonal stimuli. In many MGC-PNs, antennal stimulation with a blend of the two essential pheromone components evoked a characteristic triphasic response consisting of a brief, hyperpolarizing inhibitory potential (I1) followed by depolarization with firing of action potentials and then a delayed period of hyperpolarization (I2). MGC-PNs described in this study resolved pulsed pheromonal stimuli, up to about five pulses/second, with a distinct burst of action potentials for each pulse of odor. The larger the amplitude of I1, the higher the pulse rate an MGC-PN could follow, illustrating the importance of inhibitory synaptic input in shaping the temporal firing properties of these glomerular output neurons. In some MGC-PNs, triphasic responses were evoked by antennal stimulation with only one of the two key pheromone components. Again, the maximal pulse rate that an MGC-PN could follow with that pheromone component as sole stimulus was high in MGC-PNs that responded with a strong I1. These component-specific MGC-PNs innervated only one of the two principal glomeruli of the MGC, while MGC-PNs that were primarily excited by antennal stimulation with either key pheromone component had arborizations in both major MGC glomeruli. These observations therefore suggest that the population of antennal olfactory receptor cells responding to a single pheromone component is functionally heterogeneous: a subset of these sensory cells activates the excitatory drive to many uniglomerular MGC- PNs, while others feed onto inhibitory circuits that hyperpolarize the same PNs. This convergence of opposing inputs is a circuit property common to uniglomerular MGC-PNs branching in either of the major MGC glomeruli, and it enhances the ability of these glomerular output neurons to resolve intermittent olfactory input. Synaptic integration at the uniglomerular PN level thus contributes to the transmission of behaviorally important temporal information about each key pheromone component to higher centers in the brain.
- Hildebrand, J. G. (1999). Journal of Comparative Physiology A: Sensory, Neural, and Behavioral Physiology: Preface. Journal of Comparative Physiology - A Sensory, Neural, and Behavioral Physiology, 185(4), 289-.
- Kent, K. S., Oland, L. A., & Hildebrand, J. G. (1999). Development of the labial pit organ glomerulus in the antennal lobe of the moth Manduca sexta: The role of afferent projections in the formation of identifiable olfactory glomeruli. Journal of Neurobiology, 40(1), 28-44.More infoPMID: 10398069;Abstract: Iterated neuropil modules called glomeruli are characteristic of primary olfactory centers in both vertebrates and invertebrates. To gain insight into the developmental mechanisms underlying the formation of such structured, organized neuropil, we have examined the development of an identified glomerulus in the olfactory (antennal) lobe of the moth Manduca sexta. The labial pit organ glomerulus (LPOG) receives bilateral sensory projections from the labial pit organs in the labial palps of the mouthparts, while other glomeruli in the antennal lobe receive unilateral projections from the antenna. Here, we chronicle the development of the LPOG under normal and perturbed conditions. Our findings suggest that the sensory axons of the labial pit organ, like those of the antenna, induce and shape growth of interneuronal arborizations, but specific features of interneuronal arborizations such as the relative position of glomerular arborizations within the antennal lobe are independent of both classes of afferent innervation. Labial pit organ axons and antennal axons exhibit a high degree of specificity for their respective target regions, independent of the presence or absence of the other class of afferent axon or the route taken to the antennal lobe. Specification of glomerular position is intrinsic to the antennal lobe rather than a consequence of competition between afferent axons.
- Kent, K., Oland, L. A., Hildebrand, J. G., Kent, K., Oland, L. A., & Hildebrand, J. G. (1999). Development of the labial pit organ glomerulus in the antennal lobe of the moth Manduca sexta: The role of afferent projections in the formation of an identified olfactory glomerulus. Journal of Neurobiology, 40, 28-44.
- Roessler, W. R., Oland, L. A., Higgins, M. R., Hildebrand, J. G., & Tolbert, L. P. (1999). Development of a glia-rich axon-sorting zone in the olfactory pathway of the moth Manduca sexta. Journal of Neuroscience, 19, 9865-77.More infoPMID: 10559396
- Rössler, W., Oland, L. A., Higgins, M. R., Hildebrand, J. G., & Tolbert, L. P. (1999). Development of a glia-rich axon-sorting in the olfactory pathway of the moth Manduca sexta. Journal of Neuroscience, 19(22), 9865-9877.More infoPMID: 10559396;Abstract: Olfactory receptor cells (ORCs) of a particular odor tuning are dispersed in the olfactory epithelium, but their axons converge on distinct glomeruli in primary olfactory centers. As a consequence, axon associations must change to bring axons of ORCs with the same odor specificity together. Studies in Manduca sexta have indicated that just before they enter the antennal lobe (AL), ORC axons undergo extreme reorganization, finally entering the AL in fascicles destined for subsets of glomeruli. This axon- sorting zone is heavily populated by glial cells, and ORC axon growth cones often are in close physical contact with the glia. In moths rendered gila deficient, ORC axons fail to fasciculate in this region. Using propidium iodide to label nuclei and 5-bromo-2'-deoxyuridine to monitor proliferation, we found that the gila in the sorting zone arise from the AL, appearing shortly after the first ORC axons arrive. Experimental removal of some or all of the sensory innervation revealed that proliferation of sorting-zone glia is triggered by ORC axons. A second set of glia arises in the antenna and migrates along the antennal nerve toward the brain, populating the nerve after the establishment of the sorting zone. Development of this type of glial cell is independent of contact of the ORC axons with their central targets. We conclude that the sorting zone arises from CNS glia in response to ingrowth of ORC axons, and a critical number of glia must be present in the sorting zone for axons to correctly establish new neighbor-neighbor associations.
- Rössler, W., Randolph, P. W., Tolbert, L. P., & Hildebrand, J. G. (1999). Axons of olfactory receptor cells of transsexually grafted antennae induce development of sexually dimorphic glomeruli in Manduca sexta. Journal of Neurobiology, 38(4), 521-541.More infoPMID: 10084687;Abstract: The influence of olfactory receptor cell (ORC) axons from transsexually grafted antennae on the development of glomeruli in the antennal lobes (ALs), the primary olfactory centers, was studied in the moth Manduca sexta. Normally during metamorphic adult development, the pheromone-specific macroglomerular complex (MGC) forms only in the ALs of males, whereas two lateral female-specific glomeruli (LFGs) develop exclusively in females. A female AL innervated by ORC axons from a grafted male antenna developed an MGC with three glomeruli, like the MGC of a normal male AL. Conversely, a male AL innervated by ORC axons from a grafted female antenna lacked the MGC but exhibited LFGs. ORC axons from grafted male antenna terminated in the MGC-specific target area, even in cases when the antennal nerve (AN) entered the AL via an abnormal route. Within ectopic neuromas formed by ANs that had become misrouted and failed to enter the brain, male-specific axons were not organized and formed terminal branches in many areas. The results suggest the presence of guidance cues within the AL for male-specific ORC axons. Depending on the sex of the antennal innervation, glial borders formed in a pattern characteristic of the MGC or LFGs. The sex-specific number of projection neurons (PNs) in the medial group of AL neurons remained unaffected by the antennal graft, but significant changes occurred in the organization of PN arborizations. In gynandromorphic females, LFG-specific PNs extended processes into the induced MGC, whereas in gynandromorphic males, PNs became restricted to the LFGs. The results indicate that male-and female-specific ORC axons play important roles in determining the position, anatomical features, and innervation of sexually dimorphic glomeruli.
- Shields, V. D., & Hildebrand, J. G. (1999). Fine structure of antennal sensilla of the female sphinx moth, Manduca sexta (Lepidoptera: Sphingidae). I. Trichoid and basiconic sensilla. Canadian Journal of Zoology, 77(2), 290-301.More infoAbstract: Olfaction is the principal sensory modality through which insects locate their food sources, mates, and oviposition sites. Insects offer certain advantages as experimental models for the study of mechanisms of olfaction, and a thorough knowledge of the antennal olfactory sensory cells is essential for such studies. While the sphinx moth, Manduca sexta, has proved to be an especially favorable experimental model in studies of pheromone processing in males, little is known about the female olfactory system, including the antennal olfactory sensilla. In this and another paper in this series, we present the first thorough investigation of the structure, innervation, number, and distribution of sensilla on the antennal flagellum of female M. sexta. The flagellum of the female moth bears eight morphological types of sensilla: two trichoid, two basiconic, one auriculate, two coeloconic, and one styliform complex. In this paper, we describe the two types of trichoid and two types of basiconic sensilla. The first type of trichoid sensillum a long hairlike sensillum averaging 34 μm in length, is innervated by two bipolar sensory cells, and the second type, a shorter hairlike sensillum averaging 26 μm length, is innervated by either one or three bipolar sensory cells. The first type of basiconic sensillum is a long peg, averaging 22 μm in length, and the second is a shorter peg, averaging 15 μm in length. Both types of basiconic sensilla are innervated by three bipolar sensory cells. These trichoid and basiconic sensilla have structural features characteristics of insect olfactory sensilla.
- Shields, V. D., & Hildebrand, J. G. (1999). Fine structure of antennal sensilla of the female sphinx moth, Manduca sexta (Lepidoptera: Sphingidae). II. Auriculate, coeloconic, and styliform complex sensilla. Canadian Journal of Zoology, 77(2), 302-313.More infoAbstract: The antennal flagellum of the female sphinx moth, Manduca sexta, bears eight types of sensilla: two trichoid, two basiconic, one auriculate, two coeloconic, and one styliform complex. We previously described the fine structure of the trichoid and basiconic sensilla (Shields and Hildebrand 1999). In this paper, we described one type of auriculate, two types of coeloconic, and one type of styliform complex sensilla. The auriculate (ear-or spoon-shaped) sensillum is a small peg that averages 4 μm in length, is innervated by two bipolar sensory cells, and has structural features characteristic of an insect olfactory sensillum. Each of the two types of coeloconic sensilla is a small peg that averages 2 μm in length and is recessed in a cuticular pit. One type of coeloconic sensillum is innervated by five bipolar sensory cells and has structural features characteristic of an insect olfactory sensillum or olfactory-thermosensillum, while the other is innervated by three bipolar sensory cells and has structural features characteristic of an insect thermo-hygrosensillum. The styliform complex sensillum is a large peg that averages 38-40 μm in length and is formed by several contiguous sensilla, the number of which depends on the location of the peg on the flagellum. Each unit of the styliform complex sensillum is innervated by three bipolar sensory cells and has structural features characteristic of a thermo-hygrosensillum. We also ascertained the number and distribution of each of the eight types of sensilla on a single flagellomere (annulus) about midway along the flagellum of a female antena. A total of 2216 sensilla were found on the dorsal, ventral, and leading surfaces of that annulus.
- Strausfeld, N. J., & Hildebrand, J. G. (1999). Olfactory systems: Common design, uncommon origins?. Current Opinion in Neurobiology, 9(5), 634-639.More infoPMID: 10508748;Abstract: In both vertebrates and invertebrates, odorant molecules reach the dendrites of olfactory receptor cells through an aqueous medium, which reflects the evolutionary origin of these systems in a marine environment. Important recent advances, however, have demonstrated striking interphyletic differences between the structure of vertebrate and invertebrate olfactory receptor proteins, as well as the organization of the genes encoding them. While these disparities support independent origins for odor-processing systems in craniates and protostomes (and even between the nasal and vomeronasal systems of craniates), olfactory neuropils share close neuroanatomical and physiological characters. Whereas there is a case to be made for homology among members of the two great protostome clades (the ecdysozoans and lophotrochozoans), the position of the craniates remains ambiguous.
- Christensen, T. A., Waldrop, B. R., & Hildebrand, J. G. (1998). GABAergic mechanisms that shape the temporal response to odors in moth olfactory projection neurons. Annals of the New York Academy of Sciences, 855, 475-481.More infoPMID: 9929641;Abstract: Mitral/tufted cells in the olfactory bulb and projection neurons (PNs) in the insect antennal lobe are involved in complex synaptic interactions with inhibitory interneurons to help shape their odor-evoked responses. In the moth Manduca sexta, both γ-aminobutyric acid (GABA) and the GABA(A) receptor agonist muscimol hyperpolarize and lower input resistance in many PNs, often blocking ongoing spike traffic. The GABA response mimics a short-latency, chloride-mediated inhibitory postsynaptic potential (IPSP) evoked in PNs by electrical or odor stimulation of afferent inputs, and the classical GABA(A) receptor antagonist bicuculline methiodide (BMI) quickly and reversibly blocks this IPSP. Focal injection of BMI (100 μM) immediately preceding a GABA pulse blocks the hyperpolarization evoked by GABA, but a similar injection of BMI preceding an acetylcholine (ACh) pulse fails to block the depolarization evoked by ACh. Moreover, the temporal pattern of odor-evoked activity in moth PNs is also strongly and reversibly altered by BMI. Importantly, the temporal pattern of the response depends on the temporal characteristics of the stimulus: continuous stimulation evokes more complex, rhythmic responses, whereas a pulsatile stimulus can be copied with a discrete burst of spikes for each pulse. Collectively our results indicate that PNs in the moth antennal lobe possess GABA receptors that share certain characteristics in common with vertebrate GABA(A) receptors. These receptors are largely responsible for helping PNs integrate information about both the molecular features and the timing of olfactory input to the brain.
- Heinbockel, T., & Hildebrand, J. G. (1998). Antennal receptive fields of pheromone-responsive projection neurons in the antennal lobes of the male sphinx moth Manduca sexta. Journal of Comparative Physiology - A Sensory, Neural, and Behavioral Physiology, 183(2), 121-133.More infoPMID: 9693989;Abstract: Stimulation of the antenna of the male moth, Manduca sexta, with a key component of the female's sex pheromone and a mimic of the second key component evokes responses in projection neurons in the sexually dimorphic macroglomerular complex of the antennal lobe. Using intracellular recording and staining techniques, we studied the antennal receptive fields of 149 such projection neurons. An antennal flagellum was stimulated in six regions along its proximo-distal axis with one or both of the pheromone-related compounds while activity was recorded in projection neurons. These neurons fell mainly into two groups, based on their responses to the two-component blend: neurons with broad receptive fields that were excited when any region of the flagellum was stimulated, and neurons selectively excited by stimulation of the proximal region of the flagellum. Projection neurons that were depolarized by stimulation of one antennal region were not inhibited by stimulation of other regions, suggesting absence of antennotopic center-surround organization. In most projection neurons, the receptive field was determined by afferent input evoked by only one of the two components. Different receptive-field properties of projection neurons may be related to the roles of these neurons in sensory control of the various phases of pheromone-modulated behavior of male moths.
- Heinbockel, T., Kloppenburg, P., & Hildebrand, J. G. (1998). Pheromone-evoked potentials and oscillations in the antennal lobes of the sphinx moth Manduca sexta. Journal of Comparative Physiology - A Sensory, Neural, and Behavioral Physiology, 182(6), 703-714.More infoPMID: 9631552;Abstract: Using intra- and extracellular recording methods, we studied the activity of pheromone-responsive projection neurons in the antennal lobe of the moth Manduca sexta. Intracellularly recorded responses of neurons to antennal stimulation with the pheromone blend characteristically included both inhibitory and excitatory stages of various strengths. To observe the activity of larger groups of neurons, we recorded responses extracellularly in the macroglomerular complex of the antennal lobe. The macroglomerular complex is part of a specialized olfactory subsystem and the site of first-order central processing of sex-pheromonal information. Odors such as the pheromone blend and host-plant (tobacco) volatiles gave rise to evoked potentials that were reproducible upon repeated antennal stimulation. Evoked potentials showed overriding high-frequency oscillations when the antenna was stimulated with the pheromone blend or with either one of the two key pheromone components. The frequency of the oscillations was in the range of 30-50 Hz. Amplitude and frequency of the oscillations varied during the response to pheromonal stimulation. Recording intracellular and extracellular activity simultaneously revealed phase-locking of action potentials to potential oscillations. The results suggest that the activity of neurons of the macroglomerular complex was temporally synchronized, potentially to strengthen the pheromone signal and to improve olfactory perception.
- Hildebrand, J., Christensen, T. A., Waldrop, B. R., & Hildebrand, J. G. (1998). Multitasking in the olfactory system: context-dependent responses to odors reveal dual GABA-regulated coding mechanisms in single olfactory projection neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience, 18(15).More infoStudies of olfaction have focused mainly on neural processing of information about the chemistry of odors, but olfactory stimuli have other properties that also affect central responses and thus influence behavior. In moths, continuous and intermittent stimulation with the same odor evokes two distinct flight behaviors, but the neural basis of this differential response is unknown. Here we show that certain projection neurons (PNs) in the primary olfactory center in the brain give context-dependent responses to a specific odor blend, and these responses are shaped in several ways by a bicuculline-sensitive GABA receptor. Pharmacological dissection of PN responses reveals that bicuculline blocks GABAA-type receptors/chloride channels in PNs, and that these receptors play a critical role in shaping the responses of these glomerular output neurons. The firing patterns of PNs are not odor-specific but are strongly modulated by the temporal pattern of the odor stimulus. Brief repetitive odor pulses evoke fast inhibitory potentials, followed by discrete bursts of action potentials that are phase-locked to the pulses. In contrast, the response to a single prolonged stimulus with the same odor is a series of slow oscillations underlying irregular firing. Bicuculline disrupts the timing of both types of responses, suggesting that GABAA-like receptors underlie both coding mechanisms. These results suggest that glomerular output neurons could use more than one coding scheme to represent a single olfactory stimulus. Moreover, these context-dependent odor responses encode information about both the chemical composition and the temporal pattern of the odor signal. Together with behavioral evidence, these findings suggest that context-dependent odor responses evoke different perceptions in the brain that provide the animal with important information about the spatiotemporal variations that occur in natural odor plumes.
- Nighorn, A., Gibson, N. J., Rivers, D. M., Hildebrand, J. G., & Morton, D. B. (1998). The nitric oxide-cGMP pathway may mediate communication between sensory afferents and projection neurons in the antennal lobe of Manduca sexta. Journal of Neuroscience, 18(18), 7244-7255.More infoPMID: 9736646;Abstract: The nitric oxide (NO)-cGMP signaling system is thought to play important roles in the function of the olfactory system in both vertebrates and invertebrates. One way of studying the role of NO in the nervous system is to study the distribution and properties of NO synthase (NOS), as well as the soluble guanylyl cyclases (sGCs), which are the best characterized targets of NO. We study NOS and sGC in the relatively simple and well characterized insect olfactory system of the hawkmoth, Manduca sexta. We have cloned Manduca sexta nitric oxide synthase (MsNOS) and two sGCs (MsGCα1 and MsGCβ1), characterized their basic biochemical properties, and studied their expression in the olfactory system. The sequences of the Manduca genes are highly similar to their mammalian homologs and show similar biochemical properties when expressed in COS-7 cells. In particular, we find that MsGC functions as an obligate heterodimer that is stimulated significantly by NO. We also find that MsNOS has a Ca2+-sensitive NO-producing activity similar to that of mammalian neuronal NOS. Northern and in situ hybridization analyses show that MsNOS and the MsGCs are expressed in a complementary pattern, with MsNOS expressed at high levels in the antennae and the MsGCs expressed at high levels in a subset of antennal lobe neurons. The expression patterns of these genes suggest that the NOsGC signaling system may play a role in mediating communication between olfactory receptor neurons and projection neurons in the glomeruli of the antennal lobe.
- Rössler, W., Tolbert, L. P., & Hildebrand, J. G. (1998). Early formation of sexually dimorphic glomeruli in the developing olfactory lobe of the brain of the moth Manduca sexta. Journal of Comparative Neurology, 396(4), 415-428.More infoPMID: 9651002;Abstract: The antennal lobes (ALs), the primary olfactory centers, of the moth Manduca sexta are sexually dimorphic. Only ALs of males possess the macroglomerular complex (MGC), the site of primary processing of information about the female's sex pheromone. To understand the development of identified, odor-specific olfactory glomeruli, we investigated the cellular events involved in the morphogenesis of the MGC by means of various fluorescence staining techniques and laser-scanning confocal microscopy. The MGC lies near the entrance of the antennal nerve into the AL of the adult male and comprises three glomeruli, the globular cumulus and two toroidal structures. The MGC forms during early stages of metamorphic adult development through a stereotyped sequence of coordinated changes in MGC- specific receptor axons, glial cells, and early-ingrowing projection neurons of the medial group orAL neurons. The MGC divisions are the earliest glomeruli to form in the male AL, and their basic organization is established within about 3 days after ingrowth of the first sensory axons. Despite their special anatomical features, the MGC glomeruli develop in a manner similar to that of the ordinary glomeruli. Comparison of the ALs of males and females reveals that two relatively large and early-developing glomeruli that are situated dorsolaterally in the female AL appear to be female-specific. Development of the sexually dimorphic glomeruli diverges immediately after the ingrowth of the first olfactory receptor axons, resulting in the formation of these large glomeruli in females and the MGC in males.
- Sun, X. J., Tolbert, L. P., Hildebrand, J. G., & Meinertzhagen, I. A. (1998). A rapid method for combined laser scanning confocal microscopic and electron microscopic visualization of biocytin or Neurobiotin-labeled neurons. Journal of Histochemistry and Cytochemistry, 46(2), 263-273.More infoPMID: 9446834;Abstract: Intracellular recording and dye filling are widely used to correlate the morphology of a neuron with its physiology. With laser scanning confocal microscopy, the complex shapes of labeled neurons in three dimensions can be reconstructed rapidly, but this requires fluorescent dyes. These dyes are neither permanent nor electron dense and therefore do not allow investigation by electron microscopy. Here we report a technique that quickly and easily converts a fluorescent label into a more stable and electron-dense stain. With this technique, a neuron is filled with Neurobiotin or biocytin, reacted with fluorophore-conjugated avidin, and imaged by confocal microscopy. To permit long-term storage or EM study, the fluorescent label is then converted to a stable electron-dense material by a single-step conversion using a commercially available ABC kit. We find that the method, which apparently relies on recognition of avidin's excess biotin binding sites by the biotin- peroxidase conjugate, is both faster and less labor intensive than photo- oxidation procedures in common use. The technique is readily adaptable to immunocytochemistry with biotinylated probes, as we demonstrate using anti- serotonin as an example.
- Vickers, N. J., Christensen, T. A., & Hildebrand, J. G. (1998). Combinatorial odor discrimination in the brain: Attractive and antagonist odor blends are represented in distinct combinations of uniquely identifiable glomeruli. Journal of Comparative Neurology, 400(1), 35-56.More infoPMID: 9762865;Abstract: The rules governing the central discrimination of odors are complex and poorly understood, but a growing body of evidence supports the hypothesis that olfactory glomeruli may represent functionally distinct coding modules in the brain. Testing this hypothesis requires that both the functional characteristics and the spatial position of the glomerulus under study be uniquely identifiable. To address these questions, we examined a specialized array of glomeruli (the macroglomerular complex; MGC) in the antennal lobe of male moths that receives input from olfactory receptor cells tuned specifically to female-released odorants that either promote upwind flight (conspecific sex pheromones) or inhibit it (interspecific antagonists). By using a three-dimensional reconstruction method based on high-resolution laser-scanning confocal microscopy, we generated precise spatial maps of the MGC glomeruli in two related noctuid species with similar pheromone chemistry, Heliothis virescens and Helicoverpa zea. To determine the breadth of tuning of individual MGC glomeruli in processing information about these social signals, we used intracellular recording and staining methods to examine the responses of projection (output) neurons that innervate MGC glomeruli and that each project an axon to higher integrative centers. In both species, a close correspondence was found between the odor specificity of the projection neurons and the glomerulus (or glomeruli) supplied by them. The binary blend of pheromone components for each species was represented by neural activity in only two distinct glomeruli in both H. virescens and H. zea. Odorants that antagonize upwind flight when they are added to the respective pheromonal blends evoked excitatory activity in output neurons restricted to a third glomerulus in the MGCs of both species. In summary, these results suggest that the selective activation of different combinations of functionally distinct MGC glomeruli is a general means for discriminating these specific attractant and antagonist chemical signals in the brain.
- Christensen, T. A., & Hildebrand, J. G. (1997). Coincident stimulation with pheromone components improves temporal pattern resolution in central olfactory neurons. Journal of Neurophysiology, 77(2), 775-781.More infoPMID: 9065849;Abstract: Male moths most detect and resolve temporal discontinuities in the sex pheromonal odor signal emitted by a conspecific female moth to orient to and locate the odor source. We asked how sensory information about two key components of the pheromone influences the ability of certain sexually dimorphic projection (output) neurons in the primary olfactory center of the male moth's brain to encode the frequency and duration of discrete pulses of pheromone blends. Most of the male-specific projection neurons examined gave mixed postsynaptic responses, consisting of an early suppressive phase followed by activation of firing, to stimulation of the ipsilateral antenna with a blend of the two behaviorally essential pheromone components. Of 39 neurons tested, 33 were excited by the principal (most abundant) pheromone component but inhibited by another, less abundant but nevertheless essential component of the blend. We tested the ability of each neuron to encode intermittent pheromonal stimuli by delivering trains of 50-ms pulses of the two-component blend at progressively higher rates from 1 to 10 per second. There was a strong correlation between 1) the amplitude of the early inhibitory postsynaptic potential evoked by the second pheromone component and 2) the maximal rate of odor pulses that neuron could resolve (r = 0.92). Projection neurons receiving stronger inhibitory input encoded the temporal pattern of the stimulus with higher fidelity. With the principal, excitatory component of the pheromone alone as the stimulus, the dynamic range for encoding stimulus intermittency was reduced in nearly 60% of the neurons tested. The greatest reductions were observed in those neurons that could be shown to receive the strongest inhibitory input from the second behaviorally essential component of the blend. We also tested the ability of these neurons to encode stimulus duration. Again there was a strong correlation between the strength of the inhibitory input to a neuron mediated by the second pheromone component and that neuron's ability to encode stimulus duration. Neurons that were strongly inhibited by the second component could accurately encode pulses of the blend from 50 to 500 ms in duration (r = 0.94), but that ability was reduced in neurons receiving little or no inhibitory input (r = 0.23). This study confirms that certain olfactory projection neurons respond optimally to a particular odor blend rather than to the individual components of the blend. The key components activate opposing synaptic inputs that enable this subset of central neurons to copy the duration and frequency of intermittent odor pulses that are a fundamental feature of airborne olfactory stimuli.
- Davis, N. T., Veenstra, J. A., Feyereisen, R., & Hildebrand, J. G. (1997). Allatostatin-like-immunoreactive neurons of the tobacco Hornworm, Manduca sexta, and isolation and identification of a new neuropeptide related to cockroach allatostatins. Journal of Comparative Neurology, 385(2), 265-284.More infoPMID: 9268127;Abstract: The YXFGLamide C-terminus serves to define most members of a family of structurally related neuropeptides, the YXFGLamides. These peptides have been identified from the nervous system of various insects and include the allatostatins of cockroaches and crickets, the schistostatins of locusts, and the callatostatins of blowflies. The YXFGLamides have been shown to have various functions, including inhibition of juvenile hormone biosynthesis in cockroaches and crickets and inhibition of contraction of certain insect visceral muscles. We wanted to knew if these peptides occur in Manduca sexta and what functions they might have. A new peptide, AKSYNFGLamide, was isolated and identified from M. sexta and has been named 'lepidostatin-1'; this is the first YXFGLamide to be found in a lepidopteran, and there are indications that additional YXFGLamides occur in M. sexta. An antiserum to cockroach allatostatins (YXFGLamides) was shown to recognize lepidostatin-1 of M. sexta and was used to map YXFGLamide-immunoreactive neurons in larvae. Because immunoreactive interneurons were found to form an extensive neuropil, YXFGLamides probably function as neuromodulators in M. sexta. Neuroendocrine cells in the brain, abdominal ganglia, and their respective neurohemal organs were YXFGLamide immunereactive and appear to release YXFGLamides as neurohormones. Immunoreactivity to YXFGLamides and M. sexta diuretic hormone were found to be colocalized and appear to be coreleased in these neuroendocrine cells, indicating that YXFGLamides may be involved in regulation of fluid transport. Innervation of the corpora allata by YXFGLamide-immunoreactive processes was very sparse, suggesting that this innervation does not play an important role in allatostasis. Many thoracic motor neurons were YXFGLamide immunereactive, suggesting that YXFGLamides may have a myomodulatory or myotrophic function in larvae. However, this immunoreactivity disappeared early in metamorphosis and did not reappear in the adult. The YXFGLamide-immunoreactive neurons in the terminal abdominal ganglion were found to innervate the hindgut, indicating that YXFGLamides may be involved in the control of the rate of myogenic contractions of the larval hindgut.
- Hildebrand, J. G., & Shepherd, G. M. (1997). Mechanisms of olfactory discrimination: Converging evidence for common principles across phyla. Annual Review of Neuroscience, 20, 595-631.More infoPMID: 9056726;Abstract: Olfaction begins with the transduction of the information carried by odor molecules into electrical signals in sensory neurons. The activation of different subsets of sensory neurons to different degrees is the basis for neural encoding and further processing of the odor information by higher centers in the olfactory pathway. Recent evidence has converged on a set of transduction mechanisms, involving G-protein-coupled second-messenger systems, and neural processing mechanisms, involving modules called glomeruli, that appear to be adapted for the requirements of different species. The evidence is highlighted in this review by focusing on studies in selected vertebrates and in insects and crustaceans among invertebrates. The findings support the hypothesis that olfactory transduction and neural processing in the peripheral olfactory pathway involve basic mechanisms that are universal across most species in most phyla.
- Hildebrand, J. G., Rössler, W., & Tolbert, L. P. (1997). Postembryonic development of the olfactory system in the moth Manduca sexta: Primary-afferent control of glomerular development. Seminars in Cell and Developmental Biology, 8(2), 163-170.More infoPMID: 15001092;Abstract: The olfactory system of the sphinx moth Manduca sexta bears many similarities to its vertebrate counterpart in functional organization, physiology and development. In the moth, the antenna (the olfactory organ) and the antennal lobe (the primary olfactory center) of the brain arise during postembryonic metamorphic development and are accessible, independently manipulable, and structurally relatively simple. In addition, they house a conspicuous, sexually dimorphic subsystem specialized for detection of a specific pheromonal odor. These features make this system experimentally favorable for studies of development of olfactory glomeruli. Such studies have demonstrated the importance of regulatory interactions among sensory axons, glial cells and antennal-lobe neurons.
- Kloppenburg, P., Camazine, S. M., Sun, X. J., Randolph, P., & Hildebrand, J. G. (1997). Organization of the antennal motor system in the sphinx moth Manduca sexta. Cell and Tissue Research, 287(2), 425-433.More infoPMID: 8995213;Abstract: The antennae of the sphinx moth Manduca sexta are multimodal sense organs, each comprising three segments: scape, pedicel, and flagellum. Each antenna is moved by two systems of muscles, one controlling the movement of the scape and consisting of five muscles situated in the head capsule (extrinsic muscles), and the other system located within the scape (intrinsic muscles) and consisting of four muscles that move the pedicel. At least seven motoneurons innervate the extrinsic muscles, and at least five motoneurons innervate the intrinsic muscles. The dendritic fields of the antennal motoneurons overlap one another extensively and are located in the neuropil of the antennal mechanosensory and motor center. The density of motoneuronal arborizations is greatest in the lateral part of this neuropil region and decreases more medially. None of the motoneurons exhibits a contralateral projection. The cell bodies of motoneurons innervating the extrinsic muscles are distributed throughout an arching band of neuronal somata dorsal and dorsolateral to the neuropil of the antennal mechanosensory and motor center, whereas the cell bodies of motoneurons innervating the intrinsic muscles reside mainly among the neuronal somata situated dorsolateral to that neuropil.
- Sun, X. J., Tolbert, L. P., & Hildebrand, J. G. (1997). Synaptic organization of the uniglomerular projection neurons of the antennal lobe of the moth Manduca sexta: A laser scanning confocal and electron microscopic study. Journal of Comparative Neurology, 379(1), 2-20.More infoPMID: 9057110;Abstract: The detailed branching pattern and synaptic organization of the uniglomerular projection neurons of the antennal lobe, the first processing center of the olfactory pathway, of the moth Manduca sexta were studied with laser scanning confocal microscopy and a technique combining laser scanning confocal microscopy and electron microscopy. Uniglomerular projection neurons, identified electrophysiologically or morphologically, were stained intracellularly with neurobiotin or biocytin. Brains containing the injected neurons were treated with streptavidin-immunogold to label the injected material for electron microscopy and with Cy3-streptavidin to lable the neurons with fluorescence for laser scanning confocal microscopy, and then embedded in Epon. Labeled neurons were imaged and reconstructed with laser scanning confocal microscopy (based on the retained fluorescence of the labeled neuron in the Epon block), and thin sections were cut at selected optical levels for correlation of light microscopic data and electron microscopic detail. Each neuron had a cell body in one of the three cell- body clusters of the antennal lobe, a primary neurite that extended across the coarse neuropil at the center of the antennal lobe and then formed a dense tuft of processes within a single glomerulus, and an axon that emanated from the primary neurite and projected from the antennal lobe via the antenno-cerebral tract to the lateral horn of the ipsilateral protocerebrum and, collaterally, to the calyces of the mushroom body. In the electron microscope, the fine dendritic branches in the apical zones of the glomeruli, where sensory axons terminate, were found to receive many input synapses. In deeper layers across the glomeruli, the processes participated in both input and output synapses, and at the bases of the glomeruli, the most proximal, thickest branches formed output synapses. In both of the protocerebral areas in which axonal branches terminated, those branches formed exclusively output synapses. Our findings indicate that, in addition to conveying olfactory information to the protocerebrum, uniglomerular projection neurons in the antennal lobes of M. sexta participate in local intraglomerular synaptic circuitry.
- Christensen, T. A., Heinbockel, T., & Hildebrand, J. G. (1996). Olfactory information processing in the brain: Encoding chemical and temporal features of odors. Journal of Neurobiology, 30(1), 82-91.More infoPMID: 8727985;Abstract: A fundamental problem in studying the neural mechanisms of odor recognition and discrimination in the olfactory system lies in determining the features or 'primitives' of an odor stimulus that are analyzed by glomerular circuits at the first level of processing in the brain. Several recent studies support the idea that it is not simply the molecular features of odors that contain important information, but also the intermittent pattern of their presentation to the olfactory epithelium that helps determine the behavioral response to odor.
- Davis, N. T., Homberg, U., Teal, P. E., Altstein, M., Agricola, H., & Hildebrand, J. G. (1996). Neuroanatomy and immunocytochemistry of the median neuroendocrine cells of the subesophageal ganglion of the tobacco hawkmoth, Manduca sexta: Immunoreactivities to PBAN and other neuropeptides. Microscopy Research and Technique, 35(3), 201-229.More infoPMID: 8956271;Abstract: The median neuroendocrine cells of the subesophageal ganglion, important components of the neuroendocrine system of the tobacco hawkmoth, Manduca sexta, have not been well investigated. Therefore, we studied the anatomy of these cells by axonal backfills and characterized their peptide immunoreactivities. Both larvae and adults were examined, and developmental changes in these neuroendocrine cells were followed. Processes of the median neuroendocrine cells project to terminations in the corpora cardiaca via the third and the ventral nerves of this neurohemal organ, but the ventral nerve of the corpus cardiacum is the principal neurohemal surface for this system. Cobalt backfills of the third cardiacal nerves revealed lateral cells in the maxillary neuromere and a ventro-median pair in the labial neuromere. Backfills of the ventral cardiacal nerves revealed two ventro-median pairs of cells in the mandibular neuromere and two ventromedian triplets in the maxillary neuromere. The efferent projections of these cells are contralateral. The anatomy of the system is basically the same in larvae and adults. The three sets of median neuroendocrine cells are PBAN- and FMRFamide-immunoreactive, but only the mandibular and maxillary cells are proctolin-immunoreactive. During metamorphosis, the mandibular and maxillary cells also acquire CCK-like immunoreactivity and the labial cells become SCP- and sulfakinin-immunoreactive. Characteristics of FMRFamide-like immunostaining suggest that the median neuroendocrine cells may contain one or more of the FLRFamides that have been identified in M. sexta. The mandibular and maxillary neuroendocrine cells appear to produce the same set of hormones, and a somewhat different set of hormones is produced by the labial neuroendocrine cells. Two pairs of interneurons immunologically related to the neurosecretory cells are associated with the median maxillary neuroendocrine cells. These cells are PBAN-, FMRFamide-, SCP-, and sulfakinin-immunoreactive and project to arborizations in the brain and all ventral ganglia. These interneurons appear to have extensive modulatory functions in the CNS.
- Hildebrand, J. G. (1996). Olfactory control of behavior in moths: Central processing of odor information and the functional significance of olfactory glomeruli. Journal of Comparative Physiology A: Sensory, Neural, and Behavioral Physiology, 178(1), 5-19.More infoPMID: 8568724;
- Mercer, A. R., Kirchhof, B. S., & Hildebrand, J. G. (1996). Enhancement by serotonin of the growth in vitro of antennal lobe neurons of the sphinx moth Manduca sexta. Journal of Neurobiology, 29(1), 49-64.More infoPMID: 8748371;Abstract: Cell culture experiments have been used to examine the effects of serotonin [5-hydroxytryptamine (5-HT)] on the morphological development of antennal lobe (AL) neurons in the brain of the sphinx moth, Manduca sexta. The majority of cells used in this study were from animals at stage 5 of the 18 stages of metamorphic adult development. 5-HT did not affect the survival of M. sexta AL neurons in culture, but did increase the numbers of cells displaying features characteristic of certain cell types. Three morphologically distinct cell types were examined in detail. The principal effect of 5-HT on these neurons was enhancement of cell growth. The magnitude of responses to this amine was cell-type specific. Site-specific responses to 5-HT were apparent also in one cell type. Our results suggest that the effects of 5-HT can change during the course of metamorphic development. These changes coincide temporally with the development of fast, sodium-based action potentials.
- Mercer, A. R., Kloppenburg, P., & Hildebrand, J. G. (1996). Serotonin-induced changes in the excitability of cultured antennal-lobe neurons of the sphinx moth Manduca sexta. Journal of Comparative Physiology A: Sensory, Neural, and Behavioral Physiology, 178(1), 21-31.More infoPMID: 8568722;Abstract: The modulatory actions of 5-hydroxytryptamine (5HT or serotonin) on a morphologically identifiable class of neurons dissociated from antennal lobes of Manduca sexta at stages 9-15 of the 18 stages of metamorphic adult development were examined in vitro with whole-cell patch-clamp recording techniques. Action potentials could be elicited from approximately 20% of the cells. These cells were used to examine effects of 5HT (5 × 10-6 to 5 × 10-4 M) on cell excitability and action-potential waveform. 5HT increased the number of spikes elicited by a constant depolarizing current pulse and reduced the latency of responses. 5HT also led to broadening of action potentials in these neurons and increased cell input resistance. Modulation of potassium channels by 5HT is likely to contribute to these responses. 5HT causes reversible reduction of at least 3 distinct potassium currents, one of which is described for the first time in this study. Because effects of 5HT on antennal-lobe neurons in culture mimic those observed in situ in the brain of the adult moth, in vitro analysis should contribute to elucidation of the cellular mechanisms that underlie the modulatory effects of 5HT on central olfactory neurons in the moth.
- Tolbert, L. P., Sun, X. J., & Hildebrand, J. G. (1996). Combining laser scanning confocal microscopy and electron microscopy in studies of the insect nervous system. Journal of Neuroscience Methods, 69(1), 25-32.More infoPMID: 8912932;Abstract: Experimentally determining the synaptic interconnections between neurons in the nervous system is laborious and difficult in any animal species, but especially so in many invertebrates, including insects, where neurons generally have large, finely branching neuritic trees that form both pre- and postsynaptic specializations in dense neuropils with other neuritic trees. Electron microscopy is needed to identify synapses, but correlation of synapse type and location with the overall branching patterns of neurons, which are visible readily only in the light microscope or through extensive reconstruction of serial electron-microscope sections, is very difficult. In this paper, we present a simple method that we have developed (Sun et al. (1995) J. Histochem. Cytochem., 43: 329-335) that combines laser scanning confocal microscopy and electron microscopy for the study of synaptic relationships of neurons in the antennal lobe, the first central neuropil in the olfactory pathway, of the moth Manduca sexta. Briefly, neurons are labeled by intracellular injection with neurobiotin or biocytin, and then processed with a gold-particle tag for electron microscopic study and a fluorescent tag for confocal microscopy, and embedded in plastic. The fluorescence of the labeled neuron in the plastic blocks is imaged in three dimensions with laser scanning confocal microscopy and then the neuron is thin-sectioned at precisely chosen depths for electron microscopic study. The fluorescence pattern can be monitored repeatedly between episodes of thin-sectioning, and subtraction of a fluorescence image from the previous fluorescence image reveals which fluorescent processes have been sectioned. In this way, electron microscopic detail can be mapped onto a three-dimensional light microscopic image of the neuron.
- Christensen, T. A., & Hildebrand, J. G. (1995). Neural regulation of sex-pheromone glands in Lepidoptera. Invertebrate Neuroscience, 1(2), 97-103.More infoAbstract: Substantial progress has been made toward understanding the neuroendocrine regulation of sex-pheromone glands in Lepidoptera, but several recent studies have revealed that direct contact of the pheromone gland with blood-borne factors is not necessary to induce pheromone biosynthesis and release in some species. The nervous system provides an alternate route of activation. Evidence from several species indicates that the pheromone gland is innervated and regulated by neural activity. Electrical stimulation of efferent axons arising from the terminal abdominal ganglion results in a significant increase in pheromone production, and neural stimulation furthermore evokes the rapid release of pheromone into the surrounding air. In some heliothine moths, the biogenic monoamine octopamine stimulates pheromone production, and octopamine has also been isolated from pheromone gland tissue. Moreover, the critical period for maximal octopamine action mirrors the time when peak levels of octopamine are present in the gland. These findings suggest that octopamine is involved in the regulation of pheromone biosynthesis and/or release, but its actions depend on additional factors associated with age and photoperiod. The combined evidence using anatomical, electrophysiological, and biochemical methods indicates that the pheromone gland is innervated and regulated by neurons that arise in the terminal abdominal ganglion. Indirect evidence suggests that at least some of this innervation is octopaminergic. In these respects, the pheromone gland in Lepidoptera exhibits characteristics of other neuroeffector systems in insects. © 1995 Sheffield Academic Press.
- Christensen, T. A., Harrow, I. D., Cuzzocrea, C., Randolph, P. W., & Hildebrand, J. G. (1995). Distinct projections of two populations of olfactory receptor axons in the antennal lobe of the sphinx moth Manduca sexta. Chemical Senses, 20(3), 313-323.More infoPMID: 7552040;Abstract: The central projections of olfactory receptor cells associated with two distinct types of antennal sensilla in the sphinx moth Manduca sexta were revealed by anterograde staining. In both sexes, receptor axons that arise from sexually isomorphic, type-II trichoid sensilla (and possibly some basiconic sensilla) project to the spheroidal glomeruli in the ipsilateral antennal lobe. Each axon terminates in one glomerulus. Axons from a limited region of the antenna project to glomeruli throughout the lobe, arguing against strict topographic mapping of antennal receptor cells onto the array of glomeruli. Axons of sex-pheromone-selective receptor cells in the male-specific type-I trichoid sensilla project exclusively to the sexually dimorphic macroglomerular complex (MGC). Axons from sensilla on the dorsal surface of the antenna are biased toward the medial MGC and those from ventral sensilla, toward the lateral MGC. Some receptor-cell axons branch before reaching the MGC, but their terminals are always confined to one of the two main glomerular divisions of the MGC, the cumulus and toroid. These findings confirm that primary-afferent information about pheromonal and non-pheromonal odors is segregated in the antennal lobe and suggest that there is a functional correspondence between particular olfactory receptor cells and specific glomeruli.
- Christensen, T. A., Mustaparta, H., & Hildebrand, J. G. (1995). Chemical communication in heliothine moths - VI. Parallel pathways for information processing in the macroglomerular complex of the male tobacco budworm moth Heliothis virescens. Journal of Comparative Physiology A, 177(5), 545-557.More infoAbstract: The chemical and temporal features of the sex-pheromone emitted by Heliothis virescens females are encoded by a diverse array of output pathways from the male-specific macroglomerular complex (MGC) in the antennal lobe. Most output neurons (29 out of 32) were activated by antennal stimulation with the principal component of the sex-pheromone blend of this species, (Z)-11-hexadecenal. Six neurons were excited solely by this component, 8 neurons also responded to the second essential blend component, (Z)-9-tetradecenal, and 14 neurons displayed equivalent responses to the two. Many neurons also effectively encoded the onset and duration of the stimulus. In one additional neuron, a prolonged excitatory response (synergism) was evoked only by the blend of the two components, indicating that some MGC neurons function as 'blend detectors'. In contrast to the situation in Helicoverpa zea, none of the MGC neurons in H. virescens responded selectively to (Z)-9-tetradecenal, suggesting that these two noctuid species employ different neural strategies to encode information about their respective pheromone blends. Three MGC-output neurons responded selectively to (Z)-11-hexadecenyl acetate, an odorant released by some sympatric species that disrupts normal upwind flight to pheromones. Thus, changes in the attractant and deterrent chemical signals, as well as the physical features of these odor plumes, are encoded in the MGC across a diverse parallel array of output pathways to the protocerebrum. © 1995 Springer-Verlag.
- Hildebrand, J. G. (1995). Analysis of chemical signals by nervous systems. Proceedings of the National Academy of Sciences of the United States of America, 92(1), 67-74.More infoPMID: 7816849;PMCID: PMC42818;Abstract: Intraspecific and interspecific communication and recognition depend on olfaction in widely diverse species of animals. Olfaction, an ancient sensory modality, is based on principles of neural organization and function that appear to be remarkably similar throughout the zoosphere. Thus, the 'primitives' of olfactory stimuli that determine the input information of olfaction, the kinds of 'molecular images' formed at various levels in the olfactory pathway, and the cellular mechanisms that underlie olfactory information processing are comparable in invertebrates and vertebrates alike. A case in point is the male-specific olfactory subsystem in moths, which is specialized to detect and analyze the qualitative, quantitative, and temporal features of the conspecific females' sex-pheromonal chemical signal. This olfactory subsystem can be viewed, and is here presented, as a model in which common principles of organization and function of olfactory systems in general are exaggerated to serve the requirements of a chemical communication system that is crucial for reproductive success.
- Homberg, U., Hoskins, S. G., & Hildebrand, J. G. (1995). Distribution of acetylcholinesterase activity in the deutocerebrum of the sphinx moth Manduca sexta. Cell & Tissue Research, 279(2), 249-259.More infoPMID: 7895267;Abstract: We have used a cytochemical technique to investigate the distribution of acetylcholinesterase (AChE) activity in the deutocerebrum of the brain of the sphinx moth Manduca sexta. To distinguish between extra-and intracellular pools of the enzyme, some brains were treated prior to histochemical staining with echothiophate, an irreversible AChE inhibitor which penetrates cell membranes very slowly and, therefore, inhibits only extracellular AChE. In the antennal nerve, fascicles of presumably mechanosensory fibers show echothiophateinsensitive AChE activity. They bypass the antennal lobe and project to the antennal mechanosensory and motor center of the deutocerebrum. In the antennal lobe, fibers in the coarse neuropil, cell bodies in the lateral cell group, and all glomeruli exhibit AChE activity. In most ordinary glomeruli, echothiophate-sensitive AChE activity is concentrated in the outer cap regions, corresponding to the terminal arborizations of olfactory afferents. A previously unrecognized glomerulus in the ventro-median antennal lobe shows uniform and more intense AChE-specific staining that the other glomeruli. No AChE activity appeared to be associated with malespecific pheromone-sensitive afferents in the macro-glomerular complex. About 67 interneurons with somata in the lateral cell group of the antennal lobe show echo-thiophate-insensitive AChE activity. These neurous seem to be members of two types of antennal-lobe projection neurons with fibers passing through the outer-antenno-cerebral tract to the protocerebrum. AChE-stained arborizations of these neurons appear to invade all glomeruli, including three distinguishable subunits of the male-specific macroglomerular complex. In echothiophate-treated animals, the projections of one of these types of fiber form large terminals in the lateral horn of protocerebrum, which partly protrude into the adjacent glial cell layer. The results suggest that extracellularly accessible AChE is associated with ordinary olfactory receptor terminals but apparently not with pheromone-sensitive afferents. Intracellular AChE appears to be present in antennal mechanosensory fibers and in two types of olfactory projection neurons of the antennal lobe. The study provides further evidence for cholinergic neurotransmission of most antennal afferents. The AChE-containing interneurons might be cholinergic as well or use the enzyme for functions unrelated to hydrolysis of acetylcholine. © 1995 Springer-Verlag.
- Kloppenburg, P., & Hildebrand, J. G. (1995). Neuromodulation by 5-hydroxytryptamine in the antennal lobe of the sphinx moth Manduca sexta. Journal of Experimental Biology, 198(3), 603-611.More infoPMID: 7714450;Using intracellular recording techniques, we have begun to examine the effects of 5-hydroxytryptamine (5-HT) on antennal-lobe (AL) neurones in the brain of the adult moth Manduca sexta. 5-HT modulated the responses of local interneurones and projection neurones, which were recognized on the basis of well-established electrophysiological criteria, to primary synaptic input elicited by electrical stimulation of the ipsilateral antennal nerve. 5-HT applied at low concentration (10(-8) mol l-1) reduced the excitatory responses evoked by electrical stimulation of the antennal nerve, whereas at high concentration (10(-4) mol l-1), 5-HT enhanced the responses. At 10(-4) mol l1, 5-HT increased cell input resistance, led to broadening of action potentials and caused increased cell excitability in many AL neurones.
- Mercer, A. R., Hayashi, J. H., & Hildebrand, J. G. (1995). Modulatory effects of 5-hydroxytryptamine on voltage-activated currents in cultured antennal lobe neurones of the sphinx moth Manduca sexta. Journal of Experimental Biology, 198(3), 613-627.More infoPMID: 7714451;The modulatory effects of 5-hydroxytryptamine (5-HT or serotonin) on voltage-gated currents in central olfactory neurones of the moth Manduca sexta have been examined in vitro using whole-cell patch-clamp recording techniques. Central olfactory neurones were dissociated from the antennal lobes of animals at stage 5 of the 18 stages of metamorphic adult development. The modulatory actions of 5-HT on voltage-activated ionic currents were examined in a subset of morphologically identifiable antennal lobe neurones maintained for 2 weeks in primary cell culture. 5-HT caused reversible reduction of both a rapidly activating A-type K+ current and a relatively slowly activating K+ current resembling a delayed rectifier-type conductance. 5-HT also reduced the magnitude of voltage-activated Ca2+ influx in these cells. The functional significance of 5-HT-modulation of central neurones is discussed.
- Novak, M. G., Ribeiro, J. M., & Hildebrand, J. G. (1995). 5-Hydroxytryptamine in the salivary glands of adult female Aedes aegypti and its role in regulation of salivation. Journal of Experimental Biology, 198(1), 167-174.More infoPMID: 7891033;A dense plexus of axons, immunoreactive to antisera against 5-hydroxytryptamine (5-HT, serotonin) and surrounding the proximal medial lobe of the salivary gland of adult female Aedes aegypti mosquitoes, was demonstrated by means of whole-mount fluorescence immunocytochemistry. This innervation originates in the stomatogastric nervous system. 5-HT-immunoreactive innervation is absent in male salivary glands, suggesting that 5-HT is involved in blood-feeding. Furthermore, female mosquitoes treated with the 5-HT-depleting agent alpha-methyltryptophan (AMTP) and then allowed to feed on a rat exhibited a significantly longer mean probing period and a lower blood-feeding success rate than did control mosquitoes. When female mosquitoes were experimentally induced to salivate into mineral oil, AMTP-treated individuals secreted significantly less saliva than did control mosquitoes. These samples of saliva also contained significantly lower concentrations of apyrase, an enzyme important in blood-feeding. Injection of 5-HT into both AMTP-treated and control mosquitoes elicited significant increases in the volume of secreted saliva and/or its apyrase content. We conclude that 5-HT plays an important role in the control of salivation in adult female A. aegypti.
- Sun, X. J., Tolbert, L. P., & Hildebrand, J. G. (1995). Using laser scanning confocal microscopy as a guide for electron microscopic study: A simple method for correlation of light and electron microscopy. Journal of Histochemistry and Cytochemistry, 43(3), 329-335.More infoPMID: 7868862;Abstract: Anatomic study of synaptic connections in the nervous system is laborious and difficult, especially when neurons are large of have free branches embedded among many other processes. Although electron microscopy provides a powerful tool for such study, the correlation of fight microscopic appearance and electron microscopic detail is very time-consuming. We report here a simple method combining laser scanning confocal microscopy and electron microscopy for study of the synaptic relationships of the neurons in the antennal lobe, the first central neuropil in the olfactory pathway, of the moth Manduca sexta. Neurons were labeled intracellularly with neurobiotin or biocytin, two widely used stains. The tissue was then sectioned on a vibratome and processed with both streptavidin-nanogold (for electron microscopic study) and streptavidin-Cy3 (for confocal microscopic study) and embedded in epon/araldite. Interesting areas of the labeled neuron were imaged in the epon/araldite blocks with laser scanning confocal microscopy and then thin-sectioned at the indicated depth for electron microscopic study. This method provides an easy, reliable way to correlate three- dimensional light microscopic information with electron microscopic detail, and can be very useful in studies of synaptic connections.
- Christensen, T. A., Lashbrook, J. M., & Hildebrand, J. G. (1994). Neural activation of the sex-pheromone gland in the moth Manduca sexta: Real-time measurement of pheromone release. Physiological Entomology, 19(4), 265-270.
- Homberg, U., & Hildebrand, J. G. (1994). Postembryonic development of γ-aminobutyric acid-like immunoreactivity in the brain of the sphinx moth Manduca sexta. Journal of Comparative Neurology, 339(1), 132-149.More infoPMID: 8106658;Abstract: We have investigated the distribution of immunocytochemical staining for the neurotransmitter γ-aminobutyric acid (GABA) in the brain of the sphinx moth Manduca sexta during larval, pupal, and adult development. In the larval brain, about 300 neurons are GABA-immunoreactive. All neuropil areas except the mushroom bodies and central complex show intense immunostaining. Only minor changes in the pattern of immunoreactivity occur during larval development. During metamorphosis, changes in immunostaining occur in two phases. Beginning in wandering fifth-instar larvae (stage W2), immunoreactivity appears in numerous neurons of the central body and optic lobe and becomes more intense during early pupal stages. At the same time, GABA-like immunoreactivity disappears in most neuropil areas of the brain and becomes faint in many immunoreactive somata. Neurons with arborizations in the ventrolateral protocerebrum, however, continue to exhibit intense immunostaining during this period, and strongly immunolabeled fibers connect these areas with the ventral nerve cord. The second phase of transformation begins around pupal stage P5/P6, when faint immunostaining appears in many previously nonimmunoreactive somata and most neuropil areas of the brain. In subsequent stages (P8-P10), this immunoreactivity disappears again in most somata, but in certain cell groups, it becomes more intense and gradually develops to the adult pattern. Most larval GABA-immunoreactive neurons appear to survive through metamorphosis into the adult. Neurons in the midbrain that acquire GABA-like immunoreactivity during metamorphosis usually lie adjacent to larval immunostained neurons, suggesting common lineages. The onsets of the two developmental phases of GABA-like immunoreactivity correlate with sharp rises in hemolymph titers of ecdysteroid hormones, suggesting a role for ecdysteroids in the regulation of GABA synthesis. We hypothesize that the disappearance of GABA in many areas of the brain starting 2 days prior to pupation dramatically alters its functional circuitry and thus may account for profound changes in the behavior of the animal.
- Christensen, T. A., Waldrop, B. R., Harrow, I. D., & Hildebrand, J. G. (1993). Local interneurons and information processing in the olfactory glomeruli of the moth Manduca sexta. Journal of Comparative Physiology A, 173(4), 385-399.More infoPMID: 8254565;Abstract: Intracellular recordings were made from the major neurites of local interneurons in the moth antennal lobe. Antennal nerve stimulation evoked 3 patterns of postsynaptic activity: (i) a short-latency compound excitatory postsynaptic potential that, based on electrical stimulation of the antennal nerve and stimulation of the antenna with odors, represents a monosynaptic input from olfactory afferent axons (71 out of 86 neurons), (ii) a delayed activation of firing in response to both electrical- and odor-driven input (11 neurons), and (iii) a delayed membrane hyperpolarization in response to antennal nerve input (4 neurons). Simultaneous intracellular recordings from a local interneuron with short-latency responses and a projection (output) neuron revealed unidirectional synaptic interactions between these two cell types. In 20% of the 30 pairs studied, spontaneous and current-induced spiking activity in a local interneuron correlated with hyperpolarization and suppression of firing in a projection neuron. No evidence for recurrent or feedback inhibition of projection neurons was found. Furthermore, suppression of firing in an inhibitory local interneuron led to an increase in firing in the normally quiescent projection neuron, suggesting that a disinhibitory pathway may mediate excitation in projection neurons. This is the first direct evidence of an inhibitory role for local interneurons in olfactory information processing in insects. Through different types of multisynaptic interactions with projection neurons, local interneurons help to generate and shape the output from olfactory glomeruli in the antennal lobe. © 1993 Springer-Verlag.
- Davis, N. T., Homberg, U., Dircksen, H., Levine, R. B., & Hildebrand, J. G. (1993). Crustacean cardioactive peptide-immunoreactive neurons in the hawkmoth Manduca sexta and changes in their immunoreactivity during postembryonic development. Journal of Comparative Neurology, 338(4), 612-627.More infoPMID: 8132864;Abstract: An antiserum against crustacean cardioactive peptide was used, in indirect immunocytochemistry on whole-mounts and Vibratome sections, to map immunoreactive neurons at various stages of postembryonic development of the hawkmoth Manduca sexta. About 90 immunoreactive neurons were identified. Many of these cells are immunoreactive at hatching and persist into the adult stage; others become immunoreactive late in postembryonic development. During adult development, transient immunoreactivity is expressed in several cells in the subesophageal and thoracic ganglia. Two sets of immunoreactive neurons are found in the protocerebrum of larvae, but only one of these sets persists into the adult stage. Paired lateral interneurons and neurosecretory neurons are segmentally repeated in the abdominal ganglia and are present from the first larval stage to the adult; the abdominal interneurons project contralaterally to arborizations in adjacent ganglia, and some ascend to tritocerebral arborizations. The abdominal neurosecretory cells, which correspond to a pair of cells reported to contain bursicon, project posteriorly to neurohemal release organs. Motor neurons of dorsal external oblique abdominal muscles become immunoreactive in the fourth larval stage. Paired median neurosecretory cells of abdominal ganglia become immunoreactive during the fifth larval stage. The immunoreactive median and lateral abdominal neurosecretory cells are a subset of a group of cells known to contain cardioactive peptides. Paired lateral neurosecretory cells of the subesophageal ganglion become immunoreactive during pupation and project to the corpora cardiaca and aorta of the adult. Many of the neurons identified here are comparable to crustacean cardioactive peptide-immunoreactive cells described previously in locusts and the mealworm beetle.
- Lehman, H. K., Murgiuc, C. M., Miller, T. A., Lee, T. D., & Hildebrand, J. G. (1993). Crustacean cardioactive peptide in the sphinx moth, Manduca sexta. Peptides, 14(4), 735-741.More infoPMID: 8234018;Abstract: The isolation, identification, and actions of crustacean cardioactive peptide (CCAP) have been examined in the sphinx moth Manduca sexta. A sensitive and specific enzyme-linked immunosorbent assay (ELISA) was used to quantify CCAP-like immunoreactivity in the nervous system. The CCAP-like immunoreactivity from the abdominal CNS was then purified, and its sequence was ascertained by amino acid analysis, mass spectral analysis, and HPLC. These studies showed that the nervous system of M. sexta contains a peptide with the sequence Pro-Phe-Cys-Asn-Ala-Phe-Thr-Gly-Cys-NH2, identical to CCAP originally isolated and sequenced from the shore crab Carcinus maenas. The actions of CCAP on the isolated heart of M. sexta and the extensor-tibia muscle of Schistocerca americana were tested. Crustacean cardioactive peptide had excitatory actions on both preparations: a dose-dependent increase in the rate of contractions was observed on the heart, and an increase in the rate of the myogenic rhythm was observed in the leg muscle. Moreover, purified and synthetic CCAP had identical effects on the isolated heart. We conclude that CCAP occurs in M. sexta and exerts potent neurotransmitter or neurohormonal actions on a variety of muscles. © 1993.
- Sun, X. J., Tolbert, L. P., & Hildebrand, J. G. (1993). Ramification pattern and ultrastructural characteristics of the serotonin- immunoreactive neuron in the antennal lobe of the moth Manduca sexta: A laser scanning confocal and electron microscopic study. Journal of Comparative Neurology, 338(1), 5-16.More infoPMID: 8300899;Abstract: The two antennal lobes, the primary olfactory centers of the brain, of the moth Manduca sexta each contain one neuron that displays serotonin immunoreactivity. The neuron projects out of the antennal lobe and sends branches into ipsi- and contralateral protocerebral areas. An axon-like process extends from the contralateral protocerebrum to, and terminates in, the contralateral antennal lobe. In order to begin to investigate the possible role of this unique neuron in olfactory information processing, we have used laser scanning confocal microscopic and electron microscopic immunocytochemical techniques to study the ramification pattern, ultrastructural characteristics, and synaptic connections of the neuron in the antennal lobes of female adult Manduca sexta. The neuron ramifies extensively in the antennal lobe contralateral to the cell body. The ramifications, mainly in the base and center of each glomerulus, do not overlap with those of the sensory axons from the antenna. This finding suggests that the serotonin-immunoreactive neuron may not receive direct input from sensory neurons, and that it may modulate the activity of the neurons of the antennal lobe rather than that of the sensory neurons. In the electron microscope, the neuron exhibits large dense-cored vesicles and small, clear round vesicles. In the antennal lobe ipsilateral to the cell body, the primary neurite of the serotonin-immunoreactive neuron is unbranched and lacks detectable synaptic connections. The ramifications in the contralateral antennal lobe, however, participate in synaptic connections. At very low frequency, contralateral branches form synapses onto unlabeled processes and also receive synapses from unidentified neurons in the glomeruli, indicating that the neuron may participate directly in synaptic processing of olfactory information. The high ratio of output to input synapses made by the serotonin-immunoreactive processes in the contralateral antennal lobe is consistent with the idea that this neuron may receive synaptic input via its bilateral branches in the protocerebrum and then send information to the contralateral antennal lobe where the neuron may exert feedback or modulatory influences on olfactory information processing in the glomeruli.
- Christensen, T. A., Lehman, H. K., Teal, P. E., Itagaki, H., Tumlinson, J. H., & Hildebrand, J. G. (1992). Diel changes in the presence and physiological actions of octopamine in the female sex-pheromone glands of heliothine moths. Insect Biochemistry and Molecular Biology, 22(8), 841-849.More infoAbstract: Recent evidence suggests that the biogenic monoamine octopamine (OA) may be involved in the regulation of female sex-pheromone production in Lepidoptera. A radioenzymatic assay coupled with high performance liquid chromatography revealed the presence of OA in the innervated sex-pheromone gland of the corn earworm moth Helicoverpa (Heliothis) zea. Significantly more OA was found in glands just before the onset of scotophase (ca 320 fmol/gland), compared to levels at mid-photophase or just after the onset of scotophase (ca 160 fmol/gland). Exogenous OA had several actions on pheromone production. H. zea virgin females normally do not produce pheromone during the photophase, but highly significant levels of pheromone were induced by injection of OA into intact, day-2 photophase females. Importantly, this effect was absent in older females that showed increased levels of flight and oviposition activity. A second action of OA was revealed in isolated abdomen preparations from day-2 H. virescens females. Exogenous OA stimulated highly significant increases in pheromone production if abdomens were treated at the onset of scotophase, but not if they were treated in photophase. This critical period for OA action in these reduced preparations coincided with the time when peak levels of OA were present in the pheromone gland tissue. OA is therefore sufficient to induce pheromone production, but its actions in these short-lived insects depend on factor such as age and photoperiod. Diel fluctuations in OA levels in the pheromone gland, together with the observed phermonotropic actions of this amine, support the hypothesis that OA is involved in the regulation of pheromone production in these insects. © 1992.
- Davis, N. T., & Hildebrand, J. G. (1992). Vasopressin-immunoreactive neurons and neurohemal systems in cockroaches and mantids. Journal of Comparative Neurology, 320(3), 381-393.More infoPMID: 1613132;Abstract: Vasopressin-like neuropeptides of insects are of special interest because of their possible function as hormones and neuromodulators. Therefore, this study was undertaken by using whole-mount immunofluorescent staining by two antisera that recognize different types of vasopressin-like immunoreactive groups of neurons in the cockroaches Periplaneta americana, Leucophaea maderae, Nauphoeta cinerea, Diploptera punctata, and Blaberus discoidalis and in the mantids Litaneuria minor and Tenodera aridifolia sinensis. Using an antiserum to Arg/vasopressin, only two cells, the paired ventral paramedian (PVP) neurons, were immunostained in the central nervous system (CNS) of the cockroaches. These cells are located in the subesophageal ganglion, project throughout the CNS, and appear to be neurosecretory. Their varicose collaterals extend into the dorsal (motor) neuropil of the segmental ganglia, and this neuropil may be the principal site of the release of their neurosecretion. The PVP neurons were also stained by an antiserum to Lys/vasopressin; in addition, this antiserum stained several other groups of neurons, most of which appeared to be neurosecretory. Two pairs of Lys/vasopressin-immunoreactive cells are similar to the PVP neurons in that they are located in the subesophageal ganglion, extend through the ventral nerve cord, have collaterals in the dorsal neuropil of the segmental ganglia, and appear to be neurosecretory within the CNS. In addition, midventral and anteroventral clusters of Lys/vasopressin-immunoreactive neurosecretory neurons in the subesophageal ganglion project neurohemal release sites on the corpora allata. Other types of Lys/vasopressin-immunoreactive neurons include median and lateral neurosecretory cells of the protocerebrum and neurosecretory cells in the tritocerebrum, all of which project to the corpora cardiaca. In the abdominal ganglia there are posterolateral clusters of Lys/vasopressin neurosecretory neurons, and these cells extend to neurohemal release sites on the transverse and lateral cardiac nerves. In mantids the anti-Arg/vasopressin and anti-Lys/vasopressin antisera stained most of the same groups of neurons that these antisera recognized in cockroaches. The results of this study suggest that there are two or more vasopressin-like peptides in cockroaches and mantids and that these peptides may be released either as hormones in the blood or as neurosecretions within the CNS. Their function(s) in these insects remains to be determined.
- Hildebrand, J. G., Christensen, T. A., Harrow, I. D., Homberg, U., Matsumoto, S. G., & Waldrop, B. R. (1992). The roles of local interneurons in the processing of olfactory information in the antennal lobes of the moth Manduca sexta.. Acta Biologica Hungarica, 43(1-4), 167-174.More infoPMID: 1299109;Abstract: The antennal lobe (AL) of the sphinx month Manduca sexta is characterized by a typically glomerular neuropil and two principal classes to neurons local interneurons and projection neurons. The somata of these neurons reside in defined neural cell-body groups in the AL, and the neurons exhibit characteristic patterns of innervation of the glomeruli. Evidence gathered to date indicates that individual antennal olfactory receptor-cell axons project to single glomeruli in the ipsilateral AL and make excitatory, apparently cholinergic synapses with neurites of AL neurons (usually local neurons) innervating the target glomeruli. Much has been learned about the physiology of the projection neurons, but only recently have the physiological properties and functions of the local interneurons been examined systematically through the use of intracellular recording and staining methods. Immunocytochemical studies have shown that most of the local interneurons contain GABA as well as one or more putative neuropeptides. Physiological, pharmacological, and biochemical experiments support the view that GABAergic local interneurons are responsible for inhibitory synaptic inputs to projection neurons that predominate in shaping the activity of projection neurons conveying synaptically processed olfactory information to higher-order centers in the protocerebrum.
- Rospars, J. P., & Hildebrand, J. G. (1992). Anatomical identification of glomeruli in the antennal lobes of the male sphinx moth Manduca sexta. Cell & Tissue Research, 270(2), 205-227.More infoPMID: 1451169;Abstract: Computer-assisted neuroanatomical methods have been used to demonstrate unique identities of the glomeruli of the antennal lobes (ALs) in males of the sphinx moth Manduca sexta. The glomerular neuropil consists of the male-specific macroglomerular complex, which comprises two closely apposed bulky subunits, and 64±1 "ordinary" glomeruli arrayed in a shell around a central region of coarse neuropil. Computergenerated maps show the exact locations of all glomeruli and adjacent groups of neuronal somata in a constant Cartesian coordinate system, such that these can be accurately identified in any individual. The glomeruli belong to three classes according to the number and type of identification criteria they satisfy. The larger class comprises glomeruli (n=44) identified only in the computer-generated maps on the basis of their relative positions. The other two classes include glomeruli that were also identified in sections, either directly from their proximity to readily identifiable structures and their shape and size (n=10, including the labial-palp-pit-organ (LPO) glomerulus), or indirectly from their positions relative to the former (n=9). Two very small glomeruli were present in only one AL, demonstrating the existence of anomalous glomeruli, whereas another glomerulus had no homologue in both ALs of one individual. The true number of ordinary glomeruli (per male AL) was thus estimated to be 64. The uncertainty in delineating some glomeruli might affect this number without implying modification of the homologies proposed. The locations of tracts and cell groups, both within and near the AL, are also invariant with respect to glomeruli, as shown in the computer maps. The methods employed are general and might be useful to researchers in related fields. The results obtained call for more attention to the precise geometry of neural structures. © 1992 Springer-Verlag.
- Stengl, M., Zufall, F., Hatt, H., & Hildebrand, J. G. (1992). Olfactory receptor neurons from antennae of developing male Manduca sexta respond to components of the species-specific sex pheromone in vitro. Journal of Neuroscience, 12(7), 2523-2531.More infoPMID: 1377232;Abstract: Male-specific olfactory receptor neurons, dissociated from developing antennae of the moth Manduca sexta and grown in long-term primary cell culture, can respond to at least one component of the female moth's sex- pheromone blend with the opening of a nonspecific cation channel. This response does not require the coapplication of pheromone-binding protein.
- Christensen, T. A., Itagaki, H., E., P., Jasensky, R. D., Tumlinson, J. H., & Hildebrand, J. G. (1991). Innervation and neural regulation of the sex pheromone gland in female Heliothis moths. Proceedings of the National Academy of Sciences of the United States of America, 88(11), 4971-4975.More infoPMID: 2052579;PMCID: PMC51789;Abstract: Female Heliothis moths normally produce their species-specific male attractant (sex pheromone blend) during scotophase, and this production is stimulated by pheromone biosynthesis activating neuropeptide (PBAN), presumably carried in the hemolymph. Several lines of evidence indicate that the central nervous system plays another critical role in this regulation. Pheromone biosynthesis was induced during photophase by electrical stimulation of the ventral nerve cord or the peripheral nerves projecting from the terminal abdominal ganglion to the pheromone gland in the tip of the abdomen. Electron microscopy further revealed that axonal branches innervate the gland tissue. Nerve branches associated with pheromone gland cells are enwrapped in glia and contain dense-core vesicles, suggesting that the innervation of the gland might be neurosecretory. Finally, the biogenic monoamine octopamine was nearly as effective as purified Heliothis zea PBAN in stimulating pheromone biosynthesis when injected into intact females during mid-photophase. Furthermore, both octopamine and PBAN stimulated significant increases in the pheromone content of the glands in isolated abdomens lacking a ventral nerve cord but only when abdomens were treated at the onset of scotophase. These data suggest that the regulation of sex pheromone production in Heliothis is more complex than previously thought. Activation of the gland appears to be governed by both neural and hormonal mechanisms, and these control mechanisms depend on photoperiodic cues.
- Christensen, T. A., Mustaparta, H., & Hildebrand, J. G. (1991). Chemical communication in heliothine moths - II. Central processing of intra- and interspecific olfactory messages in the male corn earworm moth Helicoverpa zea. Journal of Comparative Physiology A, 169(3), 259-274.More infoAbstract: 1. The antennal lobes (ALs) in the brain of the corn earworm moth Helicoverpa zea (formerly Heliothis zea; Lepidoptera: Noctuidae) were examined using combined anatomical and electrophysiological methods. Like other moths, male H. zea possess a sex-specific macroglomerular complex (MGC) for processing information about the female sex-pheromone blend. Unlike other moths, however, the MGC in H. zea consists of 3 distinct glomerular structures: two situated dorsally, and a third situated ventrally (Fig. 1). 2. Intracellular recording and staining revealed a population of projection neurons that link the MGC with the protocerebrum (Figs. 4, 7, and 11). Four physiological classes of MGC neurons were identified based upon their responses to stimulation of the antenna with different components of the pheromonal blends of H. zea and Heliothis virescens (a sympatric species). One class responded selectively to the principal component in both species, Z11 16:AL (Figs. 2 and 3). A second, more broadly tuned class showed strong responses to Z11-16:AL and also to other pheromonal components (Figs. 5 and 6). A third class did not respond to Z11-16:AL but did respond to Z9-14:AL, a substance released by H. virescens females that helps attract conspecific males while it inhibits the attraction of H. zea males (Figs. 8, 9 and 10). Some of these neurons also responded to another pheromonal component required for male attraction in H. zea, Z9-16: AL. A fourth class responded in a unique fashion to a blend of Z11-16: AL and Z9-14:AL(Fig. 12). 3. Projection neurons that responded to Z11-16:AL had arborizations in all 3 MGC glomeruli (Figs. 4 and 7), whereas neurons that responded to Z9-14:AL from H. virescens had arborizations in just one of the dorsal glomeruli of the MGC (Fig. 11). Thus these two types of neurons with widely different quality-coding functions have overlapping arborizations in one dorsal glomerulus in the MGC, demonstrating that the MGC is not exclusively involved with processing species-specific (pheromonal) information. © 1991 Springer-Verlag.
- Hansson, B. S., Christensen, T. A., & Hildebrand, J. G. (1991). Functionally distinct subdivisions of the macroglomerular complex in the antennal lobe of the male sphinx moth Manduca sexta. Journal of Comparative Neurology, 312(2), 264-278.More infoPMID: 1748732;Abstract: Each antennal lobe in the brain of a male moth has a distinctive neuropil structure, the macroglomerular complex (MGC), which is specialized for primary processing of information about the conspecific female sex-pheromone blend. Olfactory interneurons with dendritic arborizations in the MGC were examined by means of tandem intracellular recording and staining with Lucifer Yellow. Neurons that responded selectively to stimulation of the antenna with the major pheromone component, (E,Z)-10,12-hexadecadienal, had arborizations that were restricted to a toroidal subdivision (the 'toroid') of the MGC. Similarly, neurons that responded selectively to antennal stimulation with (E,Z)-11,13-pentadecadienal, a more stable mimic of a second essential but chemically unstable pheromone component, (E,E,Z)-10,12,14-hexadecatrienal, had arborizations confined to a globular subdivision (the 'cumulus') of the MGC situated more proximally to the antennal nerve input. One neuron that responded to both of these stimuli clearly had arborizations in both subdivisions of the MGC. These anatomically distinct subdivisions of the MGC thus appear also to be functionally separate regions of pheromone-processing neuropil.
- Homberg, U., & Hildebrand, J. G. (1991). Histamine-immunoreactive neurons in the midbrain and suboesophageal ganglion of the sphinx moth Manduca sexta. Journal of Comparative Neurology, 307(4), 647-657.More infoPMID: 1869635;Abstract: This paper describes the distribution of histamine-like immunoreactivity in the midbrain and suboesophageal ganglion of the sphinx moth Manduca sexta. Intense immunocytochemical staining was detected in ten bilateral pairs of neurons in the median protocerebrum and in one pair of neurons in the suboesophageal ganglion. Whereas most areas of the brain and suboesophageal ganglion are innervated by one or more of these neurons, typically no immunoreactive fibers were found in the mushroom bodies, the protocerebral bridge, and the lateral horn of the protocerebrum. The 11 histamine-immunoreactive neurons were reconstructed from serial sections. Ten neurons have bilateral arborizations, often with axonal projections in symmetric areas of both hemispheres. One neuron, whose soma resides in the lateral protocerebrum, has only unilateral projections. Of the 11 neurons, 6 occur in pairs with similar morphological features. In addition to these neurons, weak histamine-like immunoreactivity was detected in 7-13 interneurons that were not reconstructed individually. The central projections of the ocellar nerves from the intracranial ocelli also exhibit histamine-like immunoreactivity. The single-cell reconstructions reveal similarities between the organization of histamine- and serotonin-immunoreactive neurons in the brain and suboesophageal ganglion of this insect.
- Homberg, U., Davis, N. T., & Hildebrand, J. G. (1991). Peptide-immunocytochemistry of neurosecretory cells in the brain and retrocerebral complex of the sphinx moth Manduca sexta. Journal of Comparative Neurology, 303(1), 35-52.More infoPMID: 1706364;Abstract: Antisera against a variety of vertebrate and invertebrate neuropeptides were used to map cerebral neurosecretory cells in the sphinx moth Manduca sexta. Intense immunoreactive staining of distinct populations of neurosecretory cells was obtained with antisera against locust adipokinetic hormone, bovine pancreatic polypeptide, FMRFamide, molluscan small cardioactive peptide (SCP(B)), leucine-enkephalin, gastrin/cholecystokinin, and crustacean β-pigment dispersing hormone (βPDH). Other antisera revealed moderate to weak staining. Each type or neurosecretory cell is immunoreactive with at least one of the antisera tested, and most of these neurons can be identified anatomically. The staining patterns provide additional information on the organization of cerebral neurosecretory cells in M. sexta. Based upon anatomical and immunocytochemical characteristics, 11 types of neurosecretory cells have been recognized in the brain, one type in the suboesophageal ganglion, and one in the corpus cardiacum. Extensive colocalization experiments show that many neurosecretory cells are immunoreactive with several different antisera. This raises the possibility that these cells may release mixtures of neuropeptides into the hemolymph, as has been demonstrated in certain other systems. The immunocytochemical data should be helpful in efforts to identify additional peptide neurohormones released from the brain of this and other insects.
- Kanzaki, R., Arbas, E. A., & Hildebrand, J. G. (1991). Physiology and morphology of descending neurons in pheromone-processing olfactory pathways in the male moth Manduca sexta. Journal of Comparative Physiology A, 169(1), 1-14.More infoPMID: 1941713;Abstract: 1. We have characterized the responses and structure of olfactory descending neurons (DNs) that reside in the protocerebrum (PC) of the brain of male sphinx moths Manduca sexta and project toward thoracic ganglia. 2. Excitatory responses of DNs to pheromone blends were of two general types: (a) brief excitation (BE) that recovered to background in
- Kanzaki, R., Arbas, E. A., & Hildebrand, J. G. (1991). Physiology and morphology of protocerebral olfactory neurons in the male moth Manduca sexta. Journal of Comparative Physiology A, 168(3), 281-298.More infoPMID: 2066906;Abstract: 1. We have used intracellular recording and staining with Lucifer Yellow, followed by reconstruction from serial sections, to characterize the responses and structure of olfactory neurons in the protocerebrum (PC) of the brain of the male sphinx moth Manduca sexta. 2. Many olfactory protocerebral neurons (PCNs) innervate a particular neuropil region lateral to the central body, the lateral accessory lobe (LAL), which appears to be important for processing olfactory information. 3. Each LAL is linked by its constituent neurons to the ipsilateral lateral PC, where projection neurons from the antennal lobe terminate, as well as to other regions of the PC. The LALs are also linked to each other by bilateral neurons with arborizations in each LAL. 4. Some PC neurons showed long-lasting excitation (LLE) that outlasted the olfactory stimuli by ≥ 1 s, and as long as 30 s in some preparations. LLE was more frequently elicited by the sex-pheromone blend than by individual pheromone components. All bilateral neurons that showed LLE had arborizations in the LALs. LLE responses were also recorded in a single local neuron innervating the mushroom body. 5. In some other PC neurons, pheromonal stimuli elicited brief excitations that recovered to background firing rates
- Zufall, F., Stengl, M., Franke, C., Hildebrand, J. G., & Hatt, H. (1991). Ionic currents of cultured olfactory receptor neurons from antennae of male manduca sexta. Journal of Neuroscience, 11(4), 956-965.More infoPMID: 1849175;Abstract: Whole-cell and single-channel voltage-clamp techniques were used to identify and characterize the ionic currents of insect olfactory receptor neurons (ORNs) in vitro. The cells were isolated from the antennae of male Manduca sexta pupae at stages 3-5 of adult development and maintained in primary cell culture. After 2-3 weeks in vitro, the presumptive ORNs had resting potentials of -62 ± 12 mV (n = 18) and expressed at least 1 type of Na+ channel and at least 3 types of K+ channels. Na+ currents, recorded in the whole-cell mode, were reversibly blocked by 0.1 μM tetrodotoxin. The predominant type of K+ channel observed was a voltage-activated K+ channel (γ = 30 pS) with characteristics similar to those of the delayed rectifier. The activity of the 30-pS K+ channel could be inhibited by the application of nucleotides to the cytoplasmic face of inside-out patches of membrane. The nucleotides had relative potencies as follows: ATP > cGMP > cAMP, with an inhibition constant for ATP of K1 = 0.18 mM. Raising the intracellular Ca2+ concentration from 0.1 to 5 μM induced the opening of a Ca2+- activated K+ channel (γ = 66 pS at 0 mV) that had a low voltage sensitivity. A third, transient type of K+ channel (γ = 12-18 pS) could be activated by depolarizing voltage steps from very negative resting potentials. Properties of this channel were similar to those of the "A-channel." These results support the conclusion that M. sexta ORNs differentiate in vitro and provide the basis for studying primary mechanisms of olfactory transduction.
- Christensen, T. A., Geoffrion, S. C., & Hildebrand, J. G. (1990). Physiology of interspecific chemical communication in Heliothis moths. Physiological Entomology, 15(3), 275-283.More infoAbstract: Discusses the pheromones (Z)-11-hexadecenal, a conspecific component, and (Z)-9-tetradecenal, a component found in the pheromone blend of a sympatric species H. virescens that inhibits attraction of H. zea males. -from Authors
- Hayashi, J. H., & Hildebrand, J. G. (1990). Insect olfactory neurons in vitro: Morphological and physiological characterization of cells from the developing antennal lobes of Manduca sexta. Journal of Neuroscience, 10(3), 848-859.More infoPMID: 2156962;Abstract: To facilitate studies of the development and membrane biophysics of cells in the central olfactory pathway of the moth Manduca sexta, we have dissociated neurons and glial cells from the antennal lobes (ALs) and cultivated them in the controlled environment of tissue culture. Cultures produced from cells of the lateral group of AL neurons alone are enriched in local interneurons (LNs), while cultures made from cells of the median and anterior groups of AL neurons contain projection neurons (PNs) but lack LNs. Cultures containing only PNs, but not cultures with both PNs and LNs, require a conditioning factor derived from a conspecific cell line in order to ensure survival. Under these conditions, we identify 5 types of cells in the "PN-only" cultures that are consistently observed and distinguishable on the basis of their morphology and characteristic whole-cell current profiles. In cultures that contain both PNs and LNs, we find 3 additional types of cells with neuronlike appearance. Whereas the PNs have whole-cell currents that suggest a neuronal phenotype, we have not yet observed similar currents in the 3 other types of cells. We tentatively identify the latter 3 cell types as LNs whose development has been arrested. The conditioning factor also has a trophic effect on phase-dark, multipolar cells that are observed in freshly dispersed cultures. Their morphology and whole-cell currents lead us to suggest that they are glial cells.
- Homberg, U., Kingan, T. G., & Hildebrand, J. G. (1990). Distribution of FMRFamide-like immunoreactivity in the brain and suboesophageal ganglion of the sphinx moth Manduca sexta and colocalization with SCPB-, BPP-, and GABA-like immunoreactivity. Cell and Tissue Research, 259(3), 401-419.More infoPMID: 2180574;Abstract: Using an antiserum against the tetrapeptide FMRFamide, we have studied the distribution of FMRFamide-like substances in the brain and suboesophageal ganglion of the sphinx moth Manduca sexta. More than 2000 neurons per hemisphere exhibit FMRFamide-like immunoreactivity. Most of these cells reside within the optic lobe. Particular types of FMRFamide-immunoreactive neurons can be identified. Among these are neurosecretory cells, putatively centrifugal neurons of the optic lobe, local interneurons of the antennal lobe, mushroom-body Kenyon cells, and small-field neurons of the central complex. In the suboesophageal ganglion, groups of ventral midline neurons exhibit FMRFamide-like immunoreactivity. Some of these cells have axons in the maxillary nerves and apparently give rise to FMRFamide-immunoreactive terminals in the sheath of the suboesophageal ganglion and the maxillary nerves. In local interneurons of the antennal lobe and a particular group of protocerebral neurons, FMRFamide-like immunoreactivity is colocalized with GABA-like immunoreactivity. This suggests that FMRFamide-like peptides may be cotransmitters of these putatively GABAergic interneurons. All FMRFamide-immunoreactive neurons are, furthermore, immunoreactive with an antiserum against bovine pancreatic polypeptide, and the vast majority is also immunoreactive with an antibody against the molluscan small cardioactive peptide SCPB. Therefore, it is possible that more than one peptide is localized within many FMRFamide-immunoreactive neurons. The results suggest that FMRFamide-related peptides are widespread within the nervous system of M. sexta and might function as neurohormones and neurotransmitters in a variety of neuronal cell types. © 1990 Springer-Verlag.
- Itagaki, H., & Hildebrand, J. G. (1990). Olfactory interneurons in the brain of the larval sphinx moth Manduca sexta. Journal of Comparative Physiology A, 167(3), 309-320.More infoPMID: 2231474;Abstract: 1. The physiology and morphology of olfactory interneurons in the brain of larval Manduca sexta were studied using intracellular recording and staining techniques. Antennal olfactory receptors were stimulated with volatile substances from plants and with pure odorants. Neurons responding to the stimuli were investigated further to reveal their response specificities, dose-response characteristics, and morphology. 2. We found no evidence of specific 'labeled-lines' among the odor-responsive interneurons, as none responded exclusively to one plant odor or pure odorant; most olfactory interneurons were broadly tuned in their response spectra. This finding is consistent with an 'across-fiber' pattern of odor coding. 3. Mechanosensory and olfactory information are integrated at early stages of central processing, appearing in the responses of some local interneurons restricted to the primary olfactory nucleus in the brain, the larval antennal center (LAC). 4. The responses of LAC projection neurons and higher-order protocerebral interneurons to a given odor were more consistent than the responses of LAC local interneurons. 5. The LAC appears to be functionally subdivided, as both local and projection neurons had arborizations in specific parts of the LAC, but none had dendrites throughout the LAC. 6. The mushroom bodies and the lateral protocerebrum contain neurons that respond to olfactory stimulation. © 1990 Springer-Verlag.
- Kingan, T. G., Teplow, D. B., Phillips, J. M., Riehm, J. P., Rao, K. R., Hildebrand, J. G., Homberg, U., Kammer, A. E., Jardine, I., Griffin, P. R., & Hunt, D. F. (1990). A new peptide in the FMRFamide family isolated from the CNS of the hawkmoth, Manduca sexta. Peptides, 11(4), 849-856.More infoPMID: 2235684;Abstract: We have purified a FMRFamide-like peptide from extracts of brain-subesophageal ganglion of the moth, Manduca sexta. The purification was monitored with a new, competitive ELISA, and accomplished with ion exchange and reverse-phase HPLC. The peptide structure was determined by a combination of tandem mass spectrometry and automated Edman degradation. The amino acid sequence of the peptide is
- Stengl, M., & Hildebrand, J. G. (1990). Insect olfactory neurons in vitro: Morphological and immunocytochemical characterization of male-specific antennal receptor cells from developing antennae of male manduca sexta. Journal of Neuroscience, 10(3), 837-847.More infoPMID: 2319305;Abstract: Sex-pheromone components released by Manduca sexta females are detected solely by male-specific olfactory receptor neurons that innervate long sensilla trichodea on the male antennae. To facilitate studies of the development and physiology of these receptor cells, we have produced primary in vitro cultures of cells dissociated from pupal male antennae. These cultures comprise several morphological types of cells, 2 of which have been characterized immunocytochemically with a pair of monoclonal antibodies that were shown previously to recognize certain antigens in olfactory receptor neurons at defined stages of development. The good correlation between in vivo and in vitro expression of these antigens suggests that the immunocytochemically recognized cells are olfactory receptor neurons that follow at least partially their normal course of differentiation in vitro.
- Stengl, M., Homberg, U., & Hildebrand, J. G. (1990). Acetylcholinesterase activity in antennal receptor neurons of the sphinx moth Manduca sexta. Cell and Tissue Research, 262(2), 245-252.More infoPMID: 2076532;Abstract: We have used a cytochemical technique to investigate the distribution of acetylcholinesterase (AChE) activity in the antenna of the sphinx moth Manduca sexta. High levels of echothiophate-insensitive (presumably intracellular) AChE activity were found in six different types of antennal receptors localized in specific regions of the three antennal segments of the adult moth. Mechanosensory organs in the scape and pedicel, the Böhm bristles and Johnston's organ, are innervated by AChE-positive neurons. In each annulus of the antennal flagellum, AChE-positive neurons are associated with six sensilla chaetica and a peg organ, probably a sensillum styloconicum. At least 112 receptor neurons (8-10 per annulus) innervating the intersegmental membranes between the 14 distalmost annuli also exhibit high levels of echothiophate-resistant AChE. In addition, each annulus has more than 30 AChE-positive somata in the epidermis of the scale-covered (back) side of the flagellum, and 4 AChE-positive somata reside within the first annulus of the flagellum. Since none of the olfactory receptor neurons show a high level of echothiophateresistant AChE activity, and all known mechanoreceptors are AChE-positive, apparently "intracellular" AChE activity in the antenna correlates well with mechanosensory functions and is consistent with the idea that these cells employ acetylcholine as a neurotransmitter. © 1990 Springer-Verlag.
- Christensen, T. A., Mustaparta, H., & Hildebrand, J. G. (1989). Discrimination of sex pheromone blends in the olfactory system of the moth. Chemical Senses, 14(3), 463-477.
- Homberg, U., & Hildebrand, J. G. (1989). Serotonin immunoreactivity in the optic lobes of the sphinx moth Manduca sexta and colocalization with FMRFamide and SCP(B) immunoreactivity. Journal of Comparative Neurology, 288(2), 243-253.More infoPMID: 2677065;Abstract: In the optic lobes (OLs) of the sphinx moth Manduca sexta, 300-350 neurons per hemisphere are immunoreactive with an antiserotonin antiserum. Two groups of weakly serotonin-immunoreactive cells (OL1) appear to be amacrine cells of the medulla, whereas more intensely immunoreactive cells (OL2) are probably centrifugal neurons that innervate the lobula, medulla, and lamina, as well as the superior protocerebrum. At least one other OL2 cells is a local optic-lobe interneuron with arborizations in the dorsal medulla and lobula. The serotonin-immunoreactive cells are also immunoreactive with an antiserum against Drosophila melanogaster DOPA decarboxylase. All OL2 cells, but not the OL1 cells, are furthermore immunoreactive with an anti-FMRFamide antiserum and an anti-SCP(B) antiserum. This suggests that neuropeptides related or identical to FMRFamide and SCP(B) are colocalized and may serve as cotransmitters with serotonin in OL2 optic-lobe interneurons.
- Homberg, U., & Hildebrand, J. G. (1989). Serotonin-immunoreactive neurons in the median protocerebrum and suboesophageal ganglion of the sphinx moth Manduca sexta. Cell and Tissue Research, 258(1), 1-24.More infoPMID: 2680097;Abstract: Serotonin-immunoreactive neurons in the median protocerebrum and suboesophageal ganglion of the sphinx moth Manduca sexta were individually reconstructed. Serotonin immunoreactivity was detected in 19-20 bilaterally symmetrical pairs of interneurons in the midbrain and 10 pairs in the suboesophageal ganglion. These neurons were also immunoreactive with antisera against DOPA decarboxylase. All major neuropil regions except the protocerebral bridge are innervated by these neurons. In addition, efferent cells are serotonin-immunoreactive in the frontal ganglion (5 neurons) and the suboesophageal ganglion (2 pairs of neurons). The latter cells probably give rise to an extensive network of immunoreactive terminals on the surface of the suboesophageal ganglion and suboesophageal nerves. Most of the serotonin-immunoreactive neurons show a gradient in the intensity of immunoreactive staining, suggesting low levels of serotonin in cell bodies and dendritic arbors and highest concentrations in axonal terminals. Serotonin-immunoreactive cells often occur in pairs with similar morphological features. With one exception, all serotonin-immunoreactive neurons have bilateral projections with at least some arborizations in identical neuropil areas in both hemispheres. The morphology of several neurons suggests that they are part of neuronal feedback circuits. The similarity in the arborization patterns of serotonin-immunoreactive neurons raises the possibility that their outgrowing neurites experienced similar forces during embryonic development. The morphological similarities further suggest that serotonin-immunoreactive interneurons in the midbrain and suboesophageal ganglion share physiological characteristics. © 1989 Springer-Verlag.
- Homberg, U., Christensen, T. A., & Hildebrand, J. G. (1989). Structure and function of the deutocerebrum in insects.. Annual Review of Entomology, 34, 477-501.More infoPMID: 2648971;
- Kanzaki, R., Arbas, E. A., Strausfeld, N. J., & Hildebrand, J. G. (1989). Physiology and morphology of projection neurons in the antennal lobe of the male moth Manduca sexta. Journal of Comparative Physiology A, 165(4), 427-453.More infoPMID: 2769606;Abstract: 1. We have used intracellular recording and staining, followed by reconstruction from serial sections, to characterize the responses and structure of projection neurons (PNs) that link the antennal lobe (AL) to other regions of the brain of the male sphinx moth Manduca sexta. 2. Dendritic arborizations of the AL PNs were usually restricted either to ordinary glomeruli or to the male-specific macroglomerular complex (MGC) within the AL neuropil. Dendritic fields in the MGC appeared to belong to distinct partitions within the MGC (Figs. 2, 3). PNs innervating the ordinary glomeruli had arborizations in a single glomerulus (uniglomerular) (Figs. 6, 7, 9, 11, 12A) or in more than one ordinary glomerulus of one AL (multiglomerular) (Figs. 12B, C, 14, 15), or in one case, in single glomeruli in both ALs (bilateral-uniglomerular) (Fig. 16). One PN innervated the MGC and many or all ordinary glomeruli of the AL (Fig. 13). 3. PNs with dendritic arborizations in the ordinary glomeruli and PNs associated with the MGC typically projected both to the calyces of the ipsilateral mushroom body and to the lateral protocerebrum, but some differences in the patterns of termination in those regions have been noted for the two classes of PNs (Figs. 2, 3, 6, 7, 9, 16). One PN conspicuously lacked branches in the calyces but did project to the lateral protocerebrum (Fig. 14). The PN innervating the MGC and many ordinary glomeruli projected to the calyces of the ipsilateral mushroom body and the superior protocerebrum (Fig. 13). 4. Crude sex-pheromone extracts excited all neurons with arborizations in the MGC, although some were inhibited by other odors (Figs. 3, 4). One P(MGC) was excited by crude sex-pheromone extract and by a mimic of one component of the pheromone blend but was inhibited by another component of the blend (Fig. 5). 5. PNs with dendritic arborizations in ordinary glomeruli were excited (Figs. 7, 8, 10) or inhibited (Figs. 9, 11) by certain non-pheromonal odors. Some of these PNs also responded to mechanosensory stimulation of the antennae (Figs. 10, 11, 15, 16). 6. The PN with dendritic arborizations in the MGC and many ordinary glomeruli was excited by crude sex-pheromone extracts and non-pheromonal odors and also responded to mechanosensory stimulation of the antenna (Fig. 13). © 1989 Springer-Verlag.
- Kingan, T. G., & Hildebrand, J. G. (1989). Sexually dimorphic polypeptides in developing antennal sensory neurons of an insect. Journal of Neuroscience, 9(6), 1951-1960.More infoPMID: 2723759;
- Waldrop, B., & Hildebrand, J. G. (1989). Physiology and pharmacology of acetylcholinergic responses of interneurons in the antennal lobes of the moth Manduca sexta. Journal of Comparative Physiology A, 164(4), 433-441.More infoPMID: 2926690;Abstract: 1. Neurons in the antennal lobe (AL) of the moth Manduca sexta respond to the application, via pressure injection into the neuropil, of acetylcholine (ACh). When synaptic transmission is not blocked, both excitatory (Fig. 2) and inhibitory (Fig. 3) responses are seen. 2. Responses to ACh appear to be receptor-mediated, as they are associated with an increase in input conductance (Figs. 2B and 3B) and are dose-dependent (Fig. 2C). 3. All neurons responsive to ACh are also excited by nicotine. Responses to nicotine are stronger and more prolonged than responses to ACh (Fig. 4C). No responses are observed to the muscarinic agonist, oxotremorine (Fig. 4B). 4. Curare blocks responses of AL neurons to applied ACh, while atropine and dexetimide are only weakly effective at reducing ACh responses (Figs. 5 and 6). 5. Curare is also more effective than atropine or dexetimide at reducing synaptically-mediated responses of AL neurons (Fig. 7). 6. In one AL neuron, bicuculline methiodide (BMI) blocked the IPSP produced by electrical stimulation of the antennal nerve, but it did not reduce the inhibitory response to application of ACh (Fig. 8). © 1989 Springer-Verlag.
- Christensen, T. A., & Hildebrand, J. G. (1988). Frequency coding by central olfactory neurons in the sphinx moth Manduca sexta. Chemical Senses, 13(1), 123-130.More infoAbstract: Sexually receptive female moths and many other insects release chemical attractants (sex pheromones) to lure conspecific mates. Recent evidence indicates, moreover, that the odor plume formed downwind from the female possesses a discontinuous structure that appears to provide the searching male with orientationcues. Using intracellular methods, we find that many central olfactory neurons in male moths (Manduca sexta) can track pulsed pheromonal stimuli precisely. The cells respond to each brief odor pulse with a similarly brief burst of action potentials, and the separation between response bursts is aided by inhibitory synaptic input. Furthermore, these neurons appear to participate in at least two levels of 'feature detection': they respond selectively to pheromonal stimuli, and they follow pulsed stimulation only in a limited range of frequencies Above the frequency limit, the cells respond as if the male is stimulated by a prolonged, uniform concentration of pheromone. The ability of these neurons to encode changes in the temporal characteristics of pheromonal stimuli may provide the male with positional cues to help him locate the pheromone source over long distances. © 1988 IRL Press Ltd.
- Hishinuma, A., Hockfield, S., McKay, R., & Hildebrand, J. G. (1988). Monoclonal antibodies reveal cell-type-specific antigens in the sexually dimorphic olfactory system of Manduca sexta. I. Generation of monoclonal antibodies and patial characterization of the antigens. Journal of Neuroscience, 8(1), 296-307.More infoPMID: 2448431;Abstract: The olfactory system of the moth Manduca sexta is sexually dimorphic. Male moths possess a male-specific olfactory 'subsystem,' comprising olfactory receptor cells (ORCs) and CNS neurons and synaptic areas associated with the detection of female sex pheromones, in addition to elements common to males and females. In order to explore the molecular differences between cells that subserve the sexual dimorphism and odor-specificity of components of the olfactory system, we generated monoclonal antibodies (Mabs) against tissue of the olfactory system of the moth. In 2 fusions, we screened 1105 hybridoma lines and obtained 272 lines that secreted antibodies against Manduca nervous tissue, as assayed immunocytochemically on sections of the primary olfactory center (the antennal lobe) in the brain of Manduca. We describe here 3 classes of Mabs exemplifying the several cell-type-specific antibodies obtained through the screening procedure. Seven hybridoma lines secrete antibodies that specifically recognize cell bodies, axons, and initial segments of dendrites of many or all ORCs of both males and females (classified as olfactory-specific antibodies, OSAs). Electron-microscopic studies of 2 of the Mabs in this class showed that they recognize antigens associated with the cell membrane and that the immunoreactive ORC axons are bundled together in fascicles in the antennal nerve. On immunoblots, one of the OSA Mabs recognizes 3 distinct protein bands of apparant M,s 42,000, 59,000, and 66,000 Da. When tissue samples enriched in either receptor cell bodies, dendrites, and initial segments of axons or in distal segments of axons and their terminals and synapses were extracted separately, different patterns of bands were detected - 42,000 and 59,000 Da bands from cell bodies and initial segments of axons and dendrites, and 42,000 and 66,000 Da bands from distal segments of axons and their terminals - suggesting that the 59,000 Da protein is modified to the 66,000 Da protein during axonal transport. The second Mab we describe here, the male olfactory-specific antibody (MOSA), selectively recognizes the sexually dimorphic ORCs that are present only in males. The antigen recognized by this antibody is found in cell bodies, dendrites, axons, and axon terminals. By electron-microscopic immunocytochemistry, the MOSA immunoreactivity is found in the cytoplasm and appears not to be associated with particular subcellular organelles. This antibody demonstrates that male-specific ORCs are molecularly distinct from other types of ORCs. The third antibody, the mechanosensory-preferring antibody (MPA), recognizes the mechanosensory receptor cells of the antenna but not the ORCs; it also recognizes some other cells in the CNS. In electron micrographs, MPA appears to bind to intermediate filaments in mechanoreceptor cells.
- Hishinuma, A., Hockfield, S., McKay, R., & Hildebrand, J. G. (1988). Monoclonal antibodies reveal cell-type-specific antigens in the sexually dimorphic olfactory system of Manduca sexta. II. Expression of antigens during postembryonic development. Journal of Neuroscience, 8(1), 308-315.More infoPMID: 3339412;Abstract: Two classes of monoclonal antibodies specific to the olfactory system of Manduca sexta have been isolated: the olfactory-specific antibody (OSA), which specifically recognizes many or all olfactory receptor cells (ORCs) in both males and females, and the male olfactory-specific antibody (MOSA), which stains male-specific receptor cells (principally or sexclusively sex-pheromone receptors present only in antennae of males; Hishinuma et al., 1988). In the investigation reported here, we examined the expression of the antigens during postembryonic development in order to correlate the presence of particular antigens with the status of differentiation of the ORCs or with their acquisition of particular functions. As assessed immunocytochemically, the OSA recognizes certain epithelial cells in the antennal imaginal disk of the fifth-instar larva. Later, during the first 70 hr of adult development, when differentiative cell divisions are occurring in the antennal epithelium to generate ORCs and the other cells that make up olfactory sensilla, no cells are stained. Immediately after this period of mitoses, the OSA immunoreactivity reappears exclusively in the ORCs, which begin to elaborate axons as an early event in their differentiation. On immunoblots, the OSA recognizes specific sets of molecules (distinguished on the basis of their apparent molecular weights): 53,000 and 59,000 Da antigens in the disk epithelial cells in the last-instar larva; 53,000, 59,000, and 66,000 Da antigens in the ORCs from 15 to 60% of metamorphic adult development; and 42,000, 59,000, and 66,000 Da antigens in the ORCs from 60 to 100% of adult development. The MOSA also recognizes a subset of the epithelial cells in the antennal disks in male and female larvae. After disappearing at the pupal molt, MOSA staining is not detected again in the epithelium of the developing male antenna until late in metamorphosis, when electroantennogram responses to female sex pheromones are first detectable. This correlation suggests that the MOSA antigen is expressed during the final maturation of the male-specific receptor cells and may be involved in some way in the olfactory functions of those cells, particularly in the detection of the female sex pheromones.
- Homberg, U., Montague, R. A., & Hildebrand, J. G. (1988). Anatomy of antenno-cerebral pathways in the brain of the sphinx moth Manduca sexta. Cell and Tissue Research, 254(2), 255-281.More infoPMID: 3197087;Abstract: In the moth Manduca sexta, the number and morphology of neuronal connections between the antennal lobes and the protocerebrum were examined. Cobalt injections revealed eight morphological types of neurons with somata adjacent to the AL neuropil that project in the inner, middle, and outer antenno-cerebral tracts to the protocerebrum. Neurons innervating the macroglomerular complex and many neurons with fibers in the inner antennocerebral tract have uniglomerular antennal-lobe arborizations. Most neurons in the middle and outer antenno-cerebral tracts, on the other hand, seem to innervate more than one glomerulus. Protocerebral areas receiving direct input from the antennal lobe include the calyces of the mushroom bodies, and circumscribed areas termed "olfactory foci" in the lateral horn of the protocerebrum and several other regions, especially areas in close proximity to the mushroom bodies. Fibers in the inner antenno-cerebral tract that innervate the male-specific macroglomerular complex have arborizations in the protocerebrum that are distinct from the projections of sexually non-specific neurons. Protocerebral neurons projecting into the antennal lobe are much less numerous than antennal-lobe output cells. Most of these protocerebral fibers enter the antennal lobe in small fiber tracts that are different from those described above. In the protocerebrum, these centrifugal cells arborize in olfactory foci and also in the inferior median protocerebrum and the lateral accessory lobes. The morphological diversity of connections between the antennal lobes and the protocerebrum, described here for the first time on a single-cell level, suggests a much greater physiological complexity of the olfactory system than has been assumed so far. © 1988 Springer-Verlag.
- Christensen, T. A., & Hildebrand, J. G. (1987). Male-specific, sex pheromone-selective projection neurons in the antennal lobes of the moth Manduca sexta. Journal of Comparative Physiology A, 160(5), 553-569.More infoPMID: 3612589;Abstract: 1. A subset of olfactory projection neurons in the brain of male Manduca sexta is described, and their role in sex pheromone information processing is examined. 2. These neurons have extensive arborizations in the macroglomerular complex (MGC), a distinctive and sexually dimorphic area of neuropil in the antennal lobe (AL), to which the axons of two known classes of antennal pheromone receptors project. Each projection neuron sends an axon from the AL into the protocerebrum (Figs. 4, 7, 9, 13 and 15). 3. Forty-one projection neurons were characterized according to their responses to electrical stimulation of the antennal nerve as well as olfactory stimulation of antennal receptors (Fig. 1). 4. All neurons exhibited strong selectivity for female sex pheromones. Other behaviorally relevant odors, such as plant volatiles, had no obvious effect on the activity of these neurons (Fig. 2). 5. Two broad physiological categories were found: (a) cells that were excited by stimulation of the ipsilateral antenna with pheromones (29 out of 41), and (b) cells that received a mixed input (inhibition and excitation) from pheromone pathways (12 out of 41). 6. Of the cells in the first category, 13 out of 29 were equally excited in response to stimulation of the antenna with either the principal natural pheromone (bombykal) or a mimic of a second unidentified pheromone ('C-15') and were similarly excited by the natural pheromone blend (Fig. 3). 7. The remaining 16 out of 29 cells responded selectively, and in some cases, in a dose-dependent manner, to stimulation of the antenna with bombykal or C-15, but not both (Figs. 5, 6 and 8). Some of these neurons had dendritic arborizations restricted to only a portion of the MGC neuropil (Fig. 9), whereas most had arborizations throughout the MGC. 8. Of the cells in the second category, 9 out of 12 were excited by bombykal, inhibited by C-l 5, and showed a mixed response to the natural pheromone blend (Figs. 11 and 12). For the other 3 out of 12 cells, the response polarity was reversed for the two chemically-identified odors (Fig. 14). 9. Two additional neurons, which were not tested with olfactory stimuli, were tonically inhibited in response to electrical stimulation of the ipsilateral antennal nerve (Fig. 15). 10. These observations suggest that some of the male-specific projection neurons may signal general pheromone-triggered arousal, whereas a smaller number can actively integrate inputs from the two known receptor classes (Bal- and C-15-selective) and may operate as 'mixture detectors' at this level of the olfactory subsystem that processes information about sex pheromones. © 1987 Springer-Verlag.
- Homberg, U., Kingan, T. G., & Hildebrand, J. G. (1987). Immunocytochemistry of GABA in the brain and suboesophageal ganglion of Manduca sexta. Cell and Tissue Research, 248(1), 1-24.More infoPMID: 3552234;Abstract: We have used specific antisera against protein-conjugated γ-aminobutyric acid (GABA) in immunocytochemical preparations to investigate the distribution of putatively GABAergic neurons in the brain and suboesophageal ganglion of the sphinx moth Manduca sexta. About 20000 neurons per brain hemisphere exhibit GABA-immunoreactivity. Most of these are optic-lobe interneurons, especially morphologically centrifugal neurons of the lamina and tangential neurons that innervate the medulla or the lobula complex. Many GABA-immunoreactive neurons, among them giant fibers of the lobula plate, project into the median protocerebrum. Among prominent GABA-immunoreactive neurons of the median protocerebrum are about 150 putatively negative-feedback fibers of the mushroom body, innervating both the calyces and lobes, and a group of large, fan-shaped neurons of the lower division of the central body. Several commissures in the supra- and suboesophageal ganglion exhibit GABA-immunoreactivity. In the suboesophageal ganglion, a group of contralaterally descending neurons shows GABA-like immunoreactivity. The frontal ganglion is innervated by immunoreactive processes from the tritocerebrum but does not contain GABA-immunoreactive somata. With few exceptions the brain nerves do not contain GABA-immunoreactive fibers. © 1987 Springer-Verlag.
- Kent, K. S., & Hildebrand, J. G. (1987). Cephalic sensory pathways in the central nervous system of larval Manduca sexta (Lepidoptera : Sphingidae).. Philosophical transactions of the Royal Society of London. Series B: Biological sciences, 315(1168), 1-36.More infoPMID: 2881311;Abstract: Central projections of neurons innervating sensory structures on the head of larval Manduca sexta were traced by using methods of anterograde cobalt-diffusion. Regions of the deutocerebrum and tritocerebrum in the brain receive input from the antenna, labrum, maxilla, labial palps, hypopharynx and other unidentified regions of the buccal cavity. Antennal, maxillary and labial inputs project to the larval antennal centre (LAC) of the deutocerebrum. Stemmatal neurons and a few antennal neurons project into the protocerebrum. The suboesophageal ganglion (SEG) receives input from mechanosensory neurons in all parts of the head and its sensory appendages. Some mechanosensory neurons project further to the first thoracic ganglion. In addition to receiving input from chemosensory neurons of the maxilla, the SEG may also receive chemosensory input from epipharyngeal sensilla of the labrum.
- Kent, K. S., Hoskins, S. G., & Hildebrand, J. G. (1987). A novel serotonin-immunoreactive neuron in the antennal lobe of the sphinx moth Manduca sexta persists throughout postembryonic life. Journal of Neurobiology, 18(5), 451-465.More infoPMID: 3309187;Abstract: A single serotonin-immunoreactive neuron in the antennal lobe (AL) of the brain of the sphinx moth Manduca sexta is present in larval, pupal, and adult stages. This neuron has a neurite that extends to the contralateral AL, where it forms sparse arborizations in each glomerulus. Other neurites from this neuron project into the ipsilateral and contralateral protocerebrum. This cell is morphologically very different from other neurons previously characterized in the adult AL. The neuron maintains the same basic profile in the adult as in the larva, although fine processes such as the arborizations within the AL neuropil appear to be restructured to conform to the larger, more anatomically differentiated regions of the adult brain.
- Waldrop, B., Christensen, T. A., & Hildebrand, J. G. (1987). GABA-mediated synaptic inhibition of projection neurons in the antennal lobes of the sphinx moth, Manduca sexta. Journal of Comparative Physiology A, 161(1), 23-32.More infoPMID: 3039128;Abstract: 1. Responses of neurons in the antennal lobe (AL) of the moth Manduca sexta to stimulation of the ipsilateral antenna by odors consist of excitatory and inhibitory synaptic potentials (Fig. 2A). Stimulation of primary afferent fibers by electrical shock of the antennal nerve causes a characteristic IPSP-EPSP synaptic response in AL projection neurons (Fig. 2B). 2. The IPSP in projection neurons reverses below the resting potential (Fig. 3), is sensitive to changes in external (Fig. 4) and internal (Fig. 5) chloride concentration, and thus is apparently mediated by an increase in chloride conductance. 3. The IPSP is reversibly blocked by 100 μM picrotoxin (Fig. 6) or bicuculline (Fig. 7). 4. Many AL neurons respond to application of GABA with a strong hyperpolarization and an inhibition of spontaneous spiking activity (Fig. 8). GABA responses are associated with an increase in neuronal input conductance (Fig. 9) and a reversal potential below the resting potential (Fig. 11). 5. Application of GABA blocks inhibitory synaptic inputs (Fig. 12 A) and reduces or blocks excitatory inputs (Fig. 12B). EPSPs can be protected from depression by application of GABA (Fig. 12B). 6. Muscimol, a GABA analog that mimics GABA responses at GABAA receptors but not at GABAB receptors in the vertebrate CNS, inhibits many AL neurons in the moth (Fig. 13). © 1987 Springer-Verlag.
- Hoskins, S. G., Homberg, U., Kingan, T. G., Christensen, T. A., & Hildebrand, J. G. (1986). Immunocytochemistry of GABA in the antennal lobes of the sphinx moth Manduca sexta. Cell and Tissue Research, 244(2), 243-252.More infoPMID: 3521878;Abstract: We have prepared and characterized specific rabbit antisera against γ-aminobutyric acid (GABA) coupled covalently to bovine serum albumin and keyhole-limpet hemocyanin. Using these antisera in immunocytochemical staining procedures, we have probed the antennal lobes and their afferent and efferent fiber tracts in the sphinx moth Manduca sexta for GABA-like immunoreactivity in order to map putatively GABAergic central neurons in the central antennal-sensory pathway. About 30% of the neuronal somata in the large lateral group of cell bodies in the antennal lobe are GABA-immunoreactive; cells in the medial and anterior groups of antennal-lobe cells did not exhibit GABA-like immunoreactivity. GABA-immunoreactive neurites had arborizations in all of the glomeruli in the antennal lobe. Double-labeling experiments involving tandem intracellular staining with Lucifer Yellow and immunocytochemical staining for GABA-like immunoreactivity demonstrated that at least some of the GABA-immunoreactive cells in the antennal lobe are amacrine local interneurons. Several fiber tracts that carry axons of antennal-lobe projection neurons exhibited GABA-immunoreactive fibers. Among the possibly GABA-containing projection neurons are several cells, with somata in the lateral group of the antennal lobe, that send their axons directly to the lateral protocerebmm. © 1986 Springer-Verlag.
- Kent, K. S., Harrow, I. D., Quartararo, P., & Hildebrand, J. G. (1986). An accessory olfactory pathway in Lepidoptera: the labial pit organ and its central projections in Manduca sexta and certain other sphinx moths and silk moths. Cell and Tissue Research, 245(2), 237-245.More infoPMID: 3742559;Abstract: In the hawkmoth, Manduca sexta, the third segment of each labial palp contains a pit, which houses a densely packed array of sensilla. We have named this structure the labial pit organ (LPO). The sensilla within the pit are typical of olfactory receptors, characterized by a grooved surface, wall pores, and pore tubules. Axons arising from receptor cells that innervate these sensilla project bilaterally to a single glomerulus in each antennal lobe. We have compared this central projection with that in three other species of Manduca (M. quinquemaculata, M. dilucida, and M. lanuginosa) and in the silkmoths Antheraea polyphemus and Bombyx mori. A bilateral projection to a single glomerulus in each antennal lobe is present in all cases. We suggest that the LPO serves as an accessory olfactory organ in adult Lepidoptera. © 1986 Springer-Verlag 1986.
- Schneiderman, A. M., Hildebrand, J. G., Brennan, M. M., & Tdieinson, J. H. (1986). Trans-sexually grafted antennae alter pheromone-directed behaviour in a moth. Nature, 323(6091), 801-803.More infoPMID: 3774007;Abstract: When tobacco hornworm moths (Manduca sexta) are tested in a wind tunnel with a source of female pheromones upwind, males but not normal females show pheromone-modulated anemotaxis and a characteristic mate-seeking behavioural sequence1-7. These behaviours are produced by stimulation of sensory neurones found only in male antennae8-10. These neurones project axons only to dendrites of pheromone-specific interneurones11,12 in the macroglomerular complex, a region of neuropil in the antennal lobe characteristic of males but not present in normal females13-15. Some interneurones in the antennal lobes of female moths that have received grafts of male antennae (gynandromorphs) respond postsynaptically to stimulation with bombykal, a major component of the pheromone16. They branch into a region resembling the macroglomerular complex16, like their counterparts in normal males. We show here that gynandromorphic females respond to pheromonal stimulation with anemotaxis. We also find that normal females display a similar sequence in response to the odour of their egg-laying site, the tobacco plant. It is likely that a common motor path is used either by pheromone-specific interneurones in the antennal lobes of males or by tobacco-specific interneurones in females. We assume that the interneurones in gynandromorphic females that branch into the macroglomerular complex induced by a grafted male antenna can activate this pathway. © 1986 Nature Publishing Group.
- Kingan, T. G., & Hildebrand, J. G. (1985). γ-aminobutyric acid in the central nervous system of metamorphosing and mature Manduca sexta. Insect Biochemistry, 15(6), 667-675.More infoAbstract: We have begun to examine the factors controlling the accumulation of the neurotransmitter γ-aminobutyric acid (GABA) in the central nervous system (CNS) of the sphinx moth Manduca sexta. Analysis of soluble amino acids in CNS structures from mature moths outlines the regional distribution of GABA. Analysis of amino acids in the antennal lobes (the primary olfactory centres) of Manduca during metamorphosis reveals that GABA accumulates gradually and continuously through most of adult development until eclosion; within 18 hr after eclosion, levels of GABA abruptly increase 27-50%. The activity of the biosynthetic enzyme glutamic acid decarboxylase (EC 4.1.1.15), assayed in extracts of antennal lobes from developing moths, does not change after eclosion. Extracts of hemolymph from mature moths contain low levels of glutamate (
- Schneiderman, A. M., & Hildebrand, J. G. (1985). Sexually dimorphic development of the insect olfactory pathway. Trends in Neurosciences, 8(C), 494-499.More infoAbstract: The antennal olfactory pathway in insects changes profoundly during postembryonic development. The immature larval system gives way to the adult pathway1-5, which is capable of detecting and processing information about odors that have behavioral significance for the adult. Notable among these important odors are the sexual pheromones, released by an insect to attract a receptive mating partner of the same species. Recent research efforts in several laboratories have focused on the pheromone-processing subsystem of the olfactory pathway of male moths and cockroaches, which controls orientation toward a female 'calling' for a mate by releasing pheromone. We have probed this subsystem to explore several aspects of neural development, asking for example how sensory cells grow to and make appropriate connections with target cells in the maturing brain. © 1985.
- Tolbert, L. P., Matsumoto, S. G., & Hildebrand, J. G. (1983). Development of synapses in the antennal lobes of the moth Manduca sexta during metamorphosis. Journal of Neuroscience, 3(6), 1158-1175.More infoPMID: 6854367;Abstract: During the metamorphosis of holometabolous insects, the larval nervous system is restructured to provide the circuitry needed by the developing adult. Prominent new centers in the brain, the antennal lobes, arise to receive olfactory afferent axons from the developing adult antennae and provide an excellent system in which to study the development of synapses in a central nervous system. We have examined the anatomy and physiology of developing synapses in the antennal lobes of the moth Manduca sexta during the 18 days of metamorphic adult development. On day 5, the neuropil of the newly emerging antennal lobe condenses into distinct glomeruli, in which intercellular junctional complexes have already begun to form. Although some junctions have associated synaptic vesicles, most complexes are desmosome-like until day 9, when the number of synaptic complexes begins to increase. Early synapses are characterized by membrane-associated densities in a least two abutting cellular processes and a small number of synaptic vesicles clustered near the membrane of one process. As adult development proceeds, the membrane-associated densities become denser and more extensive, and the number of synaptic vesicles in the clusters increases. At day 14 synapses appear ultrastructurally mature, and almost all junctions in the neuropil can be identified as synaptic. Not until day 9 do antennal lobe neurons begin to respond postsynaptically when the antennal nerve is stimulated electrically, suggesting that the earliest synapses observed in the electron microscope may not be made by antennal nerve axons. At first the postsynaptic responses are graded and fatigue rapidly. By day 11, the antennal lobe neurons respond with action potentials, but the fatigability does not decline to adult levels until day 13. Filling of antennal lobe neurons with cobalt reveals that the arborizations of both local interneurons and output neurons continue to mature morphologically until about day 13. Previous work showed that antennal sensilla do not begin to be responsive to odors until day 14. Thus the establishment of the synaptic network in the antennal lobe apparently occurs in the absence of functional olfactory input to the system.
- Hildebrand, J. G. (1982). More on peer review [5]. Chemical and Engineering News, 60(26), 74-.
- Schneiderman, A. M., Matsumoto, S. G., & Hildebrand, J. G. (1982). Trans-sexually grafted antennae influence development of sexually dimorphic neurones in moth brain. Nature, 298(5877), 844-846.More infoAbstract: Observations in insects reveal sexual differences in the central nervous system (CNS), associated with sexually dimorphic patterns of reproductive behaviour1-9. For example, in certain species of moths, including the sphinx moth Manduca sexta, only males fly towards a sexually receptive female or towards a source of the female sex pheromone10,11. In Manduca, specialized olfactory receptor cells found only on male antennae12,13 respond sensitively and selectively to the female sex pheromone (unpublished experiments with K.-E. Kaissling and R. J. O'Connell). Their axons project into the macroglomerular complex (MGC), which is characteristic of male, but not female, antennal lobes (ALs; Fig. 1b, d)1-3,5,8,9,14. These afferents to the MGC presumably synapse with male-specific AL neurones8,15 to begin the processing of phenomonal information. We have now devised a surgical procedure for producing antennal gynandromorphs of Manduca in which one of the two ALs receives sensory innervation from an antenna formed by a transplanted imaginal disk of the opposite sex. We report here that in these gynandromorphs, the physiological and morphological properties of certain AL neurones are influenced by the gender of the antennal sensory axons contacting them. In particular, neurones resembling the male-specific AL neurones appear in female ALs innervated by sensory axons from a grafted male antenna. © 1982 Nature Publishing Group.
- Maxwell, G. D., & Hildebrand, J. G. (1981). Anatomical and neurochemical consequences of deafferentation in the development of the visual system of the moth Manduca sexta. Journal of Comparative Neurology, 195(4), 667-680.More infoPMID: 6109739;Abstract: The primordium of the compound eye of the moth Manduca sexta was removed by surgery within 12 hours after the molt to the pupa. When the operated animals were examined as pharate adults just prior to emergence from the pupal cuticle, no eye tissue was present on the operated side. Histological examination of the brain at successive developmental stages showed that the volume of laminar neuropil on the operated side increased very little after deafferentation while the volume of the control-side laminar neuropil increased more than 20-fold. The impairment of development of the lamina was accompanied by a reduction in the synthesis and storage by the optic lobe of two neurotransmitter candidates, acetylcholine and 5-hydroxytryptamine. The endogenous content of 5-hydroxytryptamine was also reduced in these preparations. Excision of a section of the stemmatal nerve, which connects the primordium of the compound eye to the brain, also arrested the development of the lamina, but in this case the compound eye itself developed apparently normally without making contact with the brain. This finding supports the hypothesis that the centripetal growth of photoreceptor-cell axons normally is contact-guided. Results of neurochemical experiments on retinal tissue argue against the idea that acetylcholine, 5-hydroxytryptamine, or gamma-aminobutyric acid is a likely photoreceptor-cell neurotransmitter. From these and our previous studies of the metamorphosing brain in Manduca we conclude that different classes of neurons in the same central nervous system can exhibit widely different degrees of dependency on their normal innervation for their survival and differentiation.
- Maxwell, G. D., Moore, M. M., & Hildebrand, J. G. (1980). Metabolism of tyramine in the central nervous system of the moth Manduca sexta. Insect Biochemistry, 10(6), 657-665.More infoAbstract: Three distinct metabolites of tyramine are produced by central nervous tissue of pharate-adult Manduca sexta incubated in vitro with [3H]-tyramine. The chemical identities of these metabolites have been sought by a combination of electrophoretic, chromatographic, hydrolytic and enzymic procedures. One of the compounds is N-acetyltyramine, and the others appear to be β-alanyltyramine and a sugar conjugate, possibly of N-acetyltyramine. © 1980.
- Hildebrand, J. G., Hall, L. M., & Osmond, B. C. (1979). Distribution of binding sites for 125I labeled α bungarotoxin in normal and deafferented antennal lobes of Manduca sexta. Proceedings of the National Academy of Sciences of the United States of America, 76(1), 499-503.More infoPMID: 284367;PMCID: PMC382969;Abstract: 125I-Labeled α-bungarotoxin has been used to determine the distribution of putative acetylcholine receptors in normal and chronically deafferented antennal lobes in the brain of the moth Manduca sexta. Toxin-binding sites are confined to synaptic regions in the neuropil of normal lobes and to rudiments of these regions in deafferented lobes. These findings suggest that receptors can develop in the insect central nervous system independently of normal synaptic influences.
- Maxwell, G. D., Tait, J. F., & Hildebrand, J. G. (1978). Regional synthesis of neurotransmitter candidates in the CNS of the moth Manduca sexta. Comparative Biochemistry and Physiology. Part C, Comparative, 61(1), 109-119.More infoPMID: 30568;Abstract: 1. The ability of several defined structures in the central nervous system of the moth Manduca sexta to synthesize and store acetylcholine, γ-aminobutyric acid, 5-hydroxytryptamine, histamine, dopamine, norepinephrine, tyramine and octopamine was examined in pharate adults. With the exception of norepinephrine, synthesis and storage of all neurotransmitter candidates was detected in at least some structures. 2. The major sensory centers in the brain, the antennal and optic lobes, differ significantly in the spectrum of neurotransmitter candidates they appear able to synthesize and retain. The optic lobes can synthesize all neurotransmitter candidates examined, except norepinephrine, while the antennal lobes produce and store only acetylcholine, γ-aminobutyric acid, histamine and small amounts of tyramine. 3. Synthesis and storage of relatively large amounts of 5-hydroxytryptamine, dopamine and octopamine in segmental ganglia suggests a prominent role for these substances in the function of these ganglia. 4. Acetylcholine is produced and retained in greater quantities than all other neurotransmitter candidates, by nearly two orders of magnitude. © 1978.
- Prescott, D. J., Hildebrand, J. G., Sanes, J. R., & Jewett, S. (1977). Biochemical and developmental studies of acetylcholine metabolism in the central nervous system of the moth Manduca sexta. Comparative Biochemistry and Physiology. Part C, Comparative, 56(2), 77-84.More infoPMID: 15782;Abstract: 1. Acetylcholine and its biosynthetic and degradative enzymes (choline acetyltransferase and acetylcholinesterase, respectively) were quantified in several defined structures from the central nervous system of the tobacco hornworm Manduca sexta at various times during postembryonic development. 2. Activity of choline acetyltransferase, the key enzyme of the cholinergic pathway, changes roughly synchronously and similarly in the protocerebrum and subesophageal, prothoracic, and third abdominal ganglia, and it increases dramatically in developing antennal and optic lobes. 3. Levels of acetylcholine and of specific acetylcholinesterase, which is the principal cholinesterase in the nervous system, also rise greatly during metamorphosis of the central nervous regions. 4. The major changes in the activity of choline acetyltransferase correlate with known developmental endocrine events. © 1977.
- Sanes, J. R., Prescott, D. J., & Hildebrand, J. G. (1977). Cholinergic neurochemical development of normal and deafferented antennal lobes during metamorphosis of the moth, Manduca sexta. Brain Research, 119(2), 389-402.More infoPMID: 830392;Abstract: Sensory neurons with somata in the antennae of the moth, Manduca sexta, make synapses in the antennal lobes of the brain. These lobes develop during metamorphosis of the pupa to the adult while the antennae themselves develop and send presumably cholinergic sensory fibers into the lobes. Levels of acetylcholine, choline acetyltransferase, and acetylcholinesterase rise dramatically in the lobes as sensory axons grow from the antennae to the lobes through the antennal nerves. An [125I]α-bungarotoxin-binding activity, which may represent ACh-receptors, develops in the lobes with a time course different from that of the other cholinergic components, rising gradually throughout metamorphosis. This activity is specific to nervous tissue and is blocked by cholinergic agents (carbamylcholine, atropine, curare, and nicotine). Levels of acetylcholine, choline acetyltransferase, and acetylcholinesterase, but not of toxin-binding activity, are greatly reduced in lobes deafferented by amputation of developing antennae. © 1977.
- Sanes, J. R., & Hildebrand, J. G. (1976). Acetylcholine and its metabolic enzymes in developing antennae of the moth, Manduca sexta. Developmental Biology, 52(1), 105-120.More infoPMID: 183992;Abstract: Antennae of the moth, Manduca sexta, are thickly populated with sensory neurons, which send axons through antennal nerves to the brain. These neurons arise by cell divisions and differentiate synchronously during the 18 days of metamorphosis from pupa to adult. Biochemical studies support the hypothesis that antennal neurons use acetylcholine (ACh) as a neurotransmitter: (1) Antennae incubated with [14C]choline synthesize and store [14C]ACh; several other transmitter candidates do not accumulate detectably when appropriate radioactive precursors are supplied; (2) antennae and antennal nerves contain endogenous ACh; and (3) extracts of mature antennae contain choline acetyltransferase (ChAc) and acetylcholinesterase (AChE) with properties similar to those reported for the enzymes from other arthropods. Levels of ACh, ChAc, and AChE begin to increase in antennae soon after the sensory neurons are "born." Levels rise exponentially for over a week as the neurons differentiate and then reach a plateau, at about the time the neurons reach morphological maturity, that is maintained into adulthood. In contrast, levels of carnitine acetyltransferase, cholinesterase, and soluble protein, presumably not confined to nervous tissue, change little during metamorphosis. Levels of ACh, ChAc, and AChE rise in an intracranial segment of antennal nerve at about the same time as in the antenna, indicating that axons can transport neurotransmitter machinery at an early stage in their development. © 1976.
- Sanes, J. R., & Hildebrand, J. G. (1976). Origin and morphogenesis of sensory neurons in an insect antenna. Developmental Biology, 51(2), 300-319.More infoPMID: 955261;Abstract: Each antennal flagellum of the moth, Manduca sexta, contains about 2.5 × 105 primary sensory neurons. The neurons are components of small sensory organs (sensilla) and send axons through antennal nerves to the brain. The neurons, sensilla, and nerves differentiate as the antenna develops, during the 18 days of metamorphosis from pupa to adult. Neurons arise from divisions of epidermal cells between 25 and 60 hr after pupal ecdysis and elaborate axons and dendrites soon thereafter. Neurons have the bipolar form, ciliated dendrite, and glial sheath characteristic of the adult within a few days of their birth. The axons grow along small pupal nerves to form the adult antennal nerves, and the dendrites grow beyond the apical margin of the epidermis, where they are enveloped by a growing process of the sensilla's trichogen cell. Cuticle secreted by the trichogen cell forms the seta or sensory hair of the sensillum. Later, the neuronal somata migrate from the basal to the apical margin of the epidermis. Finally, the cytoplasm withdraws from the seta, leaving the dendrites imprisoned in a cylinder of cuticle. All of the neurons in the flagellum differentiate nearly synchronously, facilitating correlation of morphogenetic results presented here with biochemical and electrophysiological analyses of the developing neurons. © 1976.
- Sanes, J. R., & Hildebrand, J. G. (1976). Structure and development of antennae in a moth, Manduca sexta. Developmental Biology, 51(2), 282-299.More infoPMID: 955260;Abstract: The antenna of the moth, Manduca sexta, comprises two small basal segments and a long (2 cm) flagellum, which is divided into nearly 80 annuli. The annuli bear cuticular scales and small sensory organs, sensilla. A trachea, a blood vessel, and two nerve trunks run through the lumen of the antenna and into the head. Sensilla are arranged in an orderly pattern that is repeated on each flagellar annulus. Each flagellum bears about 105 sensilla, which contain about 2.5 × 105 primary sensory neurons. Clumps of undifferentiated cells (imaginal disks), present in the larva, form pupal antennae during the larval-pupal molt. During the subsequent metamorphic development of the adult, cell divisions, changes in cell shape, and cellular differentiation transform pupal into adult antennae. Sensilla and scales arise and differentiate in the antenna during metamorphosis; regions in which sensilla and scales will arise can be recognized before overt differentiation occurs. All of the flagellar annuli develop synchronously. The dense innervation and neuronal simplicity of antennal flagella, as well as their synchronous development at a late and accessible stage in the animal's life cycle, suit them for studies of neuronal differentiation. © 1976.
- Sanes, J. R., Hildebrand, J. G., & Prescott, D. J. (1976). Differentiation of insect sensory neurons in the absence of their normal synaptic targets. Developmental Biology, 52(1), 121-127.More infoPMID: 183993;Abstract: Sensory neurons in the antenna of the moth, Manduca sexta, arise and differentiate during the 18 days of metamorphosis from pupa to adult, sending axons to the brain. To assess the trophic dependence of developing antennal neurons on their targets, we studied antennae from surgically debrained animals. If the brain is removed 1 to 45 hr after pupal ecdysis (before and during the period when antennal neurons arise by cell divisions), adult development can be triggered by injection of β-ecdysone; if the brain is removed 50 to 60 hr after pupal ecdysis (before antennal axons contact the brain), metamorphosis proceeds spontaneously. Neurons proliferate normally and differentiate extensively in the antennae of debrained animals. They acquire a characteristic size and shape, elaborate axons and dendrites, migrate to appropriate positions in the sensilla, accumulate components of a neurotransmitter system (acetylcholine, choline acetyltransferase, and acetylcholinesterase), and generate electrical responses to olfactory and mechanical stimuli. Antennal sensory neurons thus differ from a variety of vertebrate neurons, which fail to mature when deprived of their synaptic targets. © 1976.
- Schweitzer, E. S., Sanes, J. R., & Hildebrand, J. G. (1976). Ontogeny of electroantennogram responses in the moth, Manduca sexta. Journal of Insect Physiology, 22(7), 955-960.More infoPMID: 947988;Abstract: The antennae of the moth, Manduca sexta, and the sensilla and sensory neurons they contain, develop during metamorphosis from pupa to adult. To determine when, during their development, antennae become capable of generating electrical responses to various stimuli, we recorded the electroantennogram (EAG), believed to be the summed extracellular record of receptor potentials, from developing and mature antennae. Antennae from male and female moths are similarly responsive to trans-2-hexenal, while only males respond to Manduca sex pheromone; these two odorants presumably stimulate separate receptors. Mechanical stimulation also elicits and EAG response. EAG responses to olfactory and mechanical stimuli are detectable several days before eclosion but not until the neurons are morphologically and biochemically quite mature. Responses increase in magnitude until the end of metamorphosis and then change little during the first 3 days after emergence of the adult. Responses to different stimuli do not develop synchronously. © 1976.
- Sanes, J. R., & Hildebrand, J. G. (1975). Nerves in the antennae of pupal Manduca sexta Johanssen (Lepidoptera: Sphingidae). Wilhelm Roux's Archives of Developmental Biology, 178(1), 71-78.More infoAbstract: The antennal rudiment of pupal Manduca sexta contains two small nerves in its lumen. Each nerve is made up of about 160 axons that run from neurons near the tip of the pupal antenna to the brain. These neurons are also present in antennae of pharate pupae, diapausing pupae, and developing adults, and are distinct from the primary sensory neurons that are associated with the cuticular sensilla of larval and adult antenna. © 1975 Springer-Verlag.
- Hildebrand, J. G., Townsel, J. G., & Kravitz, E. A. (1974). Distribution of acetylcholine, choline, choline acetyltransferase and acetylcholinesterase in regions and single identified axons of the lobster nervous system. Journal of Neurochemistry, 23(5), 951-963.More infoPMID: 4215870;Abstract: Acetylcholine, its precursor (choline), and the enzymes of its biosynthesis and degradation (choline acetyltransferase and acetylcholinesterase, respectively) have been studied and quantified in extracts of several regions of the nervous system of the lobster and in single, isolated axons of identified efferent excitatory, efferent inhibitory and afferent sensory neurons. The choline acetyltransferase is a soluble enzyme similar to that from other species. The predominant acetylcholine hydrolysing enzyme is largely membrane bound and has been characterized as a specific acetylcholinesterase. A single peak of acetylcholinesterase activity can be detected upon velocity sedimentation analysis of Triton X 100 treated extracts of all regions of the nervous system. Choline acetyltransferase distribution parallels that of sensory neural elements, and its specific activity shows nearly a 500 fold difference from the richest to the poorest neural source. Acetylcholinesterase levels span only a 23 fold range, and activity is found in all neural regions, including those free of known sensory components. A radiochemical microassay for choline and acetylcholine in the range of 20-2000 pmol is described in detail. All 3 types of axons contain comparable levels of choline (ca. 2 pmol/μg protein), but acetylcholine is asymmetrically distributed. Efferent axons contain no detectable acetylcholine, while sensory axons from abdominal muscle receptor organs have an average of 1.9 pmol/μg protein. Choline acetyltransferase is similarly distributed; sensory axons show at least 500 fold greater activity than efferent axons. Acetylcholinesterase is nearly uniformly distributed among the three types of fibres. These results are discussed in terms of a general view of transmitter accumulation in single neurons.
- Townsel, J. G., Hildebrand, J. G., & Kravitz, E. A. (1973). On transmitter accumulation in lobster neurons. Federation Proceedings, 32(3 I), 1728-.More infoAbstract: A thorough understanding of neurotransmitter accumulation requires: first, the identification of the transmitter compound used by a neuron; next, a knowledge of the substrates, enzymes and other macromolecules involved in transmitter metabolism and compartmentalization; and finally, an understanding of the genetic regulation of the production of these components. These studies are aimed at such an understanding using the lobster nervous system, from which one can isolate single physiologically identified neurons. The transmitter compounds believed to be used by three distinct lobster neuron types are: efferent inhibitory (I), GABA; efferent excitatory, glutamate; sensory (S), acetylcholine (Ach). Measurements of levels of the transmitter candidates ACh, GABA and glutamate and of the enzymes choline acetyltransferase (ChAc), acetylcholinesterase (AChE), glutamic decarboxylase (GD) and GABA glutamic transaminase (GT) have been carried out in axons of all 3 neuron types. The results demonstrate that ACh and its synthetic enzyme (ChAc) are selectively localized in S axons (enzyme Δ = 500:1), GABA and its synthetic enzyme (GD) are concentrated in I axons (enzyme Δ = 300:1), while the degradative enzymes AChE and GT are found evenly distributed amongst all 3 neuron types.
- Barker, D. L., Herbert, E., Hildebrand, J. G., & Kravitz, E. A. (1972). Acetylcholine and lobster sensory neurones.. Journal of Physiology, 226(1), 205-229.More infoPMID: 4343316;PMCID: PMC1331161;
- Hildebrand, J. G., Barker, D. L., Herbert, E., & Kravitz, E. A. (1971). Screening for neurotransmitters: a rapid radiochemical procedure.. Journal of Neurobiology, 2(3), 231-246.More infoPMID: 4400108;
- Walsh Jr., C. T., Hildebrand, J. G., & Spector, L. B. (1970). Succinyl phosphate. Its nonenzymatic hydrolysis and reaction with coenzyme A.. Journal of Biological Chemistry, 245(21), 5699-5708.More infoPMID: 5472367;
- Hildebrand, J. G., & Spector, L. B. (1969). Succinyl phosphate and the succinyl coenzyme A synthetase reaction.. Journal of Biological Chemistry, 244(10), 2606-2613.More infoPMID: 4890228;
- Hildebrand, J. G., & Law, J. H. (1964). Fatty acid distribution in bacterial phospholipids. The specificity of the cyclopropane synthetase reaction. Biochemistry, 3(9), 1304-1308.More infoPMID: 14229673;Abstract: The distribution of fatty acids in purified phospholipids isolated from several different bacteria has been examined by use of the specific phospholipase A of snake venom. In general the distribution is in accord with that found elsewhere in nature; the unsaturated acids are found in the β position and the saturated acids in the γ position. The distribution of cyclopropane fatty acids follows closely that of the unsaturated fatty acids. A notable exception was encountered with the phosphatidylethanolamine of Clostridium butyricum, in which unsaturated and cyclopropane fatty acids were found in more abundance in the γ position. The specificity of the C. butyricum cyclopropane synthetase reaction has been examined by employing as substrate a phosphatidylethanolamine with a known distribution of unsaturated fatty acids and analyzing the distribution of cyclopropane fatty acids in the phospholipid produced by the enzyme reaction. These experiments indicate that the enzyme has a definite, but not absolute, specificity for an unsaturated fatty acid in the γ position.
Presentations
- Hildebrand, J. G. (2017, August). Lecture. University of New England. Biddiford, ME.
- Hildebrand, J. G. (2017, February). James C. Smith Lecture. Florida State University. Tallahassee, Florida.
- Hildebrand, J. G. (2017, October, 2017). Plenary Lecture. Israeli Entomological Society Annual Meeting.
- Hildebrand, J. G. (2016, April, 2016). Lecture. NIH IRACDA Conference. Tucson, AZ.
- Hildebrand, J. G. (2016, September, 2016). Wigglesworth Lecturer. XXV International Congress of Entomology. Orlando FL.
- Hildebrand, J. G. (2015, Spring). Invited Lecture. Physics Teachers of Southern Arizona.
- Hildebrand, J. G. (2015, Spring). Sigma Xi Darwin Week Lecturer. College of Charleston, SC.
- Hildebrand, J. G. (2015, Spring). Sigma Xi Lecturer. Kansas State University, Manhattan, KS.
- Hildebrand, J. G. (2015, Spring). Sigma Xi Lecturer. Northern Michigan University, Marquette, MI.
- Hildebrand, J. G. (2014, Fall). Invited Seminar Speaker at West Virginia University, Dept. of Biology. N/AWest Virginia University, Dept. of Biology.
- Hildebrand, J. G. (2014, Fall). Invited Speaker at Confluence Center, University of Arizona. N/AConfluence Center, University of Arizona.
- Hildebrand, J. G. (2014, Fall). Plenary Lecturer at Chilean Society for Neuroscience Annual Meeting. Chilean Society for Neuroscience Annual MeetingChilean Society for Neuroscience Annual Meeting.
- Hildebrand, J. G. (2014, Fall). Symposium on Biologically Inspired Robotics (organizer & chair). AAAS Annual Meeting.
- Hildebrand, J. G. (2013, Fall). Invited Seminar Speaker at Louisiana State University, Dept. of Biological Sciences. N/ALouisiana State University, Dept. of Biological Sciences.
- Hildebrand, J. G. (2013, Fall). Invited Seminar Speaker at University of Chicago, Dept. of Neurobiology. N/AUniversity of Chicago, Dept. of Neurobiology.
- Hildebrand, J. G. (2013, Fall). Invited Seminar Speaker at University of Cincinnati, Dept. of Biology. N/AUniversity of Cincinnati, Dept. of Biology.
- Hildebrand, J. G. (2013, Fall). Invited Seminar Speaker at University of Sydney, Australia, School of Biological Sciences. N/A. Sydney, Australia: University of Sydney, Australia, School of Biological Sciences.
- Hildebrand, J. G. (2013, Fall). Invited Seminar Speaker at Universität Kassel, Dept. of Animal Physiology. N/AUniversität Kassel, Dept. of Animal Physiology.
- Hildebrand, J. G. (2013, Fall). Invited Seminar Speaker at Universität Würzburg, Biozentrum. N/AUniversität Würzburg, Biozentrum.
- Hildebrand, J. G. (2013, Fall). Invited Seminar Speaker at Universität zu Köln, Institute of Zoology. N/AUniversität zu Köln, Institute of Zoology.
- Hildebrand, J. G. (2013, Fall). Invited Speaker at Kuffler Symposium, Rockefeller University. Kuffler SymposiumRockefeller University .
- Hildebrand, J. G. (2013, Fall). Invited Speaker at Yale Club of Southern Arizona. N/AYale Club of Southern Arizona.
- Hildebrand, J. G. (2013, Fall). Invited Symposium Speaker at International Chemical Ecology Conference (ICEC2013). International Chemical Ecology Conference (ICEC2013). Melbourne, Australia.
- Hildebrand, J. G. (2012, Fall). Distinguished Lecturer at Max-Planck-Institut f. Neurobiologie. N/A. Munich: Max-Planck-Institut f. Neurobiologie.
- Hildebrand, J. G. (2012, Fall). Invited Seminar Speaker at Washington University, Dept. of Biology. N/A. St. Louis: Washington University, Dept. of Biology.
- Hildebrand, J. G. (2012, Fall). Invited Symposium Lecture at American Association of Anatomists annual meeting (symposium organizer & speaker). American Association of Anatomists annual meeting. San Diego.
- Hildebrand, J. G. (2012, Fall). Invited Symposium Speaker at Norwegian University of Science & Technology. N/A. Trondheim: Norwegian University of Science & Technology.
- Hildebrand, J. G. (2012, Fall). Lawrence Gilbert Distinguished Lecture, University of North Carolina, Dept. of Biology. 2012 Lawrence Gilbert Distinguished LectureUniversity of North Carolina, Dept. of Biology.
- Hildebrand, J. G. (2012, Fall). Plenary Lecturer at Latin American Association for Chemical Ecology Annual Meeting. Latin American Association for Chemical Ecology Annual Meeting.
- Hildebrand, J. G. (2012, Fall). Walter F. Heiligenberg Lecture, UC-San Diego. 2012 Walter F. Heiligenberg LectureUC-San Diego.