Richard B Levine

Interests

No activities entered.

Courses

Scholarly Contributions

Journals/Publications

• Cholanian, M., Krajweski-Hall, S., Levine, R., McMullen, N., & Rance, N. (2014). Electrophysiology of Arcuate Neurokinin B Neurons in Female Tac2-EGFP Transgenic Mice. Journal of Endocrinology.
• Powell, G. L., Levine, R. B., Frazier, A. M., & Fregosi, R. F. (2014). Influence of developmental nicotine exposure on spike timing precision & reliability in hypoglossal motoneurons. Journal of neurophysiology, jn.00838.2014.
  More info

  Smoothly graded muscle contractions depend in part on the precision and reliability of motoneuron action potential generation. Whether or not a motoneuron generates spikes precisely and reliably depends on both its intrinsic membrane properties and the nature of the synaptic input that it receives. Factors that perturb neuronal intrinsic properties and/or synaptic drive may compromise the temporal precision and the reliability of action potential generation. We have previously shown that developmental nicotine exposure (DNE) alters intrinsic properties and synaptic transmission in hypoglossal motoneurons (XIIMNs). Here we show that the effects of DNE also include alterations in spike-timing precision and reliability, and spike-frequency adaptation, in response to sinusoidal current injection. Current-clamp experiments in brainstem slices from neonatal rats show that DNE lowers the threshold for spike generation, but increases the variability of spike timing mechanisms. DNE is also associated with an increase in spike-frequency adaptation and reductions in both peak and steady-state firing rate in response to brief, square wave current injections. Taken together, our data indicate that DNE causes significant alterations in the input-output efficiency of XIIMNs. These alterations may play a role in the increased frequency of obstructive apneas and altered suckling strength and coordination observed in nicotine exposed neonatal humans.
• Pilarski, J. Q., Wakefield, H. E., Fuglevand, A. J., Levine, R. B., & Fregosi, R. F. (2012). Increased nicotinic receptor desensitization in hypoglossal motor neurons following chronic developmental nicotine exposure. Journal of Neurophysiology, 107(1), 257-264.
  More info

  PMID: 22013232;PMCID: PMC3349681;Abstract: Neuronal nicotinic acetylcholine receptors (nAChRs) are expressed on hypoglossal motor neurons (XII MNs) that innervate muscles of the tongue. Activation of XII MN nAChRs evokes depolarizing currents, which are important for regulating the size and stiffness of the upper airway. Although data show that chronic developmental nicotine exposure (DNE) blunts cholinergic neurotransmission in the XII motor nucleus, it is unclear how nAChRs are involved. Therefore, XII MN nAChR desensitization and recovery were examined in tissues from DNE or control pups using a medullary slice preparation and tight-seal whole cell patch-clamp recordings. nAChR-mediated inward currents were evoked by brief pressure pulses of nicotine or the α4β2 nAChR agonist RJR-2403. We found that, regardless of treatment, activatable nAChRs underwent desensitization, but, following DNE, nAChRs exhibited increased desensitization and delayed recovery. Similar results were produced using RJR-2403, showing that DNE influences primarily the α4β2 nAChR subtype. These results show that while some nAChRs preserve their responsiveness to acute nicotine following DNE, they more readily desensitize and recover more slowly from the desensitized state. These data provide new evidence that chronic DNE modulates XII MN nAChR function, and suggests an explanation for the association between DNE and the incidence of central and obstructive apneas. © 2012 the American Physiological Society.
• Ryglewski, S., Lance, K., Levine, R. B., & Duch, C. (2012). Ca _v2 channels mediate low and high voltage-activated calcium currents in Drosophila motoneurons. Journal of Physiology, 590(4), 809-825.
  More info

  PMID: 22183725;PMCID: PMC3381312;Abstract: Different blends of membrane currents underlie distinct functions of neurons in the brain. A major step towards understanding neuronal function, therefore, is to identify the genes that encode different ionic currents. This study combined in situ patch clamp recordings of somatodendritic calcium currents in an identified adult Drosophila motoneuron with targeted genetic manipulation. Voltage clamp recordings revealed transient low voltage-activated (LVA) currents with activation between -60 mV and -70 mV as well as high voltage-activated (HVA) current with an activation voltage around -30 mV. LVA could be fully inactivated by prepulses to -50 mV and was partially amiloride sensitive. Recordings from newly generated mutant flies demonstrated that DmαG (Ca v3 homolog) encoded the amiloride-sensitive portion of the transient LVA calcium current. We further demonstrated that the Ca v2 homolog, Dmca1A, mediated the amiloride-insensitive component of LVA current. This novel role of Ca v2 channels was substantiated by patch clamp recordings from conditional mutants, RNAi knock-downs, and following Dmca1A overexpression. In addition, we show that Dmca1A underlies the HVA somatodendritic calcium currents in vivo. Therefore, the Drosophila Ca v2 homolog, Dmca1A, underlies HVA and LVA somatodendritic calcium currents in the same neuron. Interestingly, DmαG is required for regulating LVA and HVA derived from Dmca1A in vivo. In summary, each vertebrate gene family for voltage-gated calcium channels is represented by a single gene in Drosophila, namely Dmca1D (Ca v1), Dmca1A (Ca v2) and DmαG (Ca v3), but the commonly held view that LVA calcium currents are usually mediated by Ca v3 rather than Ca v2 channels may require reconsideration. © 2012 The Authors. The Journal of Physiology © 2012 The Physiological Society.
• Srinivasan, S., Lance, K., & Levine, R. B. (2012). Contribution of EAG to excitability and potassium currents in Drosophila larval motoneurons. Journal of Neurophysiology, 107(10), 2660-2671.
  More info

  PMID: 22323637;PMCID: PMC3362287;Abstract: Diversity in the expression of K+ channels among neurons allows a wide range of excitability, growth, and functional regulation. Ether-à-go-go (EAG), a voltage-gated K+ channel, was first characterized in Drosophila mutants by spontaneous firing in nerve terminals and enhanced neurotransmitter release. Although diverse functions have been ascribed to this protein, its role within neurons remains poorly understood. The aim of this study was to characterize the function of EAG in situ in Drosophila larval motoneurons. Whole cell patch-clamp recordings performed from the somata revealed a decrease in IAv and IKv K+ currents in eag mutants and with targeted eag RNAi expression. Spontaneous spike-like events were observed in eag mutants but absent in wild-type motoneurons. Thus our results provide evidence that EAG represents a unique K+ channel contributing to multiple K+ currents in motoneurons helping to regulate excitability, consistent with previous observations in the Drosophila larval muscle. © 2012 the American Physiological Society.
• Srinivasan, S., Lance, K., & Levine, R. B. (2012). Segmental differences in firing properties and potassium currents in Drosophila larval motoneurons. Journal of Neurophysiology, 107(5), 1356-1365.
  More info

  PMID: 22157123;PMCID: PMC3311690;Abstract: Potassium currents play key roles in regulating motoneuron activity, including functional specializations that are important for locomotion. The thoracic and abdominal segments in the Drosophila larval ganglion have repeated arrays of motoneurons that innervate body-wall muscles used for peristaltic movements during crawling. Although abdominal motoneurons and their muscle targets have been studied in detail, owing, in part, to their involvement in locomotion, little is known about the cellular properties of motoneurons in thoracic segments. The goal of this study was to compare firing properties among thoracic motoneurons and the potassium currents that influence them. Whole-cell, patch-clamp recordings performed from motoneurons in two thoracic and one abdominal segment revealed both transient and sustained voltage-activated K + currents, each with Ca ++-sensitive and Ca ++-insensitive [A-type, voltage-dependent transient K + current (I Av)] components. Segmental differences in the expression of voltageactivated K + currents were observed. In addition, we demonstrate that Shal contributes to I Av currents in the motoneurons of the first thoracic segment. © 2012 the American Physiological Society.
• 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 info

  PMID: 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.
• Pilarski, J. Q., Wakefield, H. E., Fuglevand, A. J., Levine, R. B., & Fregosi, R. F. (2011). Developmental nicotine exposure alters neurotransmission and excitability in hypoglossal motoneurons. Journal of Neurophysiology, 105(1), 423-433.
  More info

  PMID: 21068261;PMCID: PMC3023378;Abstract: Hypoglossal motoneurons (XII MNs) control muscles of the mammalian tongue and are rhythmically active during breathing. Acetylcholine (ACh) modulates XII MN activity by promoting the release of glutamate from neurons that express nicotinic ACh receptors (nAChRs). Chronic nicotine exposure alters nAChRs on neurons throughout the brain, including brain stem respiratory neurons. Here we test the hypothesis that developmental nicotine exposure (DNE) reduces excitatory synaptic input to XII MNs. Voltage-clamp experiments in rhythmically active medullary slices showed that the frequency of excitatory postsynaptic currents (EPSCs) onto XII MNs from DNE animals is reduced by 61% (DNE = 1.7 ± 0.4 events/s; control = 4.4 ± 0.6 events/s; P < 0.002). We also examine the intrinsic excitability of XII MNs to test whether cells from DNE animals have altered membrane properties. Current-clamp experiments showed XII MNs from DNE animals had higher intrinsic excitability, as evaluated by measuring their response to injected current. DNE cells had high-input resistances (DNE = 131.9 ± 13.7 MΩ, control = 78.6 ± 9.7 MΩ, P < 0.008), began firing at lower current levels (DNE = 144 ± 22 pA, control = 351 ± 45 pA, P < 0.003), and exhibited higher frequency-current gain values (DNE = 0.087 ± 0.012 Hz/pA, control = 0.050 ± 0.004 Hz/pA, P < 0.02). Taken together, our data show previously unreported effects of DNE on XII MN function and may also help to explain the association between DNE and the incidence of central and obstructive apneas. Copyright © 2011 The American Physiological Society.
• Schaefer, J. E., Worrell, J. W., & Levine, R. B. (2010). Role of intrinsic properties in Drosophila motoneuron recruitment during fictive crawling. Journal of Neurophysiology, 104(3), 1257-1266.
  More info

  PMID: 20573969;PMCID: PMC2944697;Abstract: Motoneurons in most organisms conserve a division into low-threshold and high-threshold types that are responsible for generating powerful and precise movements. Drosophila 1b and 1s motoneurons may be analogous to low-threshold and high-threshold neurons, respectively, based on data obtained at the neuromuscular junction, although there is little information available on intrinsic properties or recruitment during behavior. Therefore in situ whole cell patch-clamp recordings were used to compare parameters of 1b and 1s motoneurons in Drosophila larvae. We find that resting membrane potential, voltage threshold, and delay-to-spike distinguish 1b from 1s motoneurons. The longer delay-to-spike in 1s motoneurons is a result of the shal-encoded A-type K+ current. Functional differences between 1b and 1s motoneurons are behaviorally relevant because a higher threshold and longer delay-to-spike are observed in MNISN-1s in pairwise whole cell recordings of synaptically evoked activity during bouts of fictive locomotion. Copyright © 2010 The American Physiological Society.
• Hartwig, C. L., Worrell, J., Levine, R. B., Ramaswami, M., & Sanyal, S. (2008). Normal dendrite growth in Drosophila motor neurons requires the AP-1 transcription factor. Developmental Neurobiology, 68(10), 1225-1242.
  More info

  PMID: 18548486;PMCID: PMC2719294;Abstract: During learning and memory formation, information flow through networks is regulated significantly through structural alterations in neurons. Dendrites, sites of signal integration, are key targets of activity-mediated modifications. Although local mechanisms of dendritic growth ensure synapse-specific changes, global mechanisms linking neural activity to nuclear gene expression may have profound influences on neural function. Fos, being an immediate-early gene, is ideally suited to be an initial transducer of neural activity, but a precise role for the AP-1 transcription factor in dendrite growth remains to be elucidated. Here we measure changes in the dendritic fields of identified Drosophila motor neurons in vivo and in primary culture to investigate the role of the immediate-early transcription factor AP-1 in regulating endogenous and activity-induced dendrite growth. Our data indicate that (a) increased neural excitability or depolarization stimulates dendrite growth, (b) AP-1 (a Fos, Jun heterodimer) is required for normal motor neuron dendritic growth during development and in response to activity induction, and (c) neuronal Fos protein levels are rapidly but transiently induced in motor neurons following neural activity. Taken together, these results show that AP-1 mediated transcription is important for dendrite growth, and that neural activity influences global dendritic growth through a gene-expression dependent mechanism gated by AP-I. © 2008 Wiley Periodicals, Inc.
• Levine, R., Worrell, J. W., & Levine, R. B. (2008). Characterization of voltage-dependent Ca2+ currents in identified Drosophila motoneurons in situ. Journal of neurophysiology, 100(2).
  More info

  Voltage-dependent Ca2+ channels contribute to neurotransmitter release, integration of synaptic information, and gene regulation within neurons. Thus understanding where diverse Ca2+ channels are expressed is an important step toward understanding neuronal function within a network. Drosophila provides a useful model for exploring the function of voltage-dependent Ca2+ channels in an intact system, but Ca2+ currents within the central processes of Drosophila neurons in situ have not been well described. The aim of this study was to characterize voltage-dependent Ca2+ currents in situ from identified larval motoneurons. Whole cell recordings from the somata of identified motoneurons revealed a significant influence of extracellular Ca2+ on spike shape and firing rate. Using whole cell voltage clamp, along with blockers of Na+ and K+ channels, a Ca2+-dependent inward current was isolated. The Drosophila genome contains three genes with homology to vertebrate voltage-dependent Ca2+ channels: Dmca1A, Dmca1D, and Dmalpha1G. We used mutants of Dmca1A and Dmca1D as well as targeted expression of an RNAi transgene to Dmca1D to determine the genes responsible for the voltage-dependent Ca2+ current recorded from two identified motoneurons. Our results implicate Dmca1D as the major contributor to the voltage-dependent Ca2+ current recorded from the somatodendritic processes of motoneurons, whereas Dmca1A has previously been localized to the presynaptic terminal where it is essential for neurotransmitter release. Altered firing properties in cells from both Dmca1D and Dmca1A mutants indicate a role for both genes in shaping firing properties.
• Barbee, S. A., Estes, P. S., Cziko, A., Hillebrand, J., Luedeman, R. A., Coller, J. M., Johnson, N., Howlett, I. C., Geng, C., Ueda, R., Brand, A. H., Newbury, S. F., Wilhelm, J. E., Levine, R. B., Nakamura, A., Parker, R., & Ramaswami, M. (2006). Staufen- and FMRP-Containing Neuronal RNPs Are Structurally and Functionally Related to Somatic P Bodies. Neuron, 52(6), 997-1009.
  More info

  PMID: 17178403;PMCID: PMC1955741;Abstract: Local control of mRNA translation modulates neuronal development, synaptic plasticity, and memory formation. A poorly understood aspect of this control is the role and composition of ribonucleoprotein (RNP) particles that mediate transport and translation of neuronal RNAs. Here, we show that staufen- and FMRP-containing RNPs in Drosophila neurons contain proteins also present in somatic "P bodies," including the RNA-degradative enzymes Dcp1p and Xrn1p/Pacman and crucial components of miRNA (argonaute), NMD (Upf1p), and general translational repression (Dhh1p/Me31B) pathways. Drosophila Me31B is shown to participate (1) with an FMRP-associated, P body protein (Scd6p/trailer hitch) in FMRP-driven, argonaute-dependent translational repression in developing eye imaginal discs; (2) in dendritic elaboration of larval sensory neurons; and (3) in bantam miRNA-mediated translational repression in wing imaginal discs. These results argue for a conserved mechanism of translational control critical to neuronal function and open up new experimental avenues for understanding the regulation of mRNA function within neurons. © 2006 Elsevier Inc. All rights reserved.
• Miller, J. E., & Levine, R. B. (2006). Steroid hormone activation of wandering in the isolated nervous system of Manduca sexta. Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology, 192(10), 1049-1062.
  More info

  PMID: 16788816;Abstract: Steroid hormones modulate motor circuits in both vertebrates and invertebrates. The insect Manduca sexta, with its well-characterized developmental and endocrinological history, is a useful model system in which to study these effects. Wandering is a stage-specific locomotor behavior triggered by the steroid hormone 20-hydroxyecdysone (20E), consisting of crawling and burrowing movements as the animal searches for a pupation site. This study was undertaken to determine whether the wandering motor pattern is activated by direct action of 20E on the CNS. 20E acts on the isolated larval nervous system to induce a fictive motor pattern showing features of crawling and burrowing. The latency of the response to 20E is long, suggestive of a genomic mechanism of action. The abdominal motoneurons or segmental pattern generating circuits are unlikely to be the primary targets of 20E action in inducing fictive wandering. Exposure of the segmental ganglia alone to hormone did not evoke fictive wandering. Therefore, as suggested by an earlier study, the likely site of 20E action is within the brain. © 2006 Springer-Verlag.
• Consoulas, C., Levine, R. B., & Restifo, L. L. (2005). The steroid hormone-regulated gene Broad Complex is required for dendritic growth of motoneurons during metamorphosis of Drosophila. Journal of Comparative Neurology, 485(4), 321-337.
  More info

  PMID: 15803508;Abstract: Dendrites are subject to subtle modifications as well as extensive remodeling during the assembly and maturation of neural circuits in a wide variety of organisms. During metamorphosis, Drosophila flight motoneurons MN1-MN4 undergo dendritic regression, followed by regrowth, whereas MN5 differentiates de novo (Consoulas et al. [2002] J. Neurosci. 22:4906-4917). Many cellular changes during metamorphosis are triggered and orchestrated by the steroid hormone 20-hydroxyecdysone, which initiates a cascade of coordinated gene expression. Broad Complex (BRC), a primary response gene in the ecdysone cascade, encodes a family of transcription factors (BRC-Z1-Z4) that are essential for metamorphic reorganization of the central nervous system (CNS). Using neuron-filling techniques that reveal cellular morphology with very high resolution, we tested the hypothesis that BRC is required for metamorphic development of MN1-MN5. Through a combination of loss-of-function mutant analyses, genetic mapping, and transgenic rescue experiments, we found that 2Bc function, mediated by BRC-Z3, is required selectively for motoneuron dendritic regrowth (MN1-MN4) and de novo outgrowth (MN5), as well as for soma expansion of MN5. In contrast, larval development and dendritic regression of MN1-MN4 are BRC-independent. Surprisingly, BRC proteins are not expressed in the motoneurons, suggesting that BRC-Z3 exerts its effect in a non-cell-autonomous manner. The 2Bc mutants display no gross defects in overall thoracic CNS structure, or in peripheral structures such as target muscles or sensory neurons. Candidates for mediating the effect of BRC-Z3 on dendritic growth of MN1-MN5 include their synaptic inputs and non-neuronal CNS cells that interact with them through direct contact or diffusible factors. © 2005 Wiley-Liss, Inc.
• Dulcis, D., & Levine, R. B. (2005). Glutamatergic innervation of the heart initiates retrograde contractions in adult Drosophila melanogaster. Journal of Neuroscience, 25(2), 271-280.
  More info

  PMID: 15647470;Abstract: The adult abdominal heart of Drosophila melanogaster receives extensive innervation from glutamatergic neurons at specific cardiac regions during metamorphosis. Here, we show that the neurons form presynaptic specializations, as indicated by the localization of synaptotagmin and active zone markers, adjacent to postsynaptic sites that have aggregates of glutamate IIA receptors. To determine the role of this innervation in cardiac function, we developed an optical technique, based on the movement of green fluorescent protein-labeled nerve terminals, to monitor heart beat in intact and semi-intact preparations. Simultaneous monitoring of adjacent cardiac chambers revealed the direction of contractions and allowed correlation with volume changes. The cardiac cycle is composed of an anterograde beat in alternation with a retrograde beat, which correlate respectively with systole and diastole of this multichambered heart. The periodic change in hemolymph direction is referred to as cardiac reversal. Intracellular recordings from muscles of the first abdominal cardiac chamber, the conical chamber, revealed pacemaker action potentials and the excitatory effect of local glutamate application, which initiated retrograde contractions in semi-intact preparations. Unilateral electrical stimulation of the transverse nerve containing the glutamatergic neuron that serves the conical chamber caused a chronotropic effect and initiation of retrograde contractions. This effect is distinct from that of peripheral crustacean cardioactive peptide (CCAP) neurons, which potentiate the anterograde beat. Cardiac reversal was evoked pharmacologically by sequentially applying CCAP and glutamate to the heart.
• Dulcis, D., Levine, R. B., & Ewer, J. (2005). Role of the neuropeptide CCAP in Drosophila cardiac function. Journal of Neurobiology, 64(3), 259-274.
  More info

  PMID: 15898062;Abstract: The heartbeat of adult Drosophila melanogaster displays two cardiac phases, the anterograde and retrograde beat, which occur in cyclic alternation. Previous work demonstrated that the abdominal heart becomes segmentally innervated during metamorphosis by peripheral neurons that express crustacean cardioactive peptide (CCAP). CCAP has a cardioacceleratory effect when it is applied in vitro. The role of CCAP in adult cardiac function was studied in intact adult flies using targeted cell ablation and RNA interference (RNAi). Optical detection of heart activity showed that targeted ablation of CCAP neurons selectively altered the anterograde beat, without apparently altering the cyclic cardiac reversal. Normal development of the abdominal heart and of the remainder of cardiac innervation in flies lacking CCAP neurons was confirmed by immunocytochemistry. Thus, in addition to its important role in ecdysis behavior (the behavior used by insects to shed the remains of the old cuticle at the end of the molt), CCAP may control the level of activity of the anterograde cardiac pacemaker in the adult fly. Expression of double stranded CCAP RNA in the CCAP neurons (targeted CCAP RNAi) caused a significant reduction in CCAP expression. However, this reduction was not sufficient to compromise CCAP's function in ecdysis behavior and heartbeat regulation. © 2005 Wiley Periodicals, Inc.
• Dulcis, D., & Levine, R. B. (2004). Remodeling of a larval skeletal muscle motoneuron to drive the posterior cardiac pacemaker in the adult moth, Manduca sexta. Journal of Comparative Neurology, 478(2), 126-142.
  More info

  PMID: 15349974;Abstract: During postembryonic development, a larval skeletal muscle motoneuron, MN-1 in abdominal segments 7 and 8, becomes respecified to innervate the terminal cardiac chamber of adult Manduca sexta. Neural tracing techniques and electrophysiology were used in this study to describe the anatomical and physiological remodeling of this identified motoneuron. During metamorphosis the MN-1 in segments 7 and 8 undergoes dendritic reorganization. Long new dendrites extend anteriorly in the terminal ganglion neuropil. Intracellular and extracellular recordings showed that broader action potentials, increased firing rate, and development of a bursting activity pattern accompany MN-1 respecification. Cardiac mechanograms showed that MN-1 activity bursts always correlate with the anterograde cardiac beat. Bilateral MNs-1 fire at similar times to activate and sustain the putative cardiac pacemaker activity of the terminal chamber synergistically. After remodeling, MN-1 output could be influenced rapidly by sensory inputs during evoked cardiac reversal. The effect is exerted by inhibition of MN-1 firing that, in turn, causes early blockade of the anterograde beat and reversal to the retrograde direction of beat. © 2004 Wiley-Liss, Inc.
• Dulcis, D., & Levine, R. B. (2003). Innervation of the heart of the adult fruit fly, Drosophila melanogaster. Journal of Comparative Neurology, 465(4), 560-578.
  More info

  PMID: 12975816;Abstract: The innervation of the adult abdominal heart of Drosophila melanogaster was studied by neuronal staining with green fluorescent protein and immunocytochemical techniques. The investigation was undertaken to determine whether the adult heart receives neuronal input or whether its complex activity must be considered independent from the nervous system. The larval heart lacks innervation, suggesting that the cardiac impulse is totally myogenic. At metamorphosis, segmental neural processes grow onto the myocardium. A pair of transverse nerves innervates bilaterally each cardiac chamber and its alary muscles. These nerve terminals are immunoreactive to glutamate and form unique synaptic structures on the ventral layer of longitudinal cardiac muscles of the conical chamber. This characteristic cardiac synapse may represent part of the neural mechanism controlling the retrograde heartbeat, and, thus, the cardiac reversal that is characteristic of adults. In addition, crustacean cardioactive peptide-immunoreactive fibers originating from peripheral, bipolar neurons (BpNs) fasciculate with the transverse nerve projections and terminate segmentally throughout the abdominal heart. An additional cluster composed of four large, CCAP-positive neurons innervates the terminal chamber. The cardioacceleratory effect of CCAP release at this location may modulate the properties of a pacemaker producing the anterograde heartbeat. © 2003 Wiley-Liss, Inc.
• Consoulas, C., Restifo, L. L., & Levine, R. B. (2002). Dendritic Remodeling and Growth of Motoneurons during Metamorphosis of Drosophila melanogaster. Journal of Neuroscience, 22(12), 4906-4917.
  More info

  PMID: 12077188;Abstract: Insect motoneurons display dramatic dendritic plasticity during metamorphosis. Many larval motoneurons survive to adulthood but undergo dendritic regression and outgrowth as they are incorporated into developing circuits. This study explores the dendritic remodeling and development of Drosophila motoneurons MN1-MN5, which innervate indirect flight muscles of the adult. MN1-MN5 are persistent larval neurons exhibiting two distinct metamorphic histories. MN1-MN4 are born in the embryo, innervate larval muscles, and undergo dendritic regression and regrowth during metamorphosis. MN5, which was identified through a combination of intracellular dye injection and retrograde staining at all stages, is also born embryonically but remains developmentally arrested until the onset of metamorphosis. In the larva, MN5 lacks dendrites, and its axon stops in the mesothoracic nerve without innervating a target muscle. It is dye coupled to the peripherally synapsing interneuron, which will become part of the giant fiber escape circuit of the adult fly. During pupal development, MN5 undergoes de novo dendritic growth and extension of its axon to innervate the developing target muscle. Its unique developmental history and identifiability make MN5 well suited for the study of dendritic growth using genetic and neurophysiological approaches.
• Duch, C., & Levine, R. B. (2002). Changes in calcium signaling during postembryonic dendritic growth in Manduca sexta. Journal of Neurophysiology, 87(3), 1415-1425.
  More info

  PMID: 11877516;Abstract: Activity-dependent Ca2+ influx plays crucial roles in adult and developing nervous systems through its influence on signal processing, synaptic plasticity, and neuronal differentiation. The responses to internal Ca2+ elevations vary depending on the spatial distribution of Ca2+ accumulation in different cell compartments. In this study, the mechanisms and the distribution of Ca2+ accumulation are addressed by in situ Ca2+ imaging of an identified insect motoneuron, MN5, at critical stages of postembryonic life. During metamorphosis of Manduca sexta, MN5 undergoes extensive dendritic regression followed by regrowth. The time course, amplitude, and distribution of Ca2+ accumulation within MN5 change during development. During the initial stage of rapid dendritic growth and branching, dendritic growth cones are present, and voltage-dependent Ca2+ currents are small. At this stage, activity-induced elevations of internal Ca2+ are largest in the distal dendrites, suggesting that the density of voltage-gated Ca2+ channels is highest in these regions. Later phases of dendritic growth are accompanied by the transient occurrence of prominent Ca2+ spikes. Single Ca2+ spikes cause robust Ca2+ influx of similar amplitudes and time courses in all central compartments of MN5. The resting Ca2+ levels also increase during development. Ca2+-induced Ca2+ release from intracellular stores did not contribute to the elevations measured at either stage, although Ca2+ stores are present in the dendrites. These developmental changes of the internal Ca2+ signaling are consistent with a regulatory role for activity-dependent Ca2+ influx in postembryonic dendritic growth.
• Johnston, R. M., & Levine, R. B. (2002). Thoracic leg motoneurons in the isolated CNS of adult Manduca produce patterned activity in response to pilocarpine, which is distinct from that produced in larvae. Invertebrate Neuroscience, 4(4), 175-192.
  More info

  PMID: 12488968;Abstract: In the hawkmoth, Manduca sexta, thoracic leg motoneurons survive the degeneration of the larval leg muscles to innervate new muscles of the adult legs. The same motoneurons, therefore, participate in the very different modes of terrestrial locomotion that are used by larvae (crawling) and adults (walking). Consequently, changes in locomotor behavior may reflect changes in both the CNS and periphery. The present study was undertaken to determine whether motor patterns produced by the isolated CNS of adult Manduca, in the absence of sensory feedback, would resemble adult specific patterns of coordination. Pilocarpine, which evokes a fictive crawling motor pattern from the isolated larval CNS, also evoked robust patterned activity from leg motoneurons in the isolated adult CNS. As in the larva, levator and depressor motoneurons innervating the same leg were active in antiphase. Unlike fictive crawling, however, bursts of activity in levator or depressor motoneurons of one leg alternated with bursts in the homologous motoneurons innervating the opposite leg of the same segment and the leg on the same side in the adjacent segment. The most common mode of intersegmental activity generated by the isolated adult CNS resembled an alternating tripod gait, which is displayed, albeit infrequently, during walking in intact adult Manduca. A detailed analysis revealed specific differences between the patterned motor activity that is evoked from the isolated adult CNS and activity patterns observed during walking in intact animals, perhaps indicating an important role for sensory feedback. Nevertheless, the basic similarity to adult walking and clear distinctions from the larval fictive crawling pattern suggest that changes within the CNS contribute to alterations in locomotor activity during metamorphosis.
• Bayline, R. J., Duch, C., & Levine, R. B. (2001). Nerve-muscle interactions regulate motor terminal growth and myoblast distribution during muscle development. Developmental Biology, 231(2), 348-363.
  More info

  PMID: 11237464;Abstract: Interactions between motoneurons and muscles influence many aspects of neuromuscular development in all animals. These interactions can be readily investigated during adult muscle development in holometabolous insects. In this study, the development of the dorsolongitudinal flight muscle (DLM) and its innervation is investigated in the moth, Manduca sexta, to address the specificity of neuromuscular interactions. The DLM develops from an anlage containing both regressed larval template fibers and imaginal myoblasts. In the adult, each fiber bundle (DLM1-5) is innervated by a single motoneuron (MN1-MN5), with the dorsal-most fiber bundle (DLM5) innervated by a mesothoracic motoneuron (MN5). The DLM failed to develop following complete denervation because myoblasts failed to accumulate in the DLM anlage. After lesioning MN1-4, MN5 retained its specificity for the DLM5 region of the anlage and failed to rescue DLM1-4. Thus specific innervation of the DLM fiber bundles does not depend on interactions among motoneurons. Myoblast accumulation, but not myonuclear proliferation, increased around the MN5 terminals, producing a hypertrophied adult DLM5. Therefore, motoneurons compete for uncommitted myoblasts. MN5 terminals subsequently grew more rapidly over the hypertrophied DLM5 anlage, indicating that motoneuron terminal expansion is regulated by the size of the target muscle anlage. © 2001 Academic Press.
• Consoulas, C., Duch, C., Bayline, R. J., & Levine, R. B. (2000). Behavioral transformations during metamorphosis: Remodeling of neural and motor systems. Brain Research Bulletin, 53(5), 571-583.
  More info

  PMID: 11165793;Abstract: During insect metamorphosis, neural and motor systems are remodeled to accommodate behavioral transformations. Nerve and muscle cells that are required for larval behavior, such as crawling, feeding and ecdysis, must either be replaced or respecified to allow adult emergence, walking, flight, mating and egg-laying. This review describes the types of cellular changes that occur during metamorphosis, as well as recent attempts to understand how they are related to behavioral changes and how they are regulated. Within the periphery, many larval muscles degenerate at the onset of metamorphosis and are replaced by adult muscles, which are derived from myoblasts and, in some cases, remnants of the larval muscle fibers. The terminal processes of many larval motoneurons persist within the periphery and are essential for the formation of adult muscle fibers. Although most adult sensory neurons are born postembryonically, a subset of larval proprioceptive neurons persist to participate in adult behavior. Within the central nervous system, larval neurons that will no longer be necessary die and some adult interneurons are born postembryonically. By contrast, all of the adult motoneurons, as well as some interneurons and modulatory neurons, are persistent larval cells. In accordance with their new behavioral roles, these neurons undergo striking changes in dendritic morphology, intrinsic biophysical properties, and synaptic interactions. Copyright © 2001 Elsevier Science Inc.
• Consoulas, C., Rose, U., & Levine, R. B. (2000). Remodeling of the femoral chordotonal organ during metamorphosis of the hawkmoth, Manduca sexta. Journal of Comparative Neurology, 426(3), 391-405.
  More info

  PMID: 10992245;Abstract: During metamorphosis of the moth, Manduca sexta, the larval legs degenerate and are replaced by adult legs with a diverse array of new sensory organs. The majority of the larval sensory neurons degenerate but some hair sensilla and chordotonal organ sensory neurons survive metamorphosis (Consoulas [2000] J. Comp. Neurol. 419:154-174). In the present study nerve-tracing techniques, birth-date labeling (5-bromodeoxyuridine), and electrophysiology were used to describe the remodeling of the femoral chordotonal organ (FCO) in the prothoracic legs. The larval FCO is composed of two scoloparia, which are associated with separate apodemes. At the onset of metamorphosis, some of the 13 larval neurons degenerate, together with the larval FCO apodemes. The remaining larval FCO sensory neurons persist in the imaginal leg to become the precursors of the adult femoral and tibial chordotonal organs respectively. Early in the pupal stage, 45 to 60 new sensory neurons are generated de novo and become associated with 6 persistent larval neurons in the imaginal femur to compose the adult FCO. Two clusters of persistent and new neurons are enclosed into two scoloparia with short apodemes that eventually become fused. In both larval and adult stages, the FCO contains units that respond phasically and others that respond tonically to femorotibial movements. Nerve activity from the FCO neurons can be recorded continuously during the remodeling of the organ. A persistent leg flexor motoneuron receives inputs from the FCO sensory neurons in both larval and adult stages, offering the opportunity to investigate the remodeling of the neural circuits underlying the proprioceptive control of the femorotibial joint. (C) 2000 Wiley-Liss, Inc.
• Duch, C., & Levine, R. B. (2000). Remodeling of membrane properties and dendritic architecture accompanies the postembryonic conversion of a slow into a fast motoneuron. Journal of Neuroscience, 20(18), 6950-6961.
  More info

  PMID: 10995839;Abstract: The postembryonic acquisition of behavior requires alterations in neuronal circuitry, which ultimately must be understood as specific changes in neuronal structure, membrane properties, and synaptic connectivity. This study addresses this goal by describing the postembryonic remodeling of the excitability and dendritic morphology of an identified motoneuron, MN5 which during the metamorphosis of Manduca sexta (L.) changes from a slow motoneuron that is involved in larval-crawling behavior into a fast adult flight motoneuron. A fivefold lower input resistance, a higher firing threshold, and an increase in voltage-activated K+ current contribute to a lower excitability of the adult MN5, which is a prerequisite for its newly acquired behavioral role. In addition, the adult MN5 displays larger Ca2+ currents. The dendrites of MN5 undergo extensive remodeling. Drastic regression of larval dendrites during early pupal stages is followed by rapid growth of new dendrites. Critical changes in excitability take place during the onset of adult dendrite formation. Larval Ca2+ currents are absent when dendritic remodeling is most dramatic but increase markedly during later development. Changes in Ca2+ and K+ currents follow different time courses, allowing the transient occurrence of Ca2+ spikes during pupal stages when new dendritic branching ceases. The adult MN5 can produce prolonged Ca2+ spikes after K+ currents are reduced. We suggest that alterations in Ca2+ and K+ currents are necessary for the participation of MN5 in flight behavior and that the transient production of Ca2+ spikes may influence postembryonic dendritic remodeling.
• Duch, C., Bayline, R. J., & Levine, R. B. (2000). Postembryonic development of the dorsal longitudinal flight muscle and its innervation in Manduca sexta. Journal of Comparative Neurology, 422(1), 1-17.
  More info

  PMID: 10842215;Abstract: The neuromuscular systems of holometabolous insects must be remodeled during metamorphosis to allow striking behavioral changes, such as the acquisition of flight. The fast contracting dorsal longitudinal flight muscle (DLM) of Manduca arises from an anlage containing both remnants of specific larval dorsal body wall muscles and extrinsic myoblasts. In the mesothorax, the DLM is innervated by five persisting larval motoneurons: one in the mesothoracic and four in the prothoracic ganglion. These motoneurons innervate two slowly contracting body wall muscles in the larva. 2 days before pupation, the DLM template fibers begin to degenerate, whereas other muscles remain intact until pupation. Correspondingly, the motor terminals retract from the template fibers while they remain on other muscle fibers until pupation. Accumulation and proliferation of putative myoblasts also starts 2 days before pupation in close spatial relationship to the retracted motor tufts around the degenerating larval template fibers. Proliferation increases through the early pupal stages, and is detected within the anlage until the ninth day after pupation. 2 days after pupation, the anlage splits into five bundles, each innervated by one motoneuron. Striations occur on the seventh day after pupation when the growing motor axons reach the attachment sites. Subsequently, the muscle grows in volume and higher-order motor branches are formed. Within the central nervous system, there is dramatic regression of larval dendrites followed by growth of new dendrites as the persistent motoneurons assume their new role in flight behavior. Both central and peripheral remodeling follow similar time courses. (C) 2000 Wiley-Liss, Inc.
• Rose, U., & Levine, R. B. (2000). Comparison of identified leg motoneuron structure and function between larval and adult Manduca sexta. Journal of Comparative Physiology - A Sensory, Neural, and Behavioral Physiology, 186(4), 327-336.
  More info

  PMID: 10798721;Abstract: Persistent leg motoneurons of the moth Manduca sexta were investigated in larval and adult animals to compare their dendritic structures, intrinsic electrical properties and pattern of target innervation. The study focused on two identified motoneurons of the prothoracic leg. Despite the complete remodeling of leg muscles, the motoneurons innervated pretarsal flexor muscles in both larval and adult legs. Similarly, although the central dendrites regress and regrow, the branching pattern was similar with the exception of a prominent midline branch that was not present in the adult stage. The intrinsic electrical properties of the motoneurons differed between larval and adult stages. Larval motoneurons had significantly higher membrane input resistances and more depolarized resting membrane potentials than did motoneurons in pharate adults or adults. In all stages, one motoneuron had a low maximal firing frequency, whereas the second motoneuron, which innervated the other half of the muscle, had a high maximum firing frequency. Although the two motoneurons continued to innervate the same halves of the target muscle, their relative effects on muscular contraction were reversed during metamorphosis along with concomitant changes in intrinsic properties. Pre-tarsal flexor motoneurons in pharate adults (just prior to emergence) displayed properties similar to those in emerged adults.
• Consoulas, C., Johnston, R. M., Pflüger, H., & Levine, R. B. (1999). Peripheral distribution of presynaptic sites of abdominal motor and modulatory neurons in Manduca sexta larvae. Journal of Comparative Neurology, 410(1), 4-19.
  More info

  PMID: 10397391;Abstract: Insect muscle fibers are commonly innervated by multiple motor neurons and efferent unpaired median (UM) neurons. The role of UM neurons in the modulation rather than rapid activation of muscle contraction (Evans and O'Shea [1977] Nature 270:257-259) suggests that their terminal varicosities may differ structurally and functionally from the presynaptic terminals of motor neurons. Furthermore, differences in the characteristics of UM neuron terminal varicosities may be correlated with functional differences among their diverse target muscles. Larval abdominal body wall muscles in the hawkmoth, Manduca sexta, consist of large, elongated fibers that are multiterminally innervated by one and occasionally two motor neurons (Levine and Truman [1985] J. Neurosci. 5:2424-2431). The fibers are also innervated by one of two efferent UM neurons that bifurcate to innervate targets on both sides of the abdomen (Pfluger et al. [1993] J. Comp. Neurol. 335:508-522). In this study, the intracellular tracer biocytin was used to identify the targets of the UM neurons and to distinguish their terminal axonal varicosities on the muscle fibers. An antiserum to the synaptic vesicle protein, synaptotagmin, was used to label synaptic vesicles, and the styryl dye FM1-43 was used to demonstrate release and recycling. Most of the abdominal muscles in a given hemisegment were found to be supplied by one of the two UM neurons. Terminal varicosities of the excitatory motor neurons were large (3-7 μm) and were found in rows of rosettes that extended to every aspect of the muscle fiber; these varicosities were designated as type I terminals. The UM neuron terminal varicosities also occupied every aspect of the fiber but were smaller (1-3 μm) and more separated from each other; these were designated as type II terminals. Both type I and type II terminals are synaptotagmin immunoreactive and, as shown by FM1-43 staining, are sites of synaptic vesicle recycling. The excitatory motor neuron terminals (type I) could easily be loaded and unloaded with FM1-43, which indicates their capacity for repeated vesicular exocytosis and recycling. In contrast, the dye could not as readily be unloaded from UM neuron terminals (type II), which may indicate that they have a slower turnover of synaptic vesicles.
• Johnston, R. M., Consoulas, C., Pflüger, H., & Levine, R. B. (1999). Patterned activation of unpaired median neurons during fictive crawling in Manduca sexta larvae. Journal of Experimental Biology, 202(2), 103-113.
  More info

  Abstract: The unpaired median neurons are common to the segmental ganglia of many insects. Although some of the functional consequences of their activation, among them the release of octopamine to modulate muscle contraction, have been described, less is understood about how and when these neurons are recruited during movement. The present study demonstrates that peripherally projecting unpaired median neurons in the abdominal and thoracic ganglia of the larval tobacco hornworm Manduca sexta are recruited rhythmically during the fictive crawling motor activity that is produced by the isolated central nervous system in response to pilocarpine. Regardless of the muscles to which they project, the efferent unpaired median neurons in all segmental ganglia are depolarized together during the phase of the crawling cycle when the thoracic leg levator motoneurons are active. During fictive crawling, therefore, the unpaired median neurons are not necessarily active in synchrony with the muscles to which they project. The rhythmical synaptic drive of the efferent unpaired median neurons is derived, at least in part, from a source within the subesophageal ganglion, even when the motor pattern is evoked by exposing only the more posterior ganglia to pilocarpine. In pairwise intracellular recordings from unpaired median neurons in different ganglia, prominent excitatory postsynaptic potentials, which occur with an anterior-to-posterior delay in both neurons, are seen to underlie the rhythmic depolarizations. One model consistent with these findings is that one or more neurons within the subesophageal ganglion, which project posteriorly to the segmental ganglia and ordinarily provide unpatterned synaptic inputs to all efferent unpaired median neurons, become rhythmically active during fictive crawling in response to ascending information from the segmental pattern-generating network.
• Matheson, S. F., & Levine, R. B. (1999). Steroid hormone enhancement of neurite outgrowth in identified insect motor neurons involves specific effects on growth cone form and function. Journal of Neurobiology, 38(1), 27-45.
  More info

  PMID: 10027561;Abstract: Dramatic reorganization of dendrites and axonal terminals is a hallmark of neuronal remodeling during metamorphosis in the hawkmoth, Manduca sexta. The dendritic and axonal arbors of leg motor neurons regress in late larval stages, then regrow during adult development. Ecdysteroids, the insect steroids that trigger metamorphosis, control both regression and outgrowth in vivo and stimulate neuritic growth in cultured pupal leg motor neurons. To identify subcellular targets of ecdysteroid action in these neurons, we examined the dynamic and structural features of branching and their modulation by ecdysteroids in vitro. Delayed treatment of pupal leg motor neurons with ecdysteroid led to a robust enhancement of neuritic branch accumulation accompanied by a subtle effect on total neuritic length. Repeated imaging revealed that branch formation occurred almost exclusively at the growth cone; interstitial branching was extremely rare. Ecdysteroid treatment significantly enhanced both the formation and retention of branches at the growth cone. Branches formed via two distinct processes: engorgement (of fine protrusions) and condensation (of lamellae) with the relative contributions of these mechanisms being unaltered by ecdysteroid. Confocal imaging of the cytoskeleton demonstrated that growth cones consisted of microtubule-based domains fringed by actin-based filopodia. Treated growth cones were larger and displayed increased numbers of microtubule-based branches, whereas filopodial density was unaffected. These findings indicate that ecdysteroid enhances neuritic branching by altering growth cone structure and function, and suggest that hormonal modulation of cytoskeletal interactions contributes significantly to neuritic remodeling during metamorphosis.
• Consoulas, C., & Levine, R. B. (1998). Presynaptic function during muscle remodeling in insect metamorphosis. Journal of Neuroscience, 18(15), 5817-5831.
  More info

  PMID: 9671669;Abstract: During metamorphosis the leg neuromuscular system of the moth Manduca sexta undergoes an extensive remodeling as the larval muscles degenerate and are replaced by new muscles in the adult. The terminal processes of persistent leg motoneurons undergo severe regression followed by regrowth (Consoulas et al., 1996), accompanied, as shown here, by the loss and re- establishment of functional presynaptic specializations. Before and shortly after the degeneration of the larval muscle, immunoreactivity for the Vesicular protein synaptotagmin was localized to the presynaptic varicosities of the motoneurons. Similarly localized were distinct sites of Ca2+dependent uptake of the fluorescent dye FM1-43. During myoblast migration and accumulation about the re-expanding motor axons, synaptotagmin immunoreactivity was widely distributed in axons, and specific FM1-43 staining revealed vesicle exocytosis in distal axon branches. During myoblast proliferation and fusion, and myotube formation, synaptotagmin staining remained widely distributed in nerve branches, whereas FM1-43 staining was more localized to subdomains of these nerve branches. These initial presynaptic active sites were transient and were replaced by new sites in more distal nerve processes as the muscle anlage increased in size and additional myotubes formed. After myotube separation, synaptotagmin staining disappeared from primary branches but remained distributed within secondary and high-order nerve branches. FM1-43 staining was detected in high-order branches only. During muscle fiber striation, growth, and maturation, both FM1-43 staining and synaptotagmin immunoreactivity became localized to terminal varicosities. Thus, presynaptic function can persist after the loss of the target and occurs transiently in axon shafts before becoming restricted to terminal domains as the underlying muscle fibers mature.
• Grünewald, B., & Levine, R. B. (1998). Ecdysteroid control of ionic current development in Manduca sexta motoneurons. Journal of Neurobiology, 37(2), 211-223.
  More info

  PMID: 9805268;Abstract: The steroid hormone 20-hydroxyecdysone (20-HE) regulates several processes during insect metamorphosis. We studied the effects of 20-HE on the development of voltage-sensitive ionic currents of thoracic leg motoneurons of Manduca sexta. The larval leg motoneurons persist throughout metamorphosis but undergo substantial morphological reorganization, which is under the control of 20-HE and accompanied by changes in Ca2+ and K+ current densities. To determine whether 20-HE controls the changes in Ca2+ and K+ current levels during postembryonic development, identified thoracic leg motoneurons isolated from late larval and early pupal stages were taken into primary cell culture. Whole-cell Ca2+ and K+ currents were measured after 1-4 days of steroid hormone incubation. In the presence of 20-HE, peak Ca2+ currents of pupal leg motoneurons increased from day I to day 4 in vitro. Thus, at culture day 4 the pupal Ca2+ current levels were larger in 20-HE- treated than in untreated cells. By contrast, 20-HE did not affect the Ca2+ current amplitudes of larval leg motoneurons. Whole-cell K+ currents, measured at 4 days in pupal motoneurons, consisted of a fast-activating transient current and a sustained, slowly inactivating current. 20-HE did not affect the amplitude of the transient or sustained currents after 4 days in vitro. Thus, a direct steroid hormone effect may control the proper maturation of voltage-sensitive Ca2+ currents in leg motoneurons.
• Kraft, R., Levine, R. B., & Restifo, L. L. (1998). The steroid hormone 20-hydroxyecdysone enhances neurite growth of Drosophila mushroom body neurons isolated during metamorphosis. Journal of Neuroscience, 18(21), 8886-8899.
  More info

  PMID: 9786994;Abstract: Mushroom bodies (MBs) are symmetrically paired neuropils in the insect brain that are of critical importance for associative olfactory learning and memory. In Drosophila melanogaster, the MB intrinsic neurons (Kenyon cells) undergo extensive reorganization at the onset of metamorphosis. A phase of rapid axonal degeneration without cell death is followed by axonal regeneration. This re-elaboration occurs as levels of the steroid hormone 20- hydroxyecdysone (20E) are rising during the pupal stage. Based on the known role of 20E in directing many features of CNS remodeling during insect metamorphosis, we hypothesized that the outgrowth of MB axonal processes is promoted by 20E. Using a GAL4 enhancer trap line (201Y) that drives MB- restricted reporter gene expression, we identified Kenyon cells in primary cultures dissociated from early pupal CNS. Paired cultures derived from single brains isolated before the 20E pupal peak were incubated in medium with or without 20E for 2-4 d. Morphometric analysis demonstrated that MB neurons exposed to 20E had significantly greater total neurite length and branch number compared with that of MB neurons grown without hormone. The relationship between branch number and total neurite length remained constant regardless of hormone treatment in vitro, suggesting that 20E enhances the rate of outgrowth from pupal MB neurons in a proportionate manner and does not selectively increase neuritic branching. These results implicate 20E in enhancing axonal outgrowth of Kenyon cells to support MB remodeling during metamorphosis.
• Consoulas, C., & Levine, R. B. (1997). Accumulation and proliferation of adult leg muscle precursors in Manduca are dependent on innervation. Journal of Neurobiology, 32(6), 531-553.
  More info

  PMID: 9183736;Abstract: During metamorphosis, the larval thoracic legs of the moth Manduca sexta are replaced by new adult legs. The leg motoneurons do not die after the loss of the larval muscles, but persist to innervate the new adult leg muscles (Kent and Levine, 1988). The adult muscles form from myoblasts that originate in specific production sites within the legs and migrate to the sites of muscle formation, where they accumulate, proliferate, and fuse to form myofibers (Consoulas et al., 1996b). Throughout adult leg muscle development, there is a close association between nerves and the developing muscles, suggesting a role for the nervous system in myogenesis (Consoulas et al., 1996a). This prediction was confirmed and the role of the nervous system clarified in the present study by cutting the larval leg nerves prior to metamorphosis. Although myoblasts were generated and migrated normally in the operated leg, they failed to accumulate in the appropriate regions. The myoblasts did not die, but failed to proliferate and remained in the denervated legs as dispersed cells or as aggregates in inappropriate regions. In about 26% of cases, this resulted in the formation of adult legs that lacked muscles. In the remaining cases, however, delayed regeneration of the leg nerve occurred and small muscles appeared in the more proximal segments of the denervated legs. Each muscle fiber in these operated legs bore motor terminals belonging to axons of the leg nerves which had grown out from the proximal nerve stump and invaded the leg. Following the delayed appearance of motor axons, myoblasts aggregated and underwent proliferation and differentiation into muscle fibers. In a second set of experiments, denervation was performed later, after myoblasts had aggregated to establish anlagen. Myoblast proliferation was reduced but differentiation continued. These observations suggest that motor nerves are essential for both the accumulation of myoblasts into the correct areas of muscle development and the appropriate level of proliferation.
• Consoulas, C., Anezaki, M., & Levine, R. B. (1997). Development of adult thoracic leg muscles during metamorphosis of the hawk moth Manduca sexta. Cell and Tissue Research, 287(2), 393-412.
  More info

  PMID: 8995211;Abstract: During metamorphosis, the larval thoracic legs of the hawk moth Manduca sexta are replaced by a new set of adult legs. The larval leg motoneurons persist to innervate new adult muscles, and the motor terminals remain within the developing adult legs. Here we describe the fate of the larval leg muscles and the origin of new muscles within the adult legs. During the larval instars, large and small nuclei proliferate within leg muscle fibers. Near the end of the larval stage a subset of the small nuclei undergo a wave of proliferation, as indicated by the incorporation of 5-bromodeoxyuridine, whereas other nuclei die. However, none of the larval leg muscles fibers persist to serve as templates for adult muscle formation, and there was no evidence for persistence of larval myonuclei. Migrating myoblasts that are born within aggregate to form adult muscle anlagen at specific production sites within the developing imaginal legs. Intense nuclear proliferation occurs within the anlagen during the early pupal stage, followed by muscle fiber formation and striation. We conclude that adult leg muscles form mainly, if not exclusively, from migrating myoblasts that without the involvement of larval elements.
• Lemon, W. C., & Levine, R. B. (1997). Multisegmental motor activity in the segmentally restricted gin trap behavior in Manduca sexta pupae. Journal of Comparative Physiology - A Sensory, Neural, and Behavioral Physiology, 180(6), 611-619.
  More info

  PMID: 9190044;Abstract: Stimulation of sensory neurons innervating hairs in the gin traps on the abdomen of Manduca sexta pupae evokes a rapid bending of the abdomen that is restricted to one or more of the three articulating posterior segments. However, electrical stimulation of the gin trap sensory nerve in an isolated abdominal nerve cord evokes characteristic motor neuron activity in every abdominal segment. To determine if the segmentally distributed motor activity also occurred in intact animals and how it contributed to the segmentally restricted reflex movement, mechanical stimulation of the sensory hairs in intact animals was used to evoke reflex responses that were recorded as electromyograms synchronized with video recordings of the behavior. Motor activity was monitored during movements to determine if there was activity in many segments when the movement was restricted to one segment. Coordinated muscle activity was evoked throughout the abdomen in response to stimulation of any of the three gill traps, even when movement was restricted to one segment. Differences in the timing of ipsilateral and contralateral motor activity among segments allowed the closing of gin traps to be segmentally restricted. These findings suggest that the neural circuit underlying the gin trap reflex is distributed througout the abdominal nerve cord. This network generates a complex, yet coordinated, motor pattern with muscular activity in many abdominal segments that produces a localized bending reflex.
• Lemon, W. C., & Levine, R. B. (1997). Segmentally distributed metamorphic changes in neural circuits controlling abdominal bending in the hawkmoth Manduca sexta. Journal of Comparative Physiology - A Sensory, Neural, and Behavioral Physiology, 180(6), 597-610.
  More info

  PMID: 9190043;Abstract: During the metamorphosis of Manduca sexta the larval nervous system is reorganized to allow the generation of behaviors that are specific to the pupal and adult stages. In some instances, metamorphic changes in neurons that persist from the larval stage are segment-specific and lead to expression of segment-specific behavior in later stages. At the larval-pupal transition, the larval abdominal bending behavior, which is distributed throughout the abdomen, changes to the pupal gin trap behavior which is restricted to three abdominal segments. This study suggests that the neural circuit that underlies larval bending undergoes segment specific modifications to produce the segmentally restricted gin trap behavior. We show, however, that non-gin trap segments go through a developmental change similar to that seen in gin trap segments. Pupal-specific motor patterns are produced by stimulation of sensory neurons in abdominal segments that do not have gin traps and cannot produce the gin trap behavior. In particular sensory stimulation in non-gin trap pupal segments evokes a motor response that is faster than the larval response and that displays the triphasic contralateral-ipsilateral-contralateral activity pattern that is typical of the pupal gin trap behavior. Despite the alteration of reflex activity in all segments, developmental changes in sensory neuron morphology are restricted to those segments that form gin traps. In non-gin trap segments, persistent sensory neurons do not expand their terminal arbors, as do sensory neurons in gin trap segments, yet are capable of eliciting gin trap-like motor responses.
• Consoulas, C., Kent, K. S., & Levine, R. B. (1996). Remodeling of the peripheral processes and presynaptic terminals of leg motoneurons during metamorphosis of the hawkmoth, Manduca sexta. Journal of Comparative Neurology, 372(3), 415-434.
  More info

  PMID: 8873869;Abstract: During metamorphosis of the hawkmoth, Manduca sexta, the muscles, cuticular structures, and most sensory neurons of the larval thoracic legs are replaced by new elements in the adult legs. The thoracic leg motoneurons, however, survive the loss of the larval muscles and persist to innervate new targets in the imaginal legs. Here we have used biocytin staining, immunocytochemistry, and confocal microscopy to follow the fates of the peripheral processes and presynaptic terminals of the leg motoneurons. Although the most distal processes of the motor nerves retract following the degeneration of larval leg muscles, the axon terminals always retain close association with the muscle remnants and the antigen of the new adult muscles. As the imaginal muscles differentiate and enlarge, the motor terminals expand to form adult presynaptic terminals. An antibody to the presynaptic protein, synaptotagmin, revealed its localization to the terminal varicosities in both larval and adult stages but distribution within pre- terminal branches during adult development. Electrophysiological methods revealed that functional neuromuscular transmission first occurs quite early during metamorphosis, before the differentiation of contractile elements in the muscle fibers.
• Johnston, R. M., & Levine, R. B. (1996). Crawling motor patterns induced by pilocarpine in isolated larval nerve cords of Manduca sexta. Journal of Neurophysiology, 76(5), 3178-3195.
  More info

  PMID: 8930265;Abstract: 1. Larval crawling is a bilaterally symmetrical behavior that involves an anterior moving wave of motor activity in the body wall muscles in conjunction with sequential movements of the abdominal prolegs and thoracic legs. The purpose of this study was to determine whether the larval CNS by itself and without phasic sensory feedback was capable of producing patterned activity associated with crawling. To establish the extent of similarity between the output of the isolated nerve cord and crawling, the motor activity produced in isolated larval nerve cords was compared with the motor activity from freely crawling larvae. 2. When exposed to the muscarinic receptor agonist pilocarpine (1.0 mM), isolated larval nerve cords produced long-lasting rhythmic activity in the motor neurons that supply the thoracic leg, abdominal body wall, and abdominal proleg muscles. The rhythmic activity evoked by pilocarpine was abolished reversibly and completely by bath application of the muscarinic-receptor antagonist atropine (0.01 mM) in conjunction with pilocarpine (1.0 mM), suggesting that the response was mediated by muscarinic-like acetylcholine receptors. 3. Similar to crawling in intact animals, the evoked activity in isolated nerve cords involved bilaterally symmetrical motor activity that progressed from the most posterior abdominal segment to the most anterior thoracic segment. The rhythmic activity in thoracic leg, abdominal proleg, and abdominal body wall motor neurons showed intrasegmental and intersegmental cycle-to-cycle coupling. The average cycle period for rhythmic activity in the isolated nerve cord was ~2.5 times slower than the cycle period for crawling in intact larvae, but not more variable. 4. Like crawling in intact animals, in isolated nerve cords, bursting activity in the dorsal body wall motor neurons occurred before activity in ventral/lateral body wall motor neurons within an abdominal segment. The evoked bursting activity recorded from the proleg nerve was superimposed on a high level of tonic activity. 5. In isolated nerve cords, bursts of activity in the thoracic leg levator/extensor motor neurons alternated with bursts of activity in the depressor/flexor motor neurons. The burst duration of the levator/extensor activity was brief and remained relatively steady as cycle period increased. The burst duration of the depressor/flexor activity occupied the majority of an average cycle and increased as cycle period increased. The phase of both levator/extensor motor nerve activity and depressor/flexor motor nerve activity remained relatively stable over the entire range of cycle periods. The timing and patterning of thoracic leg motor neuron activity in isolated nerve cords quantitatively resembled thoracic leg motor activity in freely crawling larvae. 6. The rhythmic motor activity generated by an isolated larval nerve cord resembled a slower version of normal crawling in intact larvae. Because of the many similarities between activity induced in the isolated nerve cord and the muscle activity and movements of thoracic and abdominal segments during crawling, we concluded that central mechanisms can establish the timing and patterning of the crawling motor pattern and that crawling may reflect the output of a central pattern generating network.
• Johnston, R. M., & Levine, R. B. (1996). Locomotory behavior in the hawkmoth Manduca sexta: Kinematic and electromyographic analyses of the thoracic legs in larvae and adults. Journal of Experimental Biology, 199(4), 759-774.
  More info

  PMID: 8788085;Abstract: During metamorphosis in Manduca sexta, muscles and most sensory structures of the thoracic legs undergo extensive changes while the motor neurons that are present in the larva persist into the adult. The main goal of this work was to identify similarities and dissimilarities in thoracic leg movements during crawling in larvae and walking in adults. This information provides a foundation for understanding the extent to which centrally located neural elements are reorganized during metamorphosis to accommodate changes in locomotion. Analysis of electromyographic activity from leg muscles synchronized with video-taped recordings of the leg movements during larval crawling and adult walking revealed differences in cycle periods as well as intersegmental and intrasegmental patterns of coordination. Larval crawling was characterized by synchronous movements of segmental pairs of legs as activity proceeded slowly from the more posterior to the more anterior segments. During crawling, antagonistic muscles maintained a strict reciprocity. In contrast, walking in adults was characterized by fast, alternating movements of the left and right prothoracic legs and more variable coordination patterns in the mesothoracic and metathoracic legs (ranging from synchrony to alternation). In adults, sensory information, possibly associated with the weight-bearing or postural demands of walking on an incline, contributed to a strong dependence between the duration of muscle activity and cycle period and to the extent that the muscle activity overlapped during walking.
• Levine, R. B., & Weeks, J. C. (1996). Cell culture approaches to understanding the actions of steroid hormones on the insect nervous system. Developmental Neuroscience, 18(1-2), 73-86.
  More info

  PMID: 8840087;Abstract: During metamorphosis of the hawkmoth, Manduca sexta, ecdysteroids regulate the dendritic remodeling and programmed death of identified motoneurons. These changes contribute to the dramatic reorganization of behavior that accompanies metamorphosis. As a step toward elucidating cellular and molecular mechanisms by which ecdysteroids affect neuronal phenotype, we have investigated the responses of Manduca motoneurons to ecdysteroids in vitro. Following dendritic regression at the end of larval life, thoracic leg motoneurons placed in culture respond to ecdysteroids by an increase in branching complexity, similar to events in vivo. Growth cone structure is affected markedly by ecdysteroids. At pupation, a rise in ecdysteroids triggers the segment-specific death of proleg motoneurons: the same segmental pattern of death is observed when motoneurons from different segments are removed from the nervous system and exposed to ecdysteroids in vitro. These studies provide strong evidence that Manduca motoneurons are direct targets of steroid action and set the stage for further studies of the specific mechanisms involved.
• Luedeman, R., & Levine, R. B. (1996). Neurons and ecdysteroids promote the proliferation of myogenic cells cultured from the developing adult legs of Manduca sexta. Developmental Biology, 173(1), 51-68.
  More info

  PMID: 8575638;Abstract: During metamorphosis in the hawkmoth Manduca sexta, larval leg motoneurons survive the degeneration of their target muscles to innervate new muscles that form during the development of the adult legs. Observations of muscle development in vivo suggest that there are close interactions between motor terminals and the muscle precursor cells at the earliest stages of muscle formation and surgical denervation compromises further development of adult muscles. Here we describe a nerve/muscle coculture system that allows further exploration of this critical developmental interaction. Muscle precursor cells derived from the developing thoracic legs of early pupae and cultured in the presence of neurons assumed a spindle-like morphology and fused to form multinucleate contractile myotubes. Contractile fibers did not form in cultures of muscle precursor cells alone. In the presence of neurons the rate of bromodeoxyuridine (BrdU) incorporation into myonuclei was significantly enhanced, suggesting that neurons promote the proliferation of myogenic cells. This effect was not unique to thoracic leg motoneurons of the early pupal stage, in that larval thoracic neurons as well as neurons from the pupal brain or abdominal ganglia were also effective at enhancing BrdU incorporation and the formation of contractile muscle fibers. Medium conditioned by neurons was ineffective at promoting BrdU incorporation, and in cocultures BrdU incorporation was enhanced only in regions of physical overlap between neurons and muscle precursor cells, suggesting that a very close-range interaction was involved. Tetrodotoxin-sensitive neuronal activity was not required for the effect on muscle development, but fixed neurons were ineffective. The insect steroid hormone 20-hydroxyecdysone enhanced BrdU incorporation into the nuclei of myogenic cells in both the presence and the absence of neurons. The results suggest that both neurons and ecdysteroids play an important regulatory role in adult muscle development, at least in part by enhancing the proliferation of myogenic cells.
• Tamarkin, D. A., & Levine, R. B. (1996). Synaptic interactions between a muscle-associated proprioceptor and body wall muscle motor neurons in larval and adult Manduca sexta. Journal of Neurophysiology, 76(3), 1597-1610.
  More info

  PMID: 8890279;Abstract: 1. Synaptic remodeling of a proprioceptive circuit during metamorphosis of the insect, Manduca sexta, is described. The stretch receptor organ is a muscle-associated proprioceptor that is innervated by a single sensory neuron. It inserts dorsolaterally in the abdomen in parallel with the intersegmental muscles of each abdominal segment. The synaptic input from the stretch receptor sensory neuron to select abdominal internal (intersegmental) and external muscle motor neurons was characterized in both the larva and adult. 2. In the larva, the sensory neuron provides excitatory synaptic input to motor neurons that innervate muscles ipsilateral to the stretch receptor organ in the body wall; the strongest excitatory synaptic input is to motor neurons that innervate targets in close proximity to the stretch receptor organ. The sensory neuron also provides excitatory synaptic input to motor neurons that innervate contralateral, dorsal targets. However, it inhibits, apparently through a polysynaptic pathway, motor neurons innervating contralateral, lateral, and ventral targets. 3. The synaptic input to intersegmental muscle motor neurons from the stretch receptor sensory neuron changes during metamorphosis. In contrast to the larva, all motor neurons recorded in the adult (both ipsilateral and contralateral) were excited by the sensory neuron. As in the larva, the adult sensory neuron provides the strongest excitatory synaptic input to motor neurons innervating targets in close proximity to the stretch receptor organ. 4. The proprioceptive input to the body wall muscle motor neurons was evaluated to determine whether the pathway is monosynaptic, as has been described in other systems. Spike- triggered signal averaging and synaptic latency measurements suggested that the strongest excitatory synaptic input to motor neurons involves a monosynaptic pathway.
• Kent, K. S., Consoulas, C., Duncan, K., Johnston, R. M., Luedeman, R., & Levine, R. B. (1995). Remodelling of neuromuscular systems during insect metamorphosis. Integrative and Comparative Biology, 35(6), 578-584.
  More info

  Abstract: During metamorphosis in the hawkmoth, Manduca sexta, the larval thoracic legs are replaced by a new set of adult legs that include new sensory neurons and muscles, and participate in new patterns of locomotor activity. Larval leg motoneurons persist to innervate the new adult leg muscles, but undergo striking changes in dendritic morphology that are regulated by the insect steroid, 20-hydroxyecdysone. In the periphery, the motor terminals regress as larval muscles degenerate, and expand as new adult muscles form from myoblasts. Evidence obtained both in vivo and in vitro suggests that the proliferation of myoblasts during metamorphosis is dependent upon innervation. © 1995 by the American Society of Zoologists.
• Levine, R. B., Morton, D. B., & Restifo, L. L. (1995). Remodeling of the insect nervous system. Current Opinion in Neurobiology, 5(1), 28-35.
  More info

  PMID: 7773002;Abstract: Our nervous systems and behavior are shaped by hormonally driven developmental changes that continue beyond the embryonic period. Key insights into this process have emerged from studies of the insect nervous system. During insect metamorphosis, the nervous system is remodeled through postembryonic neurogenesis, programmed cell death and the modification of persistent neurons. These changes are regulated to a large degree by gene cascades that are triggered by steroid hormones, the ecdysteroids. Current studies are attempting to reveal the molecular mechanisms involved in regulating these dramatic examples of developmental plasticity. © 1995.
• Weeks, J. C., & Levine, R. B. (1995). Steroid hormone effects on neurons subserving behavior. Current Opinion in Neurobiology, 5(6), 809-815.
  More info

  PMID: 8805414;Abstract: Recent advances in understanding effects of steroid hormones at the level of individual neurons have been achieved using model systems. Steroid hormone effects on dendritic morphology, synaptic function and ionic conductances have been implicated in the regulation of behavior in both vertebrates and invertebrates. Particularly exciting are studies demonstrating steroid hormone effects on specific synaptic connections and ionic currents. There also has been important progress in understanding the diversity of sites and mechanisms of hormone action, encompassing both genomic and non-genomic effects of steroids on neuronal properties.
• Kent, K. S., & Levine, R. B. (1993). Dendritic reorganization of an identified neuron during metamorphosis of the moth Manduca sexta: The influence of interactions with the periphery. Journal of Neurobiology, 24(1), 1-22.
  More info

  PMID: 8419520;Abstract: During metamorphosis of the moth, Manduca sexta, an identified leg motor neuron, the femoral extensor motor neuron (FeExt MN) undergoes dramatic reorganization. Larval dendrites occupy two distinct regions of neuropil, one in the lateral leg neuropil and a second in dorsomedial neuropil. Adult dendrites occupy a greater volume of lateral leg neuropil but do not extend to the dorsomedial region of the ganglion. The adult dendritic morphology is acquired by extreme dendritic regression followed by extensive dendritic growth. Towards the end of larval life, MN dendrites begin to regress, but the most dramatic loss of dendrites occurs in the 3 days following pupation, such that only a few sparse dendrites are retained in the lateral region of leg neuropil. Extensive dendritic growth occurs over the subsequent days such that the MN acquires an adult-like morphology between 12 and 14 days after pupation. This basic process of dendritic remodeling is not dependent upon the presence of the adult leg, suggesting that neither contact with the new target muscle nor inputs from new leg sensory neurons are necessary for triggering dendritic changes. The final distribution of MN dendrites in the adult, however, is altered when the adult leg is absent, suggesting that cues from the adult leg are involved in directing or shaping the growth of MN dendrites to specific regions of neuropil.
• Pfluger, H. J., Witten, J. L., & Levine, R. B. (1993). Fate of abdominal ventral unpaired median cells during metamorphosis of the hawkmoth, Manduca sexta. Journal of Comparative Neurology, 335(4), 508-522.
  More info

  PMID: 8227533;Abstract: Each of the unfused abdominal ganglia in the larval, pupal, and adult stages of the hawkmoth, Manduca sexta, has two large ventral median neurons with axons that bifurcate to innervate targets on both sides of the abdomen. Although the dendritic structures of the two neurons are similar, their axons branch to innervate distinct sets of target muscles. During metamorphosis both neurons undergo dendritic regression, followed by growth of new arborizations during adult development. The neurons must innervate different targets in the larva and adult, since many larval muscles degenerate and are replaced during metamorphosis. Both neurons were reactive with an antibody to the neuromodulatory compound, octopamine, in the larval and adult stages. Pairwise intracellular recordings in isolated nerve cords revealed spontaneous excitatory synaptic potentials that occurred in the ventral median neurons of each ganglion in an anterior-to-posterior sequence. The synaptic potentials were eliminated when the interganglionic connective was interrupted posterior to the subesophageal ganglion. The ventral median neurons were also excited by tactile stimulation of the body surface in larvae, pupae and adults.
• Hayashi, J. H., & Levine, R. B. (1992). Calcium and potassium currents in leg motoneurons during postembryonic development in the hawkmoth Manduca sexta.. Journal of Experimental Biology, 171, 15-42.
  More info

  PMID: 1431728;Abstract: During insect metamorphosis the nervous system is reorganized to accommodate changes in behavior. In the hawkmoth, Manduca sexta, many identified larval motoneurons persist to innervate new adult muscles, while undergoing changes in dendritic morphology and synaptic connections. The thoracic leg motoneurons, for example, innervate different sets of muscles in the larva and adult and participate in distinct types of locomotor behavior in the two stages of life. To determine whether changes in the biophysical properties of these motoneurons accompany the structural and functional modifications that have been described, we used the whole-cell voltage-clamp technique to compare the Ca2+ and K+ currents expressed by leg motoneurons isolated from the larval, pupal and adult stages of Manduca. After 24 h in culture, the somata of leg motoneurons isolated from all three stages expressed voltage-sensitive Ca2+ currents that could be blocked by Cd2+, Co2+ or Ni2+. The currents were larger with Ba2+ as the charge carrier. The Ca2+ current density was significantly lower in these motoneurons during the early pupal stage than in either the larva or adult. Similar experiments revealed both transient and sustained K+ currents in the leg motoneurons that could be blocked with Cs+. There was a significant decrease in the density of the transient, inactivating outward current in leg motoneurons isolated from the early pupal stage. Thus, the levels of some types of ionic currents are modulated during metamorphosis. These changes may be important for the developmental or behavioral changes that accompany metamorphosis.
• Prugh, J., Croce, K. D., & Levine, R. B. (1992). Effects of the steroid hormone, 20-hydroxyecdysone, on the growth of neuntes by identified insect motoneurons in vitro. Developmental Biology, 154(2), 331-347.
  More info

  PMID: 1426641;Abstract: During metamorphosis in the hawkmoth, Manduca sexta, identified larval leg motoneurons survive the degeneration of their larval targets to innervate new muscles of the adult legs. The dendrites and axon terminals of these motoneurons regress at the end of the larval stage and then regrow during adult development. Previous studies have implicated the insect steroid, 20-hydroxyecdysone (20-HE), in similar examples of dendritic reorganization during metamorphosis. The present studies were undertaken to test whether 20-HE acts directly on the leg motoneurons to regulate dendritic growth. Larval leg motoneurons were labeled with a fluorescent dye to permit their identification in culture following the dissociation of thoracic ganglia at later stages of development. Leg motoneurons isolated from early pupal stage animals (just before the normal onset of dendritic regrowth) survived in vitro and grew processes regardless of whether 20-HE was added to the culture medium. The extent of process outgrowth, however, as measured by the total length of all processes and the number of branches, was significantly greater for motoneurons maintained in the presence of 20-HE. The enhancement could be blocked by the addition of a juvenile hormone analog. By contrast, larval leg motoneurons that were isolated just before the normal period of dendritic regression did not show enhanced growth of neurites in the presence of 20-HE. The results suggest that 20-HE acts directly on the leg motoneurons to regulate the growth of processes during metamorphosis. © 1992 Academic Press, Inc.
• Waldrop, B., & Levine, R. B. (1992). Intersegmental interneurons serving larval and pupal mechanosensory reflexes in the moth Manduca sexta. Journal of Comparative Physiology A, 171(2), 195-205.
  More info

  PMID: 1432855;Abstract: 1. Intersegmental interneurons (INs) that participate in the larval bending reflex and the pupal gin trap closure reflex were identified in the isolated ventral nerve cord of Manduca sexta. INs 305, 504, and 703 show qualitatively different responses in the pupa than in the larva to electrical stimulation of sensory neurons that are retained during the larval-pupal transition to serve both reflexes. Action potentials produced by current injected into the 3 interneurons excite motor neurons that are directly involved in the larval and pupal reflexes. The excitation of the motor neurons is not associated with EPSPs at a fixed latency following action potentials in the interneurons, and thus there do not seem to be direct synaptic connections between the interneurons and the motor neurons. 2. IN 305 (Fig. 2) has a lateral soma, processes in most of the dorsal neuropil ipsilateral to the soma, and a crossing neurite that gives rise to a single contralateral descending axon. IN 305 is excited by stimulation of the sensory nerve ipsilateral to its soma in the larva and the pupa. Stimulation of the sensory nerve contralateral to its soma produces an inhibitory response in the larva, but a mixed excitatory/inhibitory response to the identical stimulus in the pupa. 3. IN 504 (Fig. 3) has a lateral soma, processes throughout most of the neuropil ipsilateral to the soma, and a crossing neurite that bifurcates to give rise to a process extending to the caudal limit of the neuropil and an ascending axon. IN 504 is excited by stimulation of the sensory nerve ipsilateral to its soma in both larvae and pupae, while the response to stimulation of the sensory nerve contralateral to its soma is inhibitory in the larva but mixed (excitatory/inhibitory) in the pupa. 4. IN 703 has a large antero-lateral soma, a neurite that extends across to the contralateral side giving rise to processes located primarily dorsally in both ipsilateral and contralateral neuropils, and two axons that ascend and descend in the connectives contralateral to the soma (Fig. 4). IN 703 responds to stimulation of the sensory nerves on either side of the ganglion, but the form of the response changes during the larval-pupal transition. In the larva, the response consists of very phasic (0-2 spikes) excitation, but in the pupa there is a prolonged excitation that greatly outlasts the stimulus (Fig. 6). 5. While the resting potential, and thus the relative spike threshold, of IN 703 appears to change during the larval-pupal transition (Fig. 9), hyperpolarizing IN 703 during a response shows that this difference can not account for the change in response properties (Fig. 10). Rather, IN 703 in the pupa is influenced by interneuronal inputs in the pupa whose effects are not expressed in the larva (Fig. 11). © 1992 Springer-Verlag.
• Levine, R. B., Fahrbach, S. E., & Weeks, J. C. (1991). Steroid hormones and the reorganization of the nervous system during insect metamorphosis. Seminars in Neuroscience, 3(6), 437-447.
  More info

  Abstract: Changes in the structure and function of identified neurons during insect metamorphosis that are linked to the gain or loss of specific elements of behavior are controlled by the steroid hormone, 20-hydroxyecdysone. Three approaches are being used to determine which cells are primary targets for steroid hormones: localized hormone manipulations, direct exposure of identified neurons to ecdysteroids in vitro and steroid hormone autoradiography. Identified neurons display an array of responses to steroids, which may have different thresholds and be subject to other regulatory influences. The current challenge is to link our understanding of the hormonally dependent cellular changes in identified neurons to emerging information about the molecular basis of ecdysteroid action and the regulation of cell fate in insects. © 1991.
• Levine, R. B., & Weeks, J. C. (1990). Hormonally mediated changes in simple reflex circuits during metamorphosis in Manduca. Journal of Neurobiology, 21(7), 1022-1036.
  More info

  PMID: 2258719;Abstract: During insect metamorphosis, the nervous system must be reorganized to allow the production of unique behaviors during each life stage. In the hawkmoth, Manduca sexta, it has been possible to follow this postembryonic phase of neuronal development at the level of identified neurons. Of particular interest in the present context are sensory neurons, motoneurons, and interneurons which persist through metamorphosis, but participate in different types of behavior at different stages of life. Many of these neurons undergo striking changes in their dendritic arborizations and axonal projection patterns, which can be correlated with changes in their synaptic interactions with other neurons. Manipulations of the ecdysteroid and juvenile hormone titers, both in vivo and in vitro, implicate these hormones in the regulation of metamorphic changes within the nervous system. Taking advantage of this endocrine control, it has been possible to create heterochronic mosaic animals that allow the relationship between specific cellular changes and behavioral alterations to be tested directly.
• Weeks, J. C., & Levine, R. B. (1990). Postembryonic neuronal plasticity and its hormonal control during insect metamorphosis. Annual Review of Neuroscience, 13(1), 183-194.
  More info

  PMID: 2183673;
• Levine, R. B. (1989). Expansion of the central arborizations of persistent sensory neurons during insect metamorphosis: The role of the steroid hormone, 20-hydroxyecdysone. Journal of Neuroscience, 9(3), 1045-1054.
  More info

  PMID: 2926478;
• Levine, R. B., Waldrop, B., & Tamarkin, D. (1989). The use of hormonally induced mosaics to study alterations in the synaptic connections made by persistent sensory neurons during insect metamorphosis.. Journal of Neurobiology, 20(5), 326-338.
  More info

  PMID: 2746201;Abstract: During the initial phase of metamorphosis in the hawkmoth, Manduca sexta, persistent mechanosensory neurons expand their terminal arborizations within the CNS and evoke a reflex response in the pupa which is different than in the larva. In an effort to determine the contribution of sensory neuron modifications to the difference in reflex responses, manipulations of juvenile hormone and 20-hydroxyecdysone were used to generate mosaic animals in which the sensory neurons were advanced or delayed developmentally with respect to the rest of the animal, including circuit components within the CNS. In the larval stage electrical stimulation of the sensory axons evokes a slow depolarization and a prolonged burst of action potentials in the ipsilateral intersegmental muscle motor neurons. By contrast, in pupal preparations the same motor neurons respond to an identical stimulus with a larger, more rapid depolarization which leads to a relatively brief, high-frequency burst of action potentials. Motor responses on the contralateral side of the body are also altered during pupal development. In mosaic animals where larval-like sensory neurons interact with a pupal CNS, a larval reflex response is generated. In the converse situation, pupal-like sensory neurons interacting with a larval or prepupal CNS evoke a motor response that is typical of larvae or prepupae. We conclude, therefore, that pupal development of the sensory neurons is necessary, but not sufficient, for the production of the pupal reflex.
• Waldrop, B., & Levine, R. B. (1989). Development of the gin trap reflex in Manduca sexta: a comparison of larval and pupal motor responses. Journal of Comparative Physiology A, 165(6), 743-753.
  More info

  PMID: 2810148;Abstract: 1. Responses of motor neurons in larvae and pupae of Manduca sexta to stimulation of tactile sensory neurons were measured in both semiintact, and isolated nerve cord preparations. These motor neurons innervate abdominal intersegmental muscles which are involved in the production of a general flexion reflex in the larva, and the closure reflex of the pupal gin traps. 2. Larval motor neurons respond to stimulation of sensory neurons innervating abdominal mechanosensory hairs with prolonged, tonic excitation ipsilaterally, and either weak excitation or inhibition contralaterally (Figs. 4A, 6). 3. Pupae respond to tactile stimulation of mechanosensory hairs within the gin traps with a rapid closure reflex. Motor neurons which innervate muscles ipsilateral to the stimulus exhibit a large depolarization, high frequency firing, and abrupt termination (Figs. 2, 4B). Generally, contralateral motor neurons fire antiphasically to the ipsilateral motor neurons, producing a characteristic triphasic firing pattern (Figs. 7, 8) which is not seen in the larva. 4. Pupal motor neurons can also respond to sensory stimulation with other types of patterns, including rotational responses (Fig. 3 A), gin trap opening reflexes (Fig. 3 B), and 'flip-flop' responses (Fig. 9). 5. Pupal motor neurons, like larval motor neurons, do not show oscillatory responses to tonic current injection, nor do motor neurons of either stage appear to interact synaptically with one another. Most pupal motor neurons also exhibit i-V properties similar to those of larval motor neurons (Table 1; Fig. 10). Some pupal motor neurons, however, show a marked non-linear response to depolarizing current injection (Fig. 11). © 1989 Springer-Verlag.
• Kent, K. S., & Levine, R. B. (1988). Neural control of leg movements in a metamorphic insect: Persistence of larval leg motor neurons to innervate the adult legs of Manduca sexta. Journal of Comparative Neurology, 276(1), 30-43.
  More info

  PMID: 3192763;Abstract: During metamorphosis of the hawkmoth Manduca sexta, the larval thoracic legs along with their associated sensory organs and muscles degenerate and new adult legs develop. The larval legs are small and relatively simple structures capable of lateral extension and medial flexion allowing them to grasp the substrate as the caterpillar crawls along. By contrast, the adult legs are used for walking with an alternating gait. They are much larger than the larval legs and articulate such that they are capable of movement in several directions. This change in form and function is accompanied by a reorganization of the neural controlling leg movements. In a previous report (Kent and Levine: J. Comp. Neurol. 271:559-576, '88) we described moter neurons innervating the prothoracic legs of the adult. Using a combination of cobalt staining methods and the persistent fluorescent dye Fluoro-Gold, we have found that some, if not all, larval leg motor neurons are retained and innervate the new adult leg muscles. Moreover, we have been able to discover the fate of individual larval leg motor neurons by marking a single larval neuron with Fluoro-Gold and using a second fluorescent dye to double the same neuron in the adult. Our results suggest that specific larval leg motor neurons innervate corresponding muscles in the adult stage, although their apparent function is significantly different. In addition, the motor neurons undergo significant remodeling of their dendritic branching patterns during metamorphosis, alterations which doubtless contribute to their new roles in adult behavior.
• Kent, K. S., & Levine, R. B. (1988). Neural control of leg movements in a metamorphic insect: Sensory and motor elements of the larval thoracic legs in Manduca sexta. Journal of Comparative Neurology, 271(4), 559-576.
  More info

  PMID: 3385017;Abstract: During the metamorphosis of the hawkmothh Manduca sexta the larval thoracic legs degenerate to be replaced in the adult by legs of very different form and function. This change must be accompanied by a reorganization of the neural circuits controlling leg movements. As an initial step in the study of this reorganization we describe here the sensory and motor elements of this circuitry in the larval stage of life. Sensory neurons innervating mechanoreceptive hairs on the thoracic surface were stained individually with cobalt. Those innervating hairs on the general thoracic surface project topographically into two ventral regions of the segmental ganglia. Sensory neurons innervating leg sensilla also map topographically to the more ventral of these regions but in addition have arborizations in a midlateral region. The density of branching within this lateral 'leg neuropil' is greatest for sensory neurons from sensilla on the more distal leg segments. Leg motor neurons were identified with intracellular recording and cobalt injection techniques. Those innervating muscles controlling distal leg segments have dense dendritic arbors in the lateral 'leg neuropil,' while motor neurons controlling more proximal segments and muscles of the ventral body wall have extensive arborizations in a dorsomedial region of the ganglion. In general, flexor motor neurons are excited by medial and inhibited by lateral leg sensilla, while the opposite is true of extensors. Distal segment motor neurons respond most strongly to sensory neurons from distal segments, thus suggesting some interaction within the lateral 'leg neuropil'. Thus, in the larval nervous system a highly ordered array of sensory and motor elements underlies the specific behavioral responses of the legs to tactile stimulation.
• Levine, R. B. (1987). Neural reorganization and its endocrine control during insect metamorphosis.. Current topics in developmental biology, 21, 341-365.
  More info

  PMID: 3308331;