Melissa Warden

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• Sidor, M. M., & Warden, M. (2015). Optogenetics. In Encyclopedia of Psychopharmacology(pp 1184-1189). New York, NY: Springer.
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  A neuromodulatory set of tools and techniques in neuroscience that involve the introduction of light-sensitive proteins, called opsins, into living cells and into the intact brain of freely moving animals to permit real-time control (both electrical and biochemical) of genetically defined populations of cells and neural circuits with both high spatial and temporal precision.

Journals/Publications

• Gu, W., Luozhong, S., Cai, S., Londhe, K., Elkasri, N., Hawkins, R., Yuan, Z., Su-Greene, K., Yin, Y., Cruz, M., Chang, Y. W., McMullen, P., Wu, C., Seo, C., Guru, A., Gao, W., Sarmiento, T., Schaffer, C., Nishimura, N., , Cerione, R., et al. (2024). Extracellular vesicles incorporating retrovirus-like capsids for the enhanced packaging and systemic delivery of mRNA into neurons. Nature biomedical engineering, 8(4), 415-426.
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  The blood-brain barrier (BBB) restricts the systemic delivery of messenger RNAs (mRNAs) into diseased neurons. Although leucocyte-derived extracellular vesicles (EVs) can cross the BBB at inflammatory sites, it is difficult to efficiently load long mRNAs into the EVs and to enhance their neuronal uptake. Here we show that the packaging of mRNA into leucocyte-derived EVs and the endocytosis of the EVs by neurons can be enhanced by engineering leucocytes to produce EVs that incorporate retrovirus-like mRNA-packaging capsids. We transfected immortalized and primary bone-marrow-derived leucocytes with DNA or RNA encoding the capsid-forming activity-regulated cytoskeleton-associated (Arc) protein as well as capsid-stabilizing Arc 5'-untranslated-region RNA elements. These engineered EVs inherit endothelial adhesion molecules from donor leukocytes, recruit endogenous enveloping proteins to their surface, cross the BBB, and enter the neurons in neuro-inflammatory sites. Produced from self-derived donor leukocytes, the EVs are immunologically inert, and enhanced the neuronal uptake of the packaged mRNA in a mouse model of low-grade chronic neuro-inflammation.
• Ho, Y., Yang, Q., Boddu, P., Bulkin, D. A., & Warden, M. (2024). Infralimbic parvalbumin neural activity facilitates cued threat avoidance. eLife.
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  The infralimbic cortex (IL) is essential for flexible behavioral responses to threatening environmental events. Reactive behaviors such as freezing or flight are adaptive in some contexts, but in others a strategic avoidance behavior may be more advantageous. IL has been implicated in avoidance, but the contribution of distinct IL neural subtypes with differing molecular identities and wiring patterns is poorly understood. Here, we study IL parvalbumin (PV) interneurons in mice as they engage in active avoidance behavior, a behavior in which mice must suppress freezing in order to move to safety. We find that activity in inhibitory PV neurons increases during movement to avoid the shock in this behavioral paradigm, and that PV activity during movement emerges after mice have experienced a single shock, prior to learning avoidance. PV neural activity does not change during movement toward cued rewards or during general locomotion in the open field, behavioral paradigms where freezing does not need to be suppressed to enable movement. Optogenetic suppression of PV neurons increases the duration of freezing and delays the onset of avoidance behavior, but does not affect movement toward rewards or general locomotion. These data provide evidence that IL PV neurons support strategic avoidance behavior by suppressing freezing.
• Ho, Y. Y., Yang, Q., Boddu, P., Bulkin, D. A., & Warden, M. (2023). Ventromedial prefrontal parvalbumin neurons are necessary for initiating cued threat avoidance. bioRxiv.
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  The ventromedial prefrontal cortex (vmPFC) is essential for regulating the balance between reactive and adaptive response. Reactive, hard-wired behaviors – such as freezing or flight – are feasible in some situations, but in others contexts an acquired, adaptive action may be more effective. Although the vmPFC has been implicated in adaptive threat avoidance, the contribution of distinct vmPFC neural subtypes with differing molecular identities and wiring patterns is poorly understood. Here, we studied vmPFC parvalbumin (PV) interneurons in mice as they learned to cross a chamber in order to avoid an impending shock, a behavior that requires both learned, adaptive action and the suppression of cued freezing. We found that vmPFC PV neural activity increased upon movement to avoid the shock, when the competing freezing response was suppressed. However, neural activity did not change upon movement toward cued rewards or during general locomotion, conditions with no competing behavior. Optogenetic suppression of vmPFC PV neurons delayed the onset of avoidance behavior and increased the duration of freezing, but did not affect movement toward rewards or general locomotion. Thus, vmPFC PV neurons support flexible, adaptive behavior by suppressing the expression of prepotent behavioral reactions.
• Lundqvist, M., Brincat, S. L., Rose, J., Warden, M., Buschman, T. J., Miller, E. K., & Herman, P. (2023). Working memory control dynamics follow principles of spatial computing. Nature communications, 14(1), 1429.
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  Working memory (WM) allows us to remember and selectively control a limited set of items. Neural evidence suggests it is achieved by interactions between bursts of beta and gamma oscillations. However, it is not clear how oscillations, reflecting coherent activity of millions of neurons, can selectively control individual WM items. Here we propose the novel concept of spatial computing where beta and gamma interactions cause item-specific activity to flow spatially across the network during a task. This way, control-related information such as item order is stored in the spatial activity independent of the detailed recurrent connectivity supporting the item-specific activity itself. The spatial flow is in turn reflected in low-dimensional activity shared by many neurons. We verify these predictions by analyzing local field potentials and neuronal spiking. We hypothesize that spatial computing can facilitate generalization and zero-shot learning by utilizing spatial component as an additional information encoding dimension.
• Mikkelsen, C. A., Charczynski, S. J., Brincat, S. K., Warden, M., Miller, E. K., & Howard, M. W. (2023). Coding of time with non-linear mixed selectivity in prefrontal cortex ensembles. bioRxiv.
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  Previous work has identified stimulus specific time cells as a potential mechanism for working memory maintenance. It has been proposed that populations of stimulus specific sequences of cells could support memory for many items in a list over long periods of time. This would require information about one stimulus to persist after the presentation of subsequent stimuli. However, it is not known if sequences triggered by one stimulus persist past the presentation of additional stimuli. It is possible that each new stimulus terminates preceding sequences, making memory for multiple stimuli impossible. To investigate this question, we utilized a data set originally published by (Warden & Miller, 2010), studying the firing of monkey prefrontal neurons during short lists of stimuli. We were able to decode “what happened when” throughout the list, using linear discriminant analysis. Additionally, we were able to decode the first stimulus after the presentation of the second stimulus. Furthermore, we found that stimulus modulated sequences of cells, with discrete temporal fields, continue after the second item was presented. Much of the information about the previous item was carried by neurons that responded to conjunctions of stimuli and the timing of late-firing cells was synchronized to the firing of the second stimulus rather than the first. These properties falsify a simple linear model of sequential time cells. These results suggest that non-linear mixed selectivity extends to continuous variables such as time, but that in this experiment at least, only the timing of the most recent stimulus was explicitly maintained in ongoing firing.
• Miller, C. H., Hillock, M. F., Yang, J., Carlson-Clarke, B., Haxhillari, K., Lee, A. Y., Warden, M., & Sheehan, M. J. (2023). Dynamic changes to signal allocation rules in response to variable social environments in house mice. Communications biology, 6(1), 297.
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  Urine marking is central to mouse social behavior. Males use depletable and costly urine marks in intrasexual competition and mate attraction. We investigate how males alter signaling decisions across variable social landscapes using thermal imaging to capture spatiotemporal marking data. Thermal recording reveals fine-scale adjustments in urinary motor patterns in response to competition and social odors. Males demonstrate striking winner-loser effects in scent mark allocation effort and timing. Competitive experience primes temporal features of marking and modulates responses to scent familiarity. Males adjust signaling effort, mark latency, and marking rhythm, depending on the scent identities in the environment. Notably, recent contest outcome affects how males respond to familiar and unfamiliar urine. Winners increase marking effort toward unfamiliar relative to familiar male scents, whereas losers reduce marking effort to unfamiliar but increase to familiar rival scents. All males adjust their scent mark timing after a contest regardless of fight outcome, and deposit marks in more rapid bursts during marking bouts. In contrast to this dynamism, initial signal investment predicts aspects of scent marking days later, revealing the possibility of alternative marking strategies among competitive males. These data show that mice flexibly update their signaling decisions in response to changing social landscapes.
• Troconis, E. L., Seo, C., Guru, A., & Warden, M. (2023). Male ejaculation phasically activates dorsal raphe serotonin neurons in mating female mice. bioRxiv.
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  Sexual stimulation triggers changes in female physiology and behavior, including sexual satiety and preparing the uterus for pregnancy. Serotonin is an important regulator of reproductive physiology and sexual receptivity, but the relationship between sexual stimulation and serotonin neural activity in females is poorly understood. Here, we investigated dorsal raphe serotonin neural activity in females during sexual behavior. We found that serotonin neural activity in mating females peaked specifically upon male ejaculation, and remained elevated above baseline until disengagement. Artificial intravaginal mechanical stimulation was sufficient to elicit increased 5-HT neural activity but the delivery of ejaculatory fluids was not. Distal penis erectile enlargement ("penile cupping") at ejaculation and forceful expulsion of ejaculatory fluid each provided sufficient mechanical stimulation to elicit serotonin neuron activation. Our study identifies a female ejaculation-specific signal in a major neuromodulatory system and shows that intravaginal mechanosensory stimulation is necessary and sufficient to drive this signal.
• Troconis, E. L., Seo, C., Guru, A., & Warden, M. (2023). Serotonin neurons in mating female mice are activated by male ejaculation. Current biology, 33(22), 4926-4936.e4.
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  Sexual stimulation triggers changes in female physiology and behavior, including sexual satiety and preparing the uterus for pregnancy. Serotonin (5-HT) is an important regulator of reproductive physiology and sexual receptivity, but the relationship between sexual stimulation and 5-HT neural activity in females is poorly understood. Here, we investigated dorsal raphe 5-HT neural activity in female mice during sexual behavior. We found that 5-HT neural activity in mating females peaked specifically upon male ejaculation and remained elevated above baseline until disengagement. Artificial intravaginal mechanical stimulation was sufficient to elicit increased 5-HT neural activity but the delivery of ejaculatory fluids was not. Distal penis expansion ("penile cupping") at ejaculation and forceful expulsion of ejaculatory fluid each provided sufficient mechanical stimulation to elicit 5-HT neuron activation. Our study identifies a female ejaculation-specific signal in a major neuromodulatory system and shows that intravaginal mechanosensory stimulation is necessary and sufficient to drive this signal.
• Lundqvist, M., Rose, J., Brincat, S. L., Warden, M., Buschman, T. J., Herman, P., & Miller, E. K. (2022). Reduced variability of bursting activity during working memory. Scientific reports, 12(1), 15050.
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  Working memories have long been thought to be maintained by persistent spiking. However, mounting evidence from multiple-electrode recording (and single-trial analyses) shows that the underlying spiking is better characterized by intermittent bursts of activity. A counterargument suggested this intermittent activity is at odds with observations that spike-time variability reduces during task performance. However, this counterargument rests on assumptions, such as randomness in the timing of the bursts, which may not be correct. Thus, we analyzed spiking and LFPs from monkeys' prefrontal cortex (PFC) to determine if task-related reductions in variability can co-exist with intermittent spiking. We found that it does because both spiking and associated gamma bursts were task-modulated, not random. In fact, the task-related reduction in spike variability could largely be explained by a related reduction in gamma burst variability. Our results provide further support for the intermittent activity models of working memory as well as novel mechanistic insights into how spike variability is reduced during cognitive tasks.
• Miller, C. H., Reichard, T. M., Yang, J., Carlson-Clarke, B., Vogt, C. C., Warden, M., & Sheehan, M. J. (2022). Reproductive state switches the valence of male urinary pheromones in female mice. bioRxiv.
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  Internal states shape responses to sensory stimuli. Mammalian female reproductive states are understudied considering they are one of the most regular state changes in the animal kingdom. Here we examine female house mouse preferences toward male odors across the reproductive states of estrus and late-stage pregnancy. In house mice, urine scent marks are salient social odors that convey information about the sex and identity of individuals by major urinary proteins (MUPs). Males secrete a sex-specific pheromonal protein called darcin (MUP20). Additionally, genetically diverse mice secrete unique combinations of MUPs used in individual recognition. Prior work has revealed that male odors are powerful social stimuli for female mice, yet we have a limited understanding of how the valence of such odors change across reproductive states. We discovered a valence shift among estrus and pregnant females toward novel male urine, in which estrus females exhibit preference and pregnant females show strong avoidance. This valence switch also occurs toward darcin alone, providing further support for darcin as a strong sexual signal. However, when presented with familiar male urine, the approach-avoidance response disappears, even when additional darcin is added. In contrast, when an existing identity protein (MUP11) is added to familiar male urine the approach-avoidance response is recovered. This indicates that darcin in the absence of other identity information denotes a novel male and that familiar identity information present in male urine is sufficient to modify responses to darcin. Our findings suggest that the sex and identity information encoded by MUPs are likely processed via distinct, and potentially opposing pathways, that modulate responses toward complex social odor blends. Furthermore, we identify a state-modulated shift in decision-making toward social odors and propose a neural circuit model for this flow of information. These data underscore the importance of physiological state and signal context for interpreting the meaning and importance of social odors.
• Post, R. J., Bulkin, D. A., Ebitz, R. B., Lee, V., Han, K., & Warden, M. (2022). Tonic activity in lateral habenula neurons acts as a neutral valence brake on reward-seeking behavior. Current biology, 32(20), 4325-4336.e5.
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  Survival requires both the ability to persistently pursue goals and the ability to determine when it is time to stop, an adaptive balance of perseverance and disengagement. Neural activity in the lateral habenula (LHb) has been linked to negative valence, but its role in regulating the balance between engaged reward seeking and disengaged behavioral states remains unclear. Here, we show that LHb neural activity is tonically elevated during minutes-long periods of disengagement from reward-seeking behavior, both when due to repeated reward omission (negative valence) and when sufficient reward has been consumed (positive valence). Furthermore, we show that LHb inhibition extends ongoing reward-seeking behavioral states but does not prompt task re-engagement. We find no evidence for similar tonic activity changes in ventral tegmental area dopamine neurons. Our findings support a framework in which tonic activity in LHb neurons suppresses engagement in reward-seeking behavior in response to both negatively and positively valenced factors.
• Guru, A., Seo, C., Post, R. J., Kullakanda, D. S., Schaffer, J. A., & Warden, M. (2020). Ramping activity in midbrain dopamine neurons signifies the use of a cognitive map. bioRxiv.
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  Journeys to novel and familiar destinations employ different navigational strategies. The first drive to a new restaurant relies on map-based planning, but after repeated trips the drive is automatic and guided by local environmental cues1,2. Ventral striatal dopamine rises during navigation toward goals and reflects the spatial proximity and value of goals3, but the impact of experience, the neural mechanisms, and the functional significance of dopamine ramps are unknown4,5. Here, we used fiber photometry6–8 to record the evolution of activity in midbrain dopamine neurons as mice learned a variety of reward-seeking tasks, starting recordings before training had commenced and continuing daily for weeks. When mice navigated through space toward a goal, robust ramping activity in dopamine neurons appeared immediately – after the first rewarded trial on the first training day in completely naïve animals. In this task spatial cues were available to guide behavior, and although ramps were strong at first, they gradually faded away as training progressed. If instead mice learned to run a fixed distance on a stationary wheel for reward, a task that required an internal model of progress toward the goal, strong dopamine ramps persisted indefinitely. In a passive task in which a visible cue and reward moved together toward the mouse, ramps appeared and then faded over several days, but in an otherwise identical task with a stationary cue and reward ramps never appeared. Our findings provide strong evidence that ramping activity in midbrain dopamine neurons reflects the use of a cognitive map9,10 – an internal model of the distance already covered and the remaining distance until the goal is reached. We hypothesize that dopamine ramps may be used to reinforce locations on the way to newly-discovered rewards in order to build a graded ventral striatal value landscape for guiding routine spatial behavior.
• Wang, T., Wu, C., Ouzounov, D. G., Gu, W., Xia, F., Kim, M., Yang, X., Warden, M., & Xu, C. (2020). Quantitative analysis of 1300-nm three-photon calcium imaging in the mouse brain. eLife, 9.
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  1300 nm three-photon calcium imaging has emerged as a useful technique to allow calcium imaging in deep brain regions. Application to large-scale neural activity imaging entails a careful balance between recording fidelity and perturbation to the sample. We calculated and experimentally verified the excitation pulse energy to achieve required for the detection of calcium transients in GCaMP6s-expressing neurons for 920 nm two-photon and 1320 nm three-photon excitation. By considering the combined effects of in-focus signal attenuation and out-of-focus background generation, we quantified the cross-over depth beyond which three-photon microscopy outpeforms two-photon microscopy in recording fidelity. Brain tissue heating by continuous three-photon imaging was simulated with Monte Carlo method and experimentally validated with immunohistochemistry. Increased immunoreactivity was observed with 150 mW excitation power at 1 and 1.2 mm imaging depths. Our analysis presents a translatable model for the optimization of three-photon calcium imaging based on experimentally tractable parameters.
• Seo, C., Guru, A., Jin, M., Ito, B., Sleezer, B. J., Ho, Y. Y., Wang, E., Boada, C., Krupa, N. A., Kullakanda, D. S., Shen, C. X., & Warden, M. (2019). Intense threat switches dorsal raphe serotonin neurons to a paradoxical operational mode. Science, 363(6426), 538-542.
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  Survival depends on the selection of behaviors adaptive for the current environment. For example, a mouse should run from a rapidly looming hawk but should freeze if the hawk is coasting across the sky. Although serotonin has been implicated in adaptive behavior, environmental regulation of its functional role remains poorly understood. In mice, we found that stimulation of dorsal raphe serotonin neurons suppressed movement in low- and moderate-threat environments but induced escape behavior in high-threat environments, and that movement-related dorsal raphe serotonin neural dynamics inverted in high-threat environments. Stimulation of dorsal raphe γ-aminobutyric acid (GABA) neurons promoted movement in negative but not positive environments, and movement-related GABA neural dynamics inverted between positive and negative environments. Thus, dorsal raphe circuits switch between distinct operational modes to promote environment-specific adaptive behaviors.
• Lundqvist, M., Herman, P., Warden, M., Brincat, S. L., & Miller, E. K. (2018). Gamma and beta bursts during working memory readout suggest roles in its volitional control. Nature communications, 9(1), 394.
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  Working memory (WM) activity is not as stationary or sustained as previously thought. There are brief bursts of gamma (~50-120 Hz) and beta (~20-35 Hz) oscillations, the former linked to stimulus information in spiking. We examined these dynamics in relation to readout and control mechanisms of WM. Monkeys held sequences of two objects in WM to match to subsequent sequences. Changes in beta and gamma bursting suggested their distinct roles. In anticipation of having to use an object for the match decision, there was an increase in gamma and spiking information about that object and reduced beta bursting. This readout signal was only seen before relevant test objects, and was related to premotor activity. When the objects were no longer needed, beta increased and gamma decreased together with object spiking information. Deviations from these dynamics predicted behavioral errors. Thus, beta could regulate gamma and the information in WM.
• Post, R. J., & Warden, M. (2018). Melancholy, anhedonia, apathy: the search for separable behaviors and neural circuits in depression. Current opinion in neurobiology, 49, 192-200.
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  Major depressive disorder can manifest as different combinations of symptoms, ranging from a profound and incapacitating sadness, to a loss of interest in daily life, to an inability to engage in effortful, goal-directed behavior. Recent research has focused on defining the neural circuits that mediate separable features of depression in patients and preclinical animal models, and connections between frontal cortex and brainstem neuromodulators have emerged as candidate targets. The development of methods permitting recording and manipulation of neural circuits defined by connectivity has enabled the investigation of prefrontal-neuromodulatory circuit dynamics in animal models of depression with exquisite precision, a systems-level approach that has brought new insights by integrating these fields of depression research.
• Wang, M., Wang, T., Wu, C., Li, B., Ouzounov, D. G., Sinefeld, D., Guru, A., Nam, H., Capecchi, M. R., Warden, M., & Xu, C. (2018). In vivo three-photon imaging of deep cerebellum. Proc. SPIE 10498, Multiphoton Microscopy in the Biomedical Sciences XVIII.
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  We demonstrate three-photon microscopy (3PM) of mouse cerebellum at 1 mm depth by imaging both blood vessels and neurons. We compared 3PM and 2PM in the mouse cerebellum for imaging green (using excitation sources at 1300 nm and 920 nm, respectively) and red fluorescence (using excitation sources at 1680 nm and 1064 nm, respectively). 3PM enabled deeper imaging than 2PM because the use of longer excitation wavelength reduces the scattering in biological tissue and the higher order nonlinear excitation provides better 3D localization. To illustrate these two advantages quantitatively, we measured the signal decay as well as the signal-to-background ratio (SBR) as a function of depth. We performed 2-photon imaging from the brain surface all the way down to the area where the SBR reaches ~ 1, while at the same depth, 3PM still has SBR above 30. The segmented decay curve shows that the mouse cerebellum has different effective attenuation lengths at different depths, indicating heterogeneous tissue property for this brain region. We compared the third harmonic generation (THG) signal, which is used to visualize myelinated fibers, with the decay curve. We found that the regions with shorter effective attenuation lengths correspond to the regions with more fibers. Our results indicate that the widespread, non-uniformly distributed myelinated fibers adds heterogeneity to mouse cerebellum, which poses additional challenges in deep imaging of this brain region.
• Lindsay, G. W., Rigotti, M., Warden, M., Miller, E. K., & Fusi, S. (2017). Hebbian Learning in a Random Network Captures Selectivity Properties of the Prefrontal Cortex. The Journal of Neuroscience, 37(45), 11021-11036.
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  Complex cognitive behaviors, such as context-switching and rule-following, are thought to be supported by the prefrontal cortex (PFC). Neural activity in the PFC must thus be specialized to specific tasks while retaining flexibility. Nonlinear "mixed" selectivity is an important neurophysiological trait for enabling complex and context-dependent behaviors. Here we investigate (1) the extent to which the PFC exhibits computationally relevant properties, such as mixed selectivity, and (2) how such properties could arise via circuit mechanisms. We show that PFC cells recorded from male and female rhesus macaques during a complex task show a moderate level of specialization and structure that is not replicated by a model wherein cells receive random feedforward inputs. While random connectivity can be effective at generating mixed selectivity, the data show significantly more mixed selectivity than predicted by a model with otherwise matched parameters. A simple Hebbian learning rule applied to the random connectivity, however, increases mixed selectivity and enables the model to match the data more accurately. To explain how learning achieves this, we provide analysis along with a clear geometric interpretation of the impact of learning on selectivity. After learning, the model also matches the data on measures of noise, response density, clustering, and the distribution of selectivities. Of two styles of Hebbian learning tested, the simpler and more biologically plausible option better matches the data. These modeling results provide clues about how neural properties important for cognition can arise in a circuit and make clear experimental predictions regarding how various measures of selectivity would evolve during animal training. The prefrontal cortex is a brain region believed to support the ability of animals to engage in complex behavior. How neurons in this area respond to stimuli-and in particular, to combinations of stimuli ("mixed selectivity")-is a topic of interest. Even though models with random feedforward connectivity are capable of creating computationally relevant mixed selectivity, such a model does not match the levels of mixed selectivity seen in the data analyzed in this study. Adding simple Hebbian learning to the model increases mixed selectivity to the correct level and makes the model match the data on several other relevant measures. This study thus offers predictions on how mixed selectivity and other properties evolve with training.
• Ferenczi, E. A., Zalocusky, K. A., Liston, C., Grosenick, L., Warden, M., Amatya, D., Katovich, K., Mehta, H., Patenaude, B., Ramakrishnan, C., Kalanithi, P., Etkin, A., Knutson, B., Glover, G. H., & Deisseroth, K. (2016). Prefrontal cortical regulation of brainwide circuit dynamics and reward-related behavior. Science, 351(6268), aac9698.
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  Motivation for reward drives adaptive behaviors, whereas impairment of reward perception and experience (anhedonia) can contribute to psychiatric diseases, including depression and schizophrenia. We sought to test the hypothesis that the medial prefrontal cortex (mPFC) controls interactions among specific subcortical regions that govern hedonic responses. By using optogenetic functional magnetic resonance imaging to locally manipulate but globally visualize neural activity in rats, we found that dopamine neuron stimulation drives striatal activity, whereas locally increased mPFC excitability reduces this striatal response and inhibits the behavioral drive for dopaminergic stimulation. This chronic mPFC overactivity also stably suppresses natural reward-motivated behaviors and induces specific new brainwide functional interactions, which predict the degree of anhedonia in individuals. These findings describe a mechanism by which mPFC modulates expression of reward-seeking behavior, by regulating the dynamical interactions between specific distant subcortical regions.
• Guru, A., Post, R. J., Ho, Y. Y., & Warden, M. (2015). Making Sense of Optogenetics. The international journal of neuropsychopharmacology, 18(11), pyv079.
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  This review, one of a series of articles, tries to make sense of optogenetics, a recently developed technology that can be used to control the activity of genetically-defined neurons with light. Cells are first genetically engineered to express a light-sensitive opsin, which is typically an ion channel, pump, or G protein-coupled receptor. When engineered cells are then illuminated with light of the correct frequency, opsin-bound retinal undergoes a conformational change that leads to channel opening or pump activation, cell depolarization or hyperpolarization, and neural activation or silencing. Since the advent of optogenetics, many different opsin variants have been discovered or engineered, and it is now possible to stimulate or inhibit neuronal activity or intracellular signaling pathways on fast or slow timescales with a variety of different wavelengths of light. Optogenetics has been successfully employed to enhance our understanding of the neural circuit dysfunction underlying mood disorders, addiction, and Parkinson's disease, and has enabled us to achieve a better understanding of the neural circuits mediating normal behavior. It has revolutionized the field of neuroscience, and has enabled a new generation of experiments that probe the causal roles of specific neural circuit components.
• Sidor, M. M., Davidson, T. J., Tye, K. M., Warden, M., Diesseroth, K., & McClung, C. A. (2015). In vivo optogenetic stimulation of the rodent central nervous system. Journal of visualized experiments, 51483.
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  The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.
• Sidor, M. M., Spencer, S. M., Dzirasa, K., Parekh, P. K., Tye, K. M., Warden, M., Arey, R. N., Enwright, J. F., Jacobsen, J. P., Kumar, S., Remillard, E. M., Caron, M. G., Deisseroth, K., & McClung, C. A. (2015). Daytime spikes in dopaminergic activity drive rapid mood-cycling in mice. Molecular Psychiatry.
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  Disruptions in circadian rhythms and dopaminergic activity are involved in the pathophysiology of bipolar disorder, though their interaction remains unclear. Moreover, a lack of animal models that display spontaneous cycling between mood states has hindered our mechanistic understanding of mood switching. Here, we find that mice with a mutation in the circadian Clock gene (ClockΔ19) exhibit rapid mood-cycling, with a profound manic-like phenotype emerging during the day following a period of euthymia at night. Mood-cycling coincides with abnormal daytime spikes in ventral tegmental area (VTA) dopaminergic activity, tyrosine hydroxylase (TH) levels and dopamine synthesis. To determine the significance of daytime increases in VTA dopamine activity to manic behaviors, we developed a novel optogenetic stimulation paradigm that produces a sustained increase in dopamine neuronal activity and find that this induces a manic-like behavioral state. Time-dependent dampening of TH activity during the day reverses manic-related behaviors in ClockΔ19 mice. Finally, we show that CLOCK acts as a negative regulator of TH transcription, revealing a novel molecular mechanism underlying cyclic changes in mood-related behavior. Taken together, these studies have identified a mechanistic connection between circadian gene disruption and the precipitation of manic episodes in bipolar disorder.
• Lammel, S., Tye, K. M., & Warden, M. (2014). Progress in understanding mood disorders: optogenetic dissection of neural circuits. Genes, Brain and Behavior.
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  Major depression is characterized by a cluster of symptoms that includes hopelessness, low mood, feelings of worthlessness and inability to experience pleasure. The lifetime prevalence of major depression approaches 20%, yet current treatments are often inadequate both because of associated side effects and because they are ineffective for many people. In basic research, animal models are often used to study depression. Typically, experimental animals are exposed to acute or chronic stress to generate a variety of depression-like symptoms. Despite its clinical importance, very little is known about the cellular and neural circuits that mediate these symptoms. Recent advances in circuit-targeted approaches have provided new opportunities to study the neuropathology of mood disorders such as depression and anxiety. We review recent progress and highlight some studies that have begun tracing a functional neuronal circuit diagram that may prove essential in establishing novel treatment strategies in mood disorders. First, we shed light on the complexity of mesocorticolimbic dopamine (DA) responses to stress by discussing two recent studies reporting that optogenetic activation of midbrain DA neurons can induce or reverse depression-related behaviors. Second, we describe the role of the lateral habenula circuitry in the pathophysiology of depression. Finally, we discuss how the prefrontal cortex controls limbic and neuromodulatory circuits in mood disorders.
• Warden, M., Cardin, J. A., & Deisseroth, K. (2014). Optical neural interfaces. Annual review of biomedical engineering, 16, 103-29.
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  Genetically encoded optical actuators and indicators have changed the landscape of neuroscience, enabling targetable control and readout of specific components of intact neural circuits in behaving animals. Here, we review the development of optical neural interfaces, focusing on hardware designed for optical control of neural activity, integrated optical control and electrical readout, and optical readout of population and single-cell neural activity in freely moving mammals.
• Kim, S. Y., Adhikari, A., Lee, S. Y., Marshel, J. H., Kim, C. K., Mallory, C. S., Lo, M., Pak, S., Mattis, J., Lim, B. K., Malenka, R. C., Warden, M., Neve, R., Tye, K. M., & Deisseroth, K. (2013). Diverging neural pathways assemble a behavioural state from separable features in anxiety. Nature, 496(7444), 219-23.
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  Behavioural states in mammals, such as the anxious state, are characterized by several features that are coordinately regulated by diverse nervous system outputs, ranging from behavioural choice patterns to changes in physiology (in anxiety, exemplified respectively by risk-avoidance and respiratory rate alterations). Here we investigate if and how defined neural projections arising from a single coordinating brain region in mice could mediate diverse features of anxiety. Integrating behavioural assays, in vivo and in vitro electrophysiology, respiratory physiology and optogenetics, we identify a surprising new role for the bed nucleus of the stria terminalis (BNST) in the coordinated modulation of diverse anxiety features. First, two BNST subregions were unexpectedly found to exert opposite effects on the anxious state: oval BNST activity promoted several independent anxious state features, whereas anterodorsal BNST-associated activity exerted anxiolytic influence for the same features. Notably, we found that three distinct anterodorsal BNST efferent projections-to the lateral hypothalamus, parabrachial nucleus and ventral tegmental area-each implemented an independent feature of anxiolysis: reduced risk-avoidance, reduced respiratory rate, and increased positive valence, respectively. Furthermore, selective inhibition of corresponding circuit elements in freely moving mice showed opposing behavioural effects compared with excitation, and in vivo recordings during free behaviour showed native spiking patterns in anterodorsal BNST neurons that differentiated safe and anxiogenic environments. These results demonstrate that distinct BNST subregions exert opposite effects in modulating anxiety, establish separable anxiolytic roles for different anterodorsal BNST projections, and illustrate circuit mechanisms underlying selection of features for the assembly of the anxious state.
• Rigotti, M., Barak, O., Warden, M., Wang, X. J., Daw, N. D., Miller, E. K., & Fusi, S. (2013). The importance of mixed selectivity in complex cognitive tasks. Nature, 497(7451), 585-90.
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  Single-neuron activity in the prefrontal cortex (PFC) is tuned to mixtures of multiple task-related aspects. Such mixed selectivity is highly heterogeneous, seemingly disordered and therefore difficult to interpret. We analysed the neural activity recorded in monkeys during an object sequence memory task to identify a role of mixed selectivity in subserving the cognitive functions ascribed to the PFC. We show that mixed selectivity neurons encode distributed information about all task-relevant aspects. Each aspect can be decoded from the population of neurons even when single-cell selectivity to that aspect is eliminated. Moreover, mixed selectivity offers a significant computational advantage over specialized responses in terms of the repertoire of input-output functions implementable by readout neurons. This advantage originates from the highly diverse nonlinear selectivity to mixtures of task-relevant variables, a signature of high-dimensional neural representations. Crucially, this dimensionality is predictive of animal behaviour as it collapses in error trials. Our findings recommend a shift of focus for future studies from neurons that have easily interpretable response tuning to the widely observed, but rarely analysed, mixed selectivity neurons.
• Tye, K. M., Mirzabekov, J. J., Warden, M., Ferenczi, E. A., Tsai, H. C., Finkelstein, J., Kim, S. Y., Adhikari, A., Thompson, K. R., Andalman, A. S., Gunaydin, L. A., Witten, I. B., & Deisseroth, K. (2013). Dopamine neurons modulate neural encoding and expression of depression-related behaviour. Nature, 493(7433), 537-541.
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  Major depression is characterized by diverse debilitating symptoms that include hopelessness and anhedonia. Dopamine neurons involved in reward and motivation are among many neural populations that have been hypothesized to be relevant, and certain antidepressant treatments, including medications and brain stimulation therapies, can influence the complex dopamine system. Until now it has not been possible to test this hypothesis directly, even in animal models, as existing therapeutic interventions are unable to specifically target dopamine neurons. Here we investigated directly the causal contributions of defined dopamine neurons to multidimensional depression-like phenotypes induced by chronic mild stress, by integrating behavioural, pharmacological, optogenetic and electrophysiological methods in freely moving rodents. We found that bidirectional control (inhibition or excitation) of specified midbrain dopamine neurons immediately and bidirectionally modulates (induces or relieves) multiple independent depression symptoms caused by chronic stress. By probing the circuit implementation of these effects, we observed that optogenetic recruitment of these dopamine neurons potently alters the neural encoding of depression-related behaviours in the downstream nucleus accumbens of freely moving rodents, suggesting that processes affecting depression symptoms may involve alterations in the neural encoding of action in limbic circuitry.
• Warden, M., Selimbeyoglu, A., Mirzabekov, J. J., Lo, M., Thompson, K. R., Kim, S. Y., Adhikari, A., Tye, K. M., Frank, L. M., & Deisseroth, K. (2012). A prefrontal cortex-brainstem neuronal projection that controls response to behavioural challenge. Nature, 492(7429), 428-32.
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  The prefrontal cortex (PFC) is thought to participate in high-level control of the generation of behaviours (including the decision to execute actions); indeed, imaging and lesion studies in human beings have revealed that PFC dysfunction can lead to either impulsive states with increased tendency to initiate action, or to amotivational states characterized by symptoms such as reduced activity, hopelessness and depressed mood. Considering the opposite valence of these two phenotypes as well as the broad complexity of other tasks attributed to PFC, we sought to elucidate the PFC circuitry that favours effortful behavioural responses to challenging situations. Here we develop and use a quantitative method for the continuous assessment and control of active response to a behavioural challenge, synchronized with single-unit electrophysiology and optogenetics in freely moving rats. In recording from the medial PFC (mPFC), we observed that many neurons were not simply movement-related in their spike-firing patterns but instead were selectively modulated from moment to moment, according to the animal's decision to act in a challenging situation. Surprisingly, we next found that direct activation of principal neurons in the mPFC had no detectable causal effect on this behaviour. We tested whether this behaviour could be causally mediated by only a subclass of mPFC cells defined by specific downstream wiring. Indeed, by leveraging optogenetic projection-targeting to control cells with specific efferent wiring patterns, we found that selective activation of those mPFC cells projecting to the brainstem dorsal raphe nucleus (DRN), a serotonergic nucleus implicated in major depressive disorder, induced a profound, rapid and reversible effect on selection of the active behavioural state. These results may be of importance in understanding the neural circuitry underlying normal and pathological patterns of action selection and motivation in behaviour.
• Anikeeva, P., Andalman, A. S., Witten, I., Warden, M., Goshen, I., Grosenick, L., Gunaydin, L. A., Frank, L. M., & Deisseroth, K. (2011). Optetrode: a multichannel readout for optogenetic control in freely moving mice. Nature neuroscience, 15(1), 163-70.
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  Recent advances in optogenetics have improved the precision with which defined circuit elements can be controlled optically in freely moving mammals; in particular, recombinase-dependent opsin viruses, used with a growing pool of transgenic mice expressing recombinases, allow manipulation of specific cell types. However, although optogenetic control has allowed neural circuits to be manipulated in increasingly powerful ways, combining optogenetic stimulation with simultaneous multichannel electrophysiological readout of isolated units in freely moving mice remains a challenge. We designed and validated the optetrode, a device that allows for colocalized multi-tetrode electrophysiological recording and optical stimulation in freely moving mice. Optetrode manufacture employs a unique optical fiber-centric coaxial design approach that yields a lightweight (2 g), compact and robust device that is suitable for behaving mice. This low-cost device is easy to construct (2.5 h to build without specialized equipment). We found that the drive design produced stable high-quality recordings and continued to do so for at least 6 weeks following implantation. We validated the optetrode by quantifying, for the first time, the response of cells in the medial prefrontal cortex to local optical excitation and inhibition, probing multiple different genetically defined classes of cells in the mouse during open field exploration.
• Warden, M., & Miller, E. K. (2010). Task-dependent changes in short-term memory in the prefrontal cortex. The Journal of neuroscience, 30(47), 15801-10.
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  The prefrontal cortex (PFC) is important for flexible, context-dependent behavioral control. It also plays a critical role in short-term memory maintenance. Though many studies have investigated these functions independently, it is unclear how these two very different processes are realized by a single brain area. To address this, we trained two monkeys on two variants of an object sequence memory task. These tasks had the same memory requirements but differed in how information was read out and used. For the "recognition" task, the monkeys had to remember two sequentially presented objects and then release a bar when a matching sequence was recognized. For the "recall" task, the monkeys had to remember the same sequence of objects but were instead required to recall the sequence and reproduce it with saccadic eye movements when presented with an array of objects. After training, we recorded the activity of PFC neurons during task performance. We recorded 222 neurons during the recognition task, 177 neurons during the recall task, and 248 neurons during the switching task (interleaved blocks of recognition and recall). Task context had a profound influence on neural selectivity for objects. During the recall task, the first object was encoded more strongly than the second object, while during the recognition task, the second object was encoded more strongly. In addition, most of the neurons encoded both the task and the objects, evidence for a single population responsible for these two critical prefrontal functions.
• Siegel, M., Warden, M., & Miller, E. K. (2009). Phase-dependent neuronal coding of objects in short-term memory. Proceedings of the National Academy of Sciences of the United States of America, 106(50), 21341-6.
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  The ability to hold multiple objects in memory is fundamental to intelligent behavior, but its neural basis remains poorly understood. It has been suggested that multiple items may be held in memory by oscillatory activity across neuronal populations, but yet there is little direct evidence. Here, we show that neuronal information about two objects held in short-term memory is enhanced at specific phases of underlying oscillatory population activity. We recorded neuronal activity from the prefrontal cortices of monkeys remembering two visual objects over a brief interval. We found that during this memory interval prefrontal population activity was rhythmically synchronized at frequencies around 32 and 3 Hz and that spikes carried the most information about the memorized objects at specific phases. Further, according to their order of presentation, optimal encoding of the first presented object was significantly earlier in the 32 Hz cycle than that for the second object. Our results suggest that oscillatory neuronal synchronization mediates a phase-dependent coding of memorized objects in the prefrontal cortex. Encoding at distinct phases may play a role for disambiguating information about multiple objects in short-term memory.
• Warden, M., & Miller, E. K. (2007). The representation of multiple objects in prefrontal neuronal delay activity. Cerebral cortex (New York, N.Y. : 1991), 17 Suppl 1, i41-50.
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  The ability to retain multiple items in short-term memory is fundamental for intelligent behavior, yet little is known about its neural basis. To explore the mechanisms underlying this ability, we trained 2 monkeys to remember a sequence of 2 objects across a short delay. We then recorded the activity of neurons from the lateral prefrontal cortex during task performance and found that most neurons had activity that depended on the identity of both objects while a minority reflected just one object. Further, the activity driven by a particular combination of objects was not a simple addition of the activity elicited by individual objects. Instead, the representation of the first object was altered by the addition of the second object to memory, and the form of this change was not systematically predictable. These results indicate that multiple objects are not stored in separate groups of prefrontal neurons. Rather, they are represented by a single population of neurons in a complex fashion. We also found that the strength of the memory trace associated with each object decayed over time, leading to a relatively stronger representation of more recently seen objects. This is a potential mechanism for representing the temporal order of objects.