Michael T. Shipley - US grants

Extensive anatomical pilot studies in 75 mice suggest a new concept of organizing sensory-limbic interrelations in the forebrain. Small HRP injections along the insulotemporal cortex (ITC) revealed four "modules": Each module receives direct, modality specific sensory inputs from thalamus and cortex and projects heavily to discrete parts of the amygdala and hippocampal region. Each module's sensory affiliation is with either the taste/visceral, somesthetic, auditory or visual system. Each module also gets inputs back from the amygdala and hippocampal region and sends outputs back to its thalamic and cortical affiliates. Thus there are substantial, organized and remarkably direct circuits over which sensory and limbic systems may functionally interact. But important questions about ITC modules' topographical and functional organization cannot be resolved in the small mouse brain. Hence, we will investigate these modules with pathway tracing methods and single unit recording in the alert, restrained rabbit--a preparation that eliminates anesthetic effects on neural activity. Anatomy: HRP and isotope injections will be used to delineate the afferent and efferent connections of all four modules. The larger rabbit brain will obviate the label spread-problem that hinders the analysis of some short ITC connections and let us search for topography and laminar organization in each module's sensory-limbic circuits. Physiology: 1. The functional organization of each ITC module will be studied by characterizing and mapping the distribution of single unit's responses to specific, natural stimuli. At the end of each recording experiment the physiologically characterized module's connections will be determined with anatomical methods. 2. Activity of units in the amygdala to stimuli found appropriate for a module will be studied before and after ITC lesions to evaluate ITC-mediation of sensory control over the amygdala. Our experiments should provide a cellular neurobiological basis for understanding how forebrain sensory and limbic systems functionally interact and cooperate to modulate hypothalamic function.

Olfactory neurons and taste cells are continuously replaced throughout life. Chemosensory systems, therefore, have evolved unique cellular and molecular strategies to maintain their functional integrity. The capacity to reinnervate the adult mammalian CNS is unique to olfactory neurons. Olfactory neurons induce the development and maintain the expression of transmitter phenotypes in the olfactory bulb. The initial induction and continued development of the bulb may be trophically dependent on neurons and other cells of the olfactory placode. Gustatory nerves induce and trophically maintain taste cells. The seven projects of this program focus on these unique developmental, regenerative and trophic properties of chemosensory cells. Novel hypotheses of the specific molecular and cell-cell interactions that regulate the expression of these unique properties will be tested. The Program has matured from its phenomenological, technique-driven beginnings to a hypothesis-based experiment-driven stage. Inter-project collaborations are far more extensive than four years ago. Antibodies and gene probes will be used to pinpoint hypothesized sequences of cell-cell, and cell-molecular interactions which underlie similarities and differences among development, turnover and regeneration of olfactory and taste cells. Neural transplants and tissue culture systems will pit olfactory cells and molecules against non-olfactory cells and molecules to determine whether the factors that specify pathway development, or permit reinnervation are unique to chemosensory cells or are properties expressed by other cells at certain stages of their development. Cell and ciliary patch and intracellular recording techniques will define the development of channels and membrane coupling events that are associated with maturation of transduction processes in cultured and dissociated cells. Immunoelectron microscope analysis of recently characterized transduction- coupling molecules will be coordinated with the membrane biophysical studies. Newly discovered genetic mutants, neural transplantation and tissue culture methods will be used to determine the specificity and molecular bases of olfactory nerve trophic actions on the development of the bulb and its transmitter phenotypes. Similar approaches will be used to isolate the role of bulb cells in the functional maturation of olfactory neurons. Understanding the developmental and regenerative strategies evolved to cope with receptor turnover, is one of the fundamental goals of chemosensory neuroscience. Achievement of this goal may provide new direction to the prevention and treatment of developmental and degenerative disorders in other parts of the CNS.

Olfactory neurons have the unique ability to reinnervate the adult CNS in mammals. The replacement of olfactory neurons throughout life suggests that mechanisms used in development, turnover and post-lesion reinnervation are fundamentally similar. However, while neurogenesis and axon outgrowth may be similar in these three processes, the target-environment presented by the olfactory bulb in development, turnover and regeneration are profoundly different. Preliminary results from this laboratory, taken with other new findings, suggest that glial cells are fundamental to the composition of the target environment and play a critical role in the development of neural connections and the formation of local circuits. By contrast, adult glial cells react to injury/deafferentation and contribute to the hostile environment that prevents reinnervation. Since olfactory axons do successfully reinnervate the bulb, bulb astrocytes, olfactory neurons or both may have unique properties. LM-EM immunohistochemical studies will characterize the properties of olfactory neurons and glia and their interactions during development, following deafferentiation and during reinnervation. Neural transplantation experiments will be used to contrast neuron-glial interactions during development and reinnervation, test the ability of bulb astrocytes to support reinnervation by ectopic neurons and determine whether olfactory neurons can grow through or modify glial scars in the spinal cord. The goal of this research is to test novel hypotheses of the developmental and regenerative capacities of olfactory neurons and their interactions with olfactory glial cells. Understanding how olfactory neurons innervate the adult CNS may suggest new approaches for treating developmental disorders and provide new clues for promoting regenerative repair of the damaged brain.

Olfactory neurons and taste cells are continuously replaced throughout life. Chemosensory systems, therefore, have evolved unique cellular and molecular strategies to maintain their functional integrity. The capacity to reinnervate the adult mammalian CNS is unique to olfactory neurons. Olfactory neurons induce the development and maintain the expression of transmitter phenotypes in the olfactory bulb. The initial induction and continued development of the bulb may be trophically dependent on neurons and other cells of the olfactory placode. Gustatory nerves induce and trophically maintain taste cells. The seven projects of this program focus on these unique developmental, regenerative and trophic properties of chemosensory cells. Novel hypotheses of the specific molecular and cell-cell interactions that regulate the expression of these unique properties will be tested. The Program has matured from its phenomenological, technique-driven beginnings to a hypothesis-based experiment-driven stage. Inter-project collaborations are far more extensive than four years ago. Antibodies and gene probes will be used to pinpoint hypothesized sequences of cell-cell, and cell-molecular interactions which underlie similarities and differences among development, turnover and regeneration of olfactory and taste cells. Neural transplants and tissue culture systems will pit olfactory cells and molecules against non-olfactory cells and molecules to determine whether the factors that specify pathway development, or permit reinnervation are unique to chemosensory cells or are properties expressed by other cells at certain stages of their development. Cell and ciliary patch and intracellular recording techniques will define the development of channels and membrane coupling events that are associated with maturation of transduction processes in cultured and dissociated cells. Immunoelectron microscope analysis of recently characterized transduction- coupling molecules will be coordinated with the membrane biophysical studies. Newly discovered genetic mutants, neural transplantation and tissue culture methods will be used to determine the specificity and molecular bases of olfactory nerve trophic actions on the development of the bulb and its transmitter phenotypes. Similar approaches will be used to isolate the role of bulb cells in the functional maturation of olfactory neurons. Understanding the developmental and regenerative strategies evolved to cope with receptor turnover, is one of the fundamental goals of chemosensory neuroscience. Achievement of this goal may provide new direction to the prevention and treatment of developmental and degenerative disorders in other parts of the CNS.

The long-range goal of this program is to understand the cellular and molecular bases for the unique trophic and regenerative capacities of chemosensory cells. Key to this is learning how chemosensory systems initially develop, as a wealth of evidence suggests that their trophic and regenerative capacities are due to the retention or re-expression of developmental mechanisms. The continued expression of developmental mechanisms in the adult must require additional, novel mechanisms because critical features of the adult system differ profoundly from that of the embryo. How do chemosensory systems deal with these differences? Knowledge of the strategies used may help us understand how chemosensory systems maintain functional constancy despite turnover and replacement of their transduction cells. The projects of this Program address these kinds of issues. The research centers on several fundamental problem areas. Olfactory transduction has begun to yield its molecular and membrane secrets. Studies in this program will determine how putative olfactory receptor gene families become organized into topographically distinct zones in the epithelium. regulatory sites conferring tissue and zonal specificity will be identified. Both olfactory and taste axons exhibit directional and target specific growth. Experiments will attempt to identify the cellular/molecular cues signals that regulate these specific growth patterns. Olfactory axons stop growing when they reach the olfactory bulb and then begin to express new families of functional molecules. The factors that regulate these target induced changes will be identified. Recent evidence indicates that the turnover and replacement of olfactory neurons is highly regulated. Findings in the last funding period suggest that speCific trophic factors regulate this process. Experiments will identify the transduction pathway of trophic factor actions, characterize the regulation of these factors and test their effectiveness in stimulating the proliferation of olfactory neurons. The mechanisms that regulate the production and orderly differentiation of ORNs have been difficult to identify and experimentally manipulate. The development, in the last funding period, of culture conditions that support the genesis and differentiation of ORNs opens the door to experiments that can determine the mechanisms of ORN differentiation, including their ability to respond selectively to specific odors. Olfactory neurons appear to induce the early development of the olfactory bulb and later, the expression of transmitters in bulb neurons. This trophic dependence continues throughout life as many bulb neurons cease to express their transmitters/peptides after olfactory nerve deafferentation. What is the nature of these regulatory actions? New studies will determine the roles of neural activity and target derived trophic factors in the regulation of dopamine (DA), which is contained approximately 300,000 juxtaglomerular neurons. Taste cells and taste nerves exhibit a spectrum of cell surface molecules, including some of those expressed on olfactory neurons. Are these molecules regulated by signals specific to the location of these cells in the tongue, or by the taste nerves innervating them? The answers to such questions will provide clues about the mechanisms that regulate the orderly turnover and replacement of taste cells New evidence in the last funding period suggests for the first time that there is continuous neural remodeling in normal gustatory brain areas. Experiments here will determine if this remodeling is in response to changes in taste cell-taste nerve connections that result from normal taste cell turnover and replacement.

One of the most striking examples of a "trophic" dependence of the olfactory bulb on the epithelium is the downregulation of dopamine (DA) in bulb juxtaglomerular neurons following deafferentation or nares occlusion. Work in the last funding period extended this phenomenon of "transneuronal regulation of transmitter phenotype" to two new peptides (CCK & CRF) and two new classes of neurons (other tufted cells & mitral cells), thus indicating that it is a general phenomenon. In the last funding period we discovered that the olfactory receptor neurons (ORNs) are both afferent to and the target of the DA cells which act via DA D2 receptors on the terminals of ORNs. This leads to the hypothesis that DA presynaptically regulates olfactory nerve terminals. Preliminary electrophysiological data support this hypothesis. Electrophysiological experiments using a newly developed rat olfactory bulb slice preparation and in vivo recordings will test the novel hypothesis that the DA neurons function to presynaptically regulate the excitability of olfactory nerve terminals. If this hypothesis is correct it will be important to learn how DA release is regulated. In vivo microdialysis experiments will test the hypothesis that activity in the olfactory nerve modulates the tonic release of DA. Previous studies suggest that the dependence of DA on the olfactory nerve is due either to [a] loss of an afferent trophic factor or [b] loss of afferent activity onto the DA cells. The finding that the DA cells "target" the ORN terminals suggests the hypothesis that target- derived trophic factors maintain the DA phenotype. This "target hypothesis" will be tested in a series of tissue culture and neural transplantation experIments. Nares occlusion experiments have been taken as evidence that afferent neural activity drives the DA phenotype. However, there is now strong evidence that increased neural activity causes a rapid increase in the expression of target derived trophic factors. Thus, reducing neural activity in the olfactory nerve by nasal occlusion could reduce target derived trophic factor. Tissue culture experiments with Pixley will be done to dissociate neural activity from trophic effects to determine which factor controls the DA phenotype. Together, these experiments will: [1] test novel hypotheses about the functions of one of the largest populations of neurons in the olfactory bulb, and [2] identify the mechanism(s) of transneuronal regulation of the DA phenotype; this will provide evidence for trophic functions of the olfactory nerve.

DESCRIPTION: Despite advances in our understanding of the cellular actions of norepinephrine (NE) in the CNS, we do not know how the LC-NE system modulates neural network function. The main olfactory bulb (MOB) is a relatively simple, well characterized cortical network that functions to process olfactory information. LC densely innervates this network with a higher degree of laminar specificity than in any other forebrain target. This laminar organization leads to specific predictions about the neurons targeted by NE synapses and to equally specific predictions, supported by new pilot studies, of LC-NE actions on olfactory bulb network function. We propose in vivo and in vitro electrophysiological experiments to characterize the action of the LC-NE system on the MOB at both the cellular and circuit levels. Research during the present funding cycle demonstrated that LC activation in vivo has a dramatic 'modulatory' action increasing mitral cells (MCs) responses to weak but not strong olfactory nerve (ON) shocks. This modulatory action can be reproduced in vitro, where it is mimicked by a1 agonists and blocked by a1 antagonists. We also found that MCs exhibit bistability, alternating between two membrane potentials separated by 10 mV which seems to be determined by the balance between an outward K+ current and a non-inactivating Na* current. MC responses to ON input or current injection differ dramatically between these to states. Preliminary findings suggest that NE, via a1 receptors depolarizes MCs by reducing the outward K+ current. This effect is "amplified' by bistability making the MCs more likely to discharge in response to weak ON inputs. Tufted (T) cells, by contrast, exhibit a "bursting" firing pattern and are not bistable. If NE has a similar a1 effect on T as MCs, the depolarization would not be amplified by bistability. Thus, NE may differentially modulate the responses of M and T cells - the two output neurons of the bulb. We will test this hypothesis using whole cell current/voltage-clamp studies. Synapses from M/T cell lateral dendrites excite granule (G) cell dendrites, which in turn, provide GABAergic inhibition back onto M/T cell dendrites. NE fibers heavily target the granule cell layer but nothing is known about the actions of NE on G cells. We will test hypothetical sites of NE modulation of M/T-to-G and G-to-M/T synapses using patch clamping and Ca2+ imaging. Finally, we wifi determine how NE modulates the primary function of the bulb, odor sensory processing. The influence of synaptically released NE on odor processing is unknown. We wifi close this gap. Since LC activation in vivo increases mitral cell responses to weak ON shocks. Therefore, we predict that LC activation will selectively increases mitral cell responses evoked by low concentrations of odors. We will directly test this prediction by measuring the effect of LC activation on MC responses to odors in anesthetized rats.

Description: (from the abstract) Because of their characteristic anatomical organization, it has long been suspected that glomeruli are sites of convergence for functionally related olfactory receptor neurons (ORNs). This was supported by classical 2-DG experiments showing that different odors activate different glomeruli. However, the obscure nature of olfactory reception-transduction made it difficult to move beyond this relatively general notion. Progress in the biophysics and molecular biology of olfactory transduction and the discovery of a large, multigene family of putative olfactory receptor genes (ORGs) have revolutionized our thinking about olfactory reception. The mammalian olfactory epithelium is composed of four anatomically distinct zones within which ORNs express ORGs from only one of four subgroups of the multigene family. Within these "expression zones" individual ORNs that express the same ORGs are randomly dispersed. However, recent findings suggest that axons from ORNs expressing the same ORG converge en route to the bulb and terminate in the same few glomeruli. This implies that glomeruli are sites of convergence for ORNs that "see" the same molecular sub-domains present on odor molecules. To the extent that this is correct, it follows that the extraction of information about specific odor molecules is the result of neural computation. The initial stage of this computation occurs in glomeruli. ORNs transfer information to the rest of the brain via mitral/tufted cells, the output neurons of the bulb. This transfer is initially regulated by the interneuronal network of the glomerulus. The major premise of this research is that almost nothing is known about the functional organization of glomeruli. Classical anatomical studies have provided some information about the synaptic organization, but very little is known about the functional characteristics of distinct juxtaglomerular (JG) interneuronal cell types, the pharmacology of glomerular synapses, or the dynamics and plasticity of intraglomerular synaptic processing. Interglomerular interactions have long seen suspected, and are theoretically implied by current notions of ORN-glomerular convergence, but rigorous analyses of glomerular interactions are lacking. The major impediment to research on glomeruli is the small size of JG cells, which has rendered them virtually inaccessible to classical physiological approaches. To overcome this obstacle the PI has developed the first mammalian olfactory bulb slice preparation. This preparation is physiologically robust and provides access to the functionally intact microcircuitry of the glomerulus. The PI has already obtained novel insights about functionally distinct JG cell types and synaptic plasticity (LTP). The goal of the proposed research is to use extra- and intracellular and whole-cell perforated-patch recording methods and intracellular labeling to characterize membrane, synaptic, and network mechanisms of glomerular processing and functional interactions among neighboring glomeruli.

sensation; chemoreceptors; neuroanatomy;

The proposed research aims to elucidate the integrative mechanisms of olfactory bulb mitral cells, particularly as they relate to glomerular pattern analysis in the olfactory bulb. Two novel properties of mitral cells, membrane potential bistability and long lasting depolarizations (LLDs), are the specific focus of biophysical and pharmacological measurements to determine the underlying intrinsic currents and synaptic mechanisms producing these two properties of mitral cells. A novel slice preparation of early postnatal mouse brain which retains connections between olfactory neurons and olfactory bulb will be used to test specific hypotheses concerning the integrative functions of bistability and LLDs. Voltage clamp protocols will be used to characterize the activation and inactivation curves of intrinsic and synaptically activated currents in mitral cells elicited by antidromic stimulation of the lateral olfactory tract and orthodromic stimulation of the olfactory nerve. Dual intracellular recordings from the apical dendrite and soma of a mitral cell are proposed as are pairwise recordings from two mitral cells receiving input from the same glomerulus. Cross correlation techniques will be applied to test whether LLDs lead to synchronization of the mitral cell population with apical dendrites in the same glomerulus. Dual mitral cell recordings will also be used to test the hypothesis that bistability regulates the sensitivity of mitral cells to olfactory nerve inputs and functions as a temporal amplifier of lateral inhibition. The development of a viable olfactory bulb slice preparation from postnatal mouse makes possible the visualization of mitral cells with DIC/IR optics so that biophysical and integrative synaptic mechanisms can be made routinely with dual intracellular recordings. Normal and transgenic mice will be used to address questions of neurotransmitter receptor roles in mitral cell integrative mechanisms. Preliminary data show that the proposed techniques are working in the PI?s laboratory.

[unreadable] DESCRIPTION (provided by applicant): The glomeruli are the initial site of synaptic integration in the olfactory pathway. Olfactory nerve (ON) axons from olfactory receptor neurons expressing the same odorant receptor project to the same one or few glomeruli in the main olfactory bulb, where they synapse on the apical dendrites of mitral/tufted cells (MC/TCs) and local juxtaglomerular (JG) interneurons. Each glomerulus, hence, can be considered a functional unit for processing sensory input. We hypothesize that each glomerulus is comprised of a small set of stereotyped neuron sub-types, specifically organized to perform discrete network operations. These network operations determine the transfer function from ON terminals to MCs and TCs, the output neurons of MOB. We further hypothesize that these glomerular transfer functions operate primarily on the magnitude of ON activity in each glomerulus. Analysis of patterned activity across the glomerular ensemble occurs downstream of the glomerular network. [unreadable] [unreadable] Aim 1 will identify the transmitters and receptors underlying the synaptic relationships of JG neurons. Aims 2-4 test hypotheses about three specific glomerular functions and the cellular-network mechanisms that perform these operations. Aim 2 will (i) test the hypothesis that DA- and GABAergic JG cells presynaptically inhibit ON terminals by altering Ca2+-meditaed transmitter release, and (ii) will use in vivo Ca2+ imaging and odor stimuli to test the hypothesis that presynaptic inhibition functions to scale the dynamic response range of glomerular output. Aim 3 will use in vitro and in vivo experiments to test the hypothesis that rhythmically bursting external tufted cells function to amplify and synchronize glomerular output responses to odor stimulation. Aim 4 will test the hypothesis that short axon JG cells function to mediate local, interglomerular lateral inhibition. This lateral inhibition provides interglomerular contrast enhancement that is proportional to the magnitude of ON input to each glomerulus. [unreadable] [unreadable] This renewal application builds on achievements of the previous award and uses novel approaches to clarify the organization and functions of the neural network that initiates the computation of odors.

DESCRIPTION (provided by applicant): Sensory inputs to the olfactory system are initially processed by neural circuits in olfactory bulb (OB) glomeruli. This circuitry is targeted by centrifugal inputs from neuromodulatory centers. OB output via mitral/tufted (MT) cells shows remarkable modulation of sensory responses in the awake animal. Knowing how these earliest stages of odor information processing are modulated is critical to fully understanding olfactory system function. This project will investigate - for the first time - how acetylcholine (ACh) and serotonin (5HT) modulate sensory processing in glomerular circuits. The research builds on recent advances in our understanding of these circuits and their importance in shaping OB output. The project will integrate information from OB slices and anesthetized and awake head-fixed mice. This multidimensional approach aims to link the modulation of glomerular circuits to the activation of specific neuromodulatory systems - cholinergic inputs from the diagonal band and serotonergic inputs from the raphe nuclei. Our working hypothesis is that ACh and 5HT differentially modulate glomerular processing to shape both pre- and postsynaptic inhibition as well as MT cell excitability. We further hypothesize that ACh modulation reduces the impact of sensory input while at the same time increasing the excitability of MT cells. This could limit odor detection but increase discrimination ability. The proposed research is a highly integrated effort from two established investigators to understand the modulation of early olfactory processing at levels ranging from cells and circuits to single neurons in anesthetized and unanesthetized animals.

DESCRIPTION (provided by applicant): Glomerular circuits are important. They transform sensory input into output signals that impact all downstream olfactory networks. Inhibition is key to these transforms. Intraglomerular inhibition presynaptically regulates sensory synapses and provides feedback and feed forward inhibition that shapes the strength and temporal structure of M/TC responses to sensory signals. Less is known about interglomerular inhibition. Exciting new data show that short axon cells, which form the interglomerular circuit, co-release GABA and DA. These co-transmitters trigger a temporally biphasic inhibitory-excitatory response in external tufted cells, neurons critical to glomerular circuit function. They also generate strong, temporally asymmetric, direct inhibition of M/TCs such that early-activated glomeruli inhibit their later activated neighbors, leading to The early bird gets the worm hypothesis. These discoveries were made using optogenetics combined with recordings from identified gene-targeted cell-type specific neurons. Little is known about DA release or it's metabolism in the bulb. Fast Scan Cyclic Voltammetry will be used to define the kinetics of DA release in vitro and in vivo. New data suggest that FDA-approved COMT inhibitors may enhance bulb DA function; this could benefit Parkinson's patients. We will test this hypothesis. Inhibitory-inhibitory interactions among inter- and intraglomerular neurons have not been explored. Pilot data using optogenetics combined with identified cell recordings indicate robust interactions. We will define inhibitory-inhibitory interactions, their synaptic dynamics and how they shape intra- and interglomerular signaling.

PROJECT SUMMARY Sensory systems, including olfaction, are heavily dependent on signals from higher brain regions that regulate behavioral states of alertness and attention. A major center for these modulatory inputs is the basal forebrain. The basal forebrain (BF) sends axonal projections throughout the brain and has been implicated in dynamic modulation of cortical as well as sensory circuits during behavior. BF is classically thought of as a center for cholinergic projections throughout the brain, whose chief functional role is to mediate attentional modulation of information processing. However, the recent advent of novel tools for probing neural circuits in vivo has enabled a deeper and more nuanced understanding of basal forebrain organization and role in cognition. BF projections to neocortex are in fact neurochemically diverse and precisely organized with respect to projection target. Further, different BF subpopulations are linked to distinct aspects of behavior and are implicated in diverse cognitive functions including reward signaling, behavioral responding to sensory cues and task learning. Despite these advances, however, we still know little about BF impact on the initial processing of sensory inputs on their way to cortex. The olfactory bulb (OB) is advantageous for investigating basal forebrain function, as it is the only primary (pre-cortical) sensory processing area receiving BF inputs and because olfaction is a primary modality driving behavior in rodents. BF sends massive projections to the OB, with terminations in all OB layers. Many of these projections are cholinergic. However, as for neocortex, BF projections to OB are neurochemically diverse with substantial numbers of GABAergic neurons. The importance of this diversity has only recently begun to be appreciated for cortex, but almost entirely unexamined with respect to OB circuitry. A recent study showed that GABAergic basal forebrain projections to the olfactory bulb target distinct neuronal subpopulations and can, independent of cholinergic projections, modulate OB circuits to affect odor perception. However, our understanding of the respective roles played by BF cholinergic and GABAergic in modulating OB circuits and odor perception remains rudimentary. Fundamental unanswered questions include whether cholinergic and GABAergic basal forebrain projections target distinct components of olfactory bulb circuits, whether they have complementary or opposing effects on odor representations at the level of olfactory bulb output, what are the activity patterns of cholinergic and GABAergic basal forebrain neurons during behavior, and what role these projections play in odor perception or odor-guided behaviors? The overall goal of this proposal is to investigate how the basal forebrain neuromodulatory inputs affect olfactory bulb function in a multi-disciplinary study spanning investigation at the level of single neurons through to animal behavior utilizing advances in electrophysiology, opto-/pharmacogenetics, and high resolution activity imaging and microendcosopy in behaving animals.