Vikaas Singh Sohal, MD, PhD - US grants

I am interested in how cellular and synaptic properties of neurons affect emergent patterns of activity such as network oscillations, and how these patterns in turn affect the function of neural circuits. Although myriad properties of cells and synapses are known to be altered in neuropsychiatric diseases such as schizophrenia, it has been difficult to understand exactly how these alterations cause the circuit dysfunction thought to produce clinical symptoms. My goal is to be the principal investigator of a laboratory which (1) identifies cellular and synaptic lesions in animal models of psychiatric disease using in vitro electrophysiology, (2) uses in vitro and in silico experiments to measure circuit functions that are deficient as a result of these lesions, and (3) tests whether restoring these circuit functions can rescue pathological behaviors in vivo. To complement my knowledge of electrophysiology and computational neuroscience, I propose learning to use optogenetic stimulation in vitro and in vivo, and to study rodent behavioral phenotypes relevant to neuropsychiatric disease. I will be mentored by Karl Deisseroth, whose laboratory has pioneered optogenetic technology. Using optogenetic tools to precisely control patterns of stimulation in prefrontal microcircuits, and information theory to quantitatively measure information processing, we have already elucidated mechanisms by which brain rhythms enhance information processing in prefrontal microcircuits. Now, I propose to study the effects of dopamine and manipulations that model aspects of schizophrenia to answer the following questions: (1) Do Dl and D2 receptor stimulation have opposing effects on the signal-to-noise ratio in prefrontal microcircuits? (2) Does blocking NMDA receptors and/or disrupting DISCI suppress gamma-frequency synchronization or alter information processing in prefrontal microcircuits? (3) Can rhythmic optogenetic stimulation of prefrontal neurons ameliorate effects of PCP and DISCI disruption on working memory? We believe that these experiments will not only shed light on the workings of prefrontal microcircuits and possible modes of dysfunction in schizophrenia, but also establish powerful new ways to study circuit dysfunction in neuropsychiatric disease.

DESCRIPTION (Provided by the applicant) Abstract: Schizophrenia is a devastating illness, and dysfunction of the prefrontal cortex (PFC) is widely believed to underlie cognitive impairment and other debilitating aspects of this disorder. Current treatments are limited at best, and although we have identified numerous genetic, molecular, cellular, and synaptic alterations related to schizophrenia, we have not been able to translate these findings into more effective treatments. The central reason for this is tha we simply do not know how circuits in the PFC (or elsewhere in the brain) work - we do not know how the properties of cells or their interactions give rise to patterns of activity that enabl these brain regions to carry out functions, including the ones impaired in psychiatric illnesses. Here we propose a novel strategy to reverse engineer recurrent circuits in the cortex, i.e. to measure their activity in a way that makes it possible to infer both their overall function, and th contributions that individual components (e.g. specific cell types) make to this function. Our approach is widely applicable, and we will demonstrate its utility by answering specific questions about the recurrent layer V network in prefrontal cortex, because dopaminergic modulation of this network plays a critical role in schizophrenia. Activity in this network may converge to stabl states or progress through a deterministic sequence of states in order to store information in working memory. Alternatively, this network may generate noisy activity that makes prefrontal output more variable in order to facilitate behavioral adaptation. Such noise could cause prefrontal dysfunction under pathological conditions. Our approach will determine which of these hypothesized functions the layer V prefrontal network carries out when various dopamine receptors are activated. In the process, we will define prefrontal noise and related concepts. We will isolate layer V from external inputs using a brain slice preparation, and record simultaneous activity from many neurons in layer V of the PFC using the genetically encoded calcium indicator GCaMP3. We present examples of network activity recording using GCaMP3, and describe how statistical methods such as Hidden Markov Models can decode this activity and infer the function of the network. We also show that layer V contains multiple subtypes of pyramidal neurons, and illustrate how we can distinguish these subtypes while recording activity with GCaMP3. Dopamine D2 receptors are only expressed in one of these subtypes, and we will test the hypothesis that D2 receptor activation generates prefrontal noise through specific effects on these neurons. Finally, we describe ways to extend our approach by combining optogenetic stimulation with GCaMP3 imaging. These experiments will answer long-standing questions about prefrontal networks and demonstrate approaches that may deconstruct networks throughout the cortex. Furthermore, by reverse engineering the black box of prefrontal circuitry that is commonly invoked to connect genetic or developmental lesions with their behavioral consequences, this study may open up fundamentally new ways of thinking about psychiatric illness, making it possible to design novel therapies that target circuit dysfunction. Public Health Relevance: Schizophrenia affects approximately 1% of the population worldwide, causing distress for patients and their families, and in most cases, life-long disabilit. Many of the most disabling symptoms of schizophrenia are thought to involve dopamine and dysfunction of the prefrontal cortex. Here we propose to study how dopamine modulates the function of the prefrontal cortex in order to identify critical mechanisms that are likely to be disrupted in schizophrenia and related conditions.

DESCRIPTION (provided by applicant): The prefrontal cortex (PFC) plays a key role in aspects of cognition including working memory, behavioral flexibility, and decision making. Conversely, dysfunction of this brain region causes cognitive deficits, including major aspects of psychiatric disorders such as schizophrenia. Here, we will elucidate how the neuromodulator dopamine regulates activity in a specific class of neurons within the PFC. This is important because dopamine is believed to regulate both the normal and pathological function of the PFC. In fact, a major hypothesis in schizophrenia research is that abnormal dopaminergic modulation causes PFC dysfunction and some symptoms of schizophrenia. However, specific mechanisms through which prefrontal dopamine receptors exert their normal and pathological effects remain largely unknown. Our recent publication in the Journal of Neuroscience describes new effects of dopamine receptors on a specific population of neurons in the PFC. This proposal will focus on this subpopulation of prefrontal neurons, which we refer to as type A neurons. We propose that different classes of dopamine receptors produce opposing effects on the excitability of these neurons, and that aberrant activity in these neurons, which may be driven by excessive activation of certain dopamine receptors, can produce schizophrenia-like behaviors in mice. First, we will identify specific ion channels and other mechanisms that mediate the effects of dopamine receptors on type A neurons. Then, we will determine how, by altering the excitability of type A neurons, dopamine receptors can alter their responses to synaptic input. We will specifically determine whether dopamine receptors produce distinct effects on synaptic inputs that arise from different sources. Finally, we will deliver various patterns of stimulation to fibes that release dopamine in the PFC. These experiments will test the hypothesis that different patterns of activity in these fibers will activate different dopamine receptors, producing distinct effects on type A neurons. Many of our experiments will utilize new optogenetic technologies, which make it possible to stimulate specific neurons or neural connections, with light. This proposal will focus on how dopamine receptors modulate the activity of type A neurons. Our long-term goal is to relate these changes in type A neuron activity to effects on PFC-dependent behaviors, including pathological behaviors that occur in schizophrenia and other psychiatric disorders.

? DESCRIPTION (provided by applicant): Molecular definitions of neural cell types largely depend on the expression of RNAs or proteins as assessed by in situ hybridization, RNA array and sequencing, and immunohistochemistry. However, recent studies are demonstrating that gene regulatory elements, such as enhancers, can have highly specific spatial and temporal activity patterns in the developing brain. Thus, enhancer activity can be used to define neural cell types, and importantly, also have other broad applications. First, they can be used as tools to drive gene expression in specific cell types, which can then be used to visualize and/or purify the cells (GFP), modify gene expression in the cells (Cre), modify electrical activity (channel rhodopsin), and visualize electrical activity in the cells (GCaMP). Secondly, knowledge about the nature and position of enhancers enables geneticists to identify disease alleles that map in extra-exonic genomic space. Herein, we will focus on identifying enhancers that are active in cortical interneurons, during development and in the mature state. We choose to study cortical GABAergic interneurons because of their central role in cortical function and diseases. We will focus on these GABAergic neurons derived from the medial ganglionic eminence (MGE), which generates the majority of cortical interneurons. Our aim is to identify novel enhancers that are active in cortical interneurons using three assays: 1) histone (H3K27Ac) ChIP-seq (marker of active enhancers); 2) transcription factor (TF) ChIP- seq using TFs that regulate cortical interneurons development and function (Arx, Dlx2 and Lhx6); 3) a newly developed in vivo assay of enhancer function based on viral transduction of the enhancer driving Cre and GFP in immature cortical interneurons. We will then use a series of methods to define the interneuron subtypes that have enhancer activity. Together, our unique approach should define interneuronal cell types (developmental and adult), and simultaneously generate a powerful toolkit that will enable new ways to assess neural function and disease.

? DESCRIPTION (provided by applicant): Synchronized, rhythmic activity within the prefrontal cortex (PFC) and at frequencies in the gamma range (~30- 120 Hz) is believed to contribute to many cognitive processes. A specific class of GABAergic interneurons (FSINs) that can be identified based either on their fast-spiking properties, or based on their expression of the calcium-binding protein parvalbumin (PV), play a critical role in these oscillations. Both prefrontal FSINs and gamma oscillations are abnormal in schizophrenia, suggesting that gamma oscillations may represent an important link between interneuron dysfunction and cognitive deficits; similar mechanisms may contribute to other neuropsychiatric disorders such as autism. Here, we will build on a recent study from our laboratory which found that stimulating interneurons at gamma frequencies can produce long-lasting improvements in cognitive flexibility in mice. Specifically, we studied mutant mice which model abnormalities in FSINs and gamma oscillations, as well as deficits in cognitive flexibility that are associated with schizophrenia. We found that using light sensitive proteins to active interneurons in the PFC at gamma frequencies enables these mice to perform normally on a task that measures cognitive flexibility. By contrast, activating these interneurons at other frequencies was ineffective. Now, we propose to explore whether activating PFC interneurons at gamma frequencies can enhance cognition in other contexts. For example, we will test whether activating PFC interneurons at gamma frequencies can rescue cognitive deficits in other mutant mice, in which interneuron function is not the sole or primary defect, or can produce supra-normal levels of performance in normal mice. We will also explore whether activating PFC interneurons at gamma frequencies can improve performance in tasks that measure other aspects of cognition, e.g. working memory. A second direction will be testing the idea that activating PFC interneurons at gamma-frequencies might enhance cognition by facilitating interactions between the PFC and other brain structures. In order to test this idea, we will measure how stimulating interneurons affects the degree to which activity in the PFC is synchronized with other structures. We will also determine whether activating PFC interneurons at gamma frequencies alters activity in other brain structures that are involved in cognitive flexibility. The long-term goal of this project is o determine whether enhancing interneuron-driven gamma oscillations in the PFC can improve cognitive deficits associated with schizophrenia and related disorders, and to identify possible mechanisms through which this might occur.

PROJECT SUMMARY / ABSTRACT (PARENT GRANT) The hippocampus (HPC) and prefrontal cortex (PFC) are implicated in anxiety disorders. In rodents, the ventral HPC (vHPC) and medial PFC (mPFC) form a circuit that regulates anxiety-related behavior in the elevated plus maze (EPM). The vHPC and mPFC synchronize in the theta-frequency (4-12 Hz) range during exploration of anxiogenic regions, and disrupting communication between the vHPC and mPFC prevents mice from avoiding the anxiety-provoking open arms of the EPM. Here we propose to reveal the detailed circuit interactions between the vHPC and mPFC that are crucial for anxiety-related behavior. In particular, our preliminary data has shown that populations of vHPC neurons are recruited during exploration of the open arms in the EPM, and inhibiting vHPC neurons disrupts open arm avoidance. However it is not known whether certain classes of mPFC neurons have preferential access to anxiety-related input from the vHPC. To answer this question, we will study vHPC neurons which project to specific classes of neurons in the mPFC, as mice explore the EPM. We will also study mPFC neurons which project to specific targets, e.g., the basolateral amygdala, to determine how they encode anxiety-related information. We hypothesize that classes of mPFC projection neurons which receive anxiety-related input from the vHPC will also encode anxiety-related information. Finally, we will study how prefrontal interneurons respond to theta-frequency input from the vHPC and regulate the anxiety-related responses of mPFC projection neurons. Our preliminary studies have shown that specific inhibitory neurons in the mPFC regulate prefrontal responses to vHPC input and contribute to anxiety-related avoidance. Here, we will test the hypothesis that in the EPM, theta-frequency input from the vHPC recruits these interneurons, thereby enhancing prefrontal responses to anxiety-related input from the vHPC and anxiety-related avoidance. Specifically, we will examine whether prefrontal inhibitory neurons synchronize to theta-frequency input from the vHPC during exploration of the EPM. Finally, we will examine how prefrontal interneurons regulate anxiety-related activity within specific classes of mPFC projection neurons. Together, these studies will elucidate cell-type specific and circuit-level mechanisms underlying the role of hippocampal- prefrontal networks in a commonly studied anxiety behavior. They will also answer general questions about how complex brain circuits operate. E.g., can one source of input differentially transmit emotionally-relevant information to various neuronal subtypes in a downstream target? How do inhibitory interneurons organize their activity in response to a rhythmic input? Do interneurons regulate emotional representations selectively, within specific projection neurons, or nonspecifically, across a network? The answers to these questions will reveal novel targets for disrupting pathological brain states associated with anxiety disorders.

PROJECT SUMMARY Cognitive deficits represent the major cause of disability in schizophrenia but are refractory to all existing treatments. EEG oscillations in the gamma-frequency range are recruited by many cognitive tasks, and task- evoked gamma oscillations are deficient in schizophrenia. Furthermore, gamma oscillations are generated by parvalbumin interneurons, which are abnormal in schizophrenia. This suggests that gamma oscillations may be biomarkers for cognitive deficits and parvalbumin interneuron dysfunction in schizophrenia. In fact, many studies suggest that gamma oscillations may actively contribute to cortical circuit functions that are necessary for cognition. Indeed, our previous work has shown that optogenetically restoring interneuron-generated gamma oscillations in the prefrontal cortex can rescue cognitive deficits in mutant mice. However, there are many ways to measure gamma oscillations ? some of these capture the strength of gamma oscillations at a single site whereas others reflect synchronization across sites. Our recent work suggests that long-range synchronization of gamma-frequency activity in PV interneurons, rather than just gamma-frequency activity at a single site, may be required for prefrontal cortex-dependent cognitive flexibility. Furthermore, we have developed new ways of measuring signals from genetically encoded voltage indicators in order to measure gamma-frequency synchronization between specific cell-types at different locations. We will now leverage these advances to: (1) use our novel analyses and GEVIs to directly measure cell-type specific gamma- frequency synchronization in behaving rodents; (2) determine which particular ways of quantifying EEG gamma oscillations best capture this synchronization; (3) evaluate how well these EEG measures correlate with changes in PV interneuron synchronization and behavioral performance elicited by several pharmacological manipulations including some which are known to rescue deficits in gamma oscillations and prefrontal-dependent cognition in mutant mice, and (4) validate, via optogenetics, that these EEG measures are sensitive and specific indicators for changes in PV interneuron function. This project will define particular EEG measures that reflect cell-type specific patterns of long-range synchronization underlying specific aspects of cognition.

PROJECT SUMMARY Rhythmic fluctuations of electrical activity in the brain are frequently observed during cognitive tasks. In many cases these oscillations are synchronized across brain regions. Synchronization in the gamma-frequency (~30- 100 Hz) range has been hypothesized to promote communication between brain regions, thereby facilitating cognitive functions. Conversely, deficits in gamma synchrony have been hypothesized to contribute to cognitive deficits at the heart of schizophrenia, Alzheimer?s disease, and related disorders. However, whether gamma synchrony actually contributes to brain function remains highly controversial. The specific circuit-level mechanisms through which gamma synchrony acts are also unclear. This proposal will take advantage of two recent developments in our laboratory. First, we have developed a new method for analyzing signals from genetically encoded voltage indicators in order to quantify changes in gamma synchrony within freely behaving mice. Second, using this method and optogenetics, we have found that interhemispheric gamma synchrony between parvalbumin (PV) interneurons in the prefrontal cortex plays a key role when mice learn new cue- reward associations. We hypothesize that: 1) gamma-frequency activity in PV interneurons entrains activity in prefrontal neurons which project to specific targets; 2) the activity of these projection neurons encodes key information related to learning; 3) thus, gamma-frequency synchronization allows prefrontal output to converge constructively in specific downstream targets, facilitating the transmission of critical task-relevant information across an extended prefrontal network that mediates learning. This proposal will test these hypotheses by studying whether gamma synchrony is transmitted from prefrontal PV interneurons to various classes of prefrontal projection neurons which encode task-relevant information and/or to downstream regions. We will then construct a computational model to test which hypothesized functions of gamma synchrony are consistent with our experimental observations. This will reveal circuit-level mechanisms whereby gamma synchrony is transmitted across neural networks in ways that can facilitate inter-regional communication and learning.

PROJECT SUMMARY (PARENT GRANT) Rhythmic fluctuations of electrical activity in the brain are frequently observed during cognitive tasks. In many cases these oscillations are synchronized across brain regions. Synchronization in the gamma-frequency (~30- 100 Hz) range has been hypothesized to promote communication between brain regions, thereby facilitating cognitive functions. Conversely, deficits in gamma synchrony have been hypothesized to contribute to cognitive deficits at the heart of schizophrenia, Alzheimer?s disease, and related disorders. However, whether gamma synchrony actually contributes to brain function remains highly controversial. The specific circuit-level mechanisms through which gamma synchrony acts are also unclear. This proposal will take advantage of two recent developments in our laboratory. First, we have developed a new method for analyzing signals from genetically encoded voltage indicators in order to quantify changes in gamma synchrony within freely behaving mice. Second, using this method and optogenetics, we have found that interhemispheric gamma synchrony between parvalbumin (PV) interneurons in the prefrontal cortex plays a key role when mice learn new cue- reward associations. We hypothesize that: 1) gamma-frequency activity in PV interneurons entrains activity in prefrontal neurons which project to specific targets; 2) the activity of these projection neurons encodes key information related to learning; 3) thus, gamma-frequency synchronization allows prefrontal output to converge constructively in specific downstream targets, facilitating the transmission of critical task-relevant information across an extended prefrontal network that mediates learning. This proposal will test these hypotheses by studying whether gamma synchrony is transmitted from prefrontal PV interneurons to various classes of prefrontal projection neurons which encode task-relevant information and/or to downstream regions. We will then construct a computational model to test which hypothesized functions of gamma synchrony are consistent with our experimental observations. This will reveal circuit-level mechanisms whereby gamma synchrony is transmitted across neural networks in ways that can facilitate inter-regional communication and learning.