Hirofumi Morishita, M.D., Ph.D. - US grants

? DESCRIPTION (provided by applicant): Early temporary windows of heightened brain plasticity called critical periods sculpt neural circuits and contribute to adult behaviors. Such heightened plasticity declines into adulthood. A clinically central issue is that this limits recovry of function later in life. The long-term goal of this study is to discover new regulatory mechanisms of plasticity to provide therapeutic targets to re-open plastic windows in the adult brain. One of the best-studied models of a critical period is the enduring loss of responsiveness in primary visual cortex (V1) to an eye deprived of vision, resulting in amblyopia (a loss of visua acuity), affecting 2-4% of the population and without a known cure in adulthood. Recently, the nicotinic acetylcholine receptor (nAChR) system and its endogenous modulators that belong to Lynx family have emerged as key regulators of plasticity in adult V1. Previous work showed that Lynx1 increases in expression after the critical period and acts as a brake to limit V1 plasticity in the adult brain. In preliminary studies, Lypd6, another Lynx family protein, was foun to act as a positive modulator of adult plasticity. However, the specific mechanisms of plasticity regulated by the Lynx family members, such as responsible cell-types, nAChR subtypes, and neural circuits regulated, etc, are totally unknown. The objective of this study is to identify novl mechanisms of plasticity by focusing on Lynx1 and Lypd6, which represent a new class of plasticity regulators that modulate nAChR signals. Strikingly, these two Lynx family members have opposite expression patterns in two major subtypes of GABAergic interneurons: while Lynx1 is primarily expressed in parvalbumin interneurons, Lypd6 is exclusively expressed in somatostatin interneurons. Furthermore, viral manipulations of Lynx1 and Lypd6 in GABAergic neurons but not in glutamatergic neurons modulate adult V1 plasticity, making them unique targets to dissect the mechanisms of plasticity linking the nAChR and GABAergic systems. We hypothesize that Lynx1 and Lypd6 have distinct roles on GABAergic interneurons to regulate adult plasticity through modulation of specific nAChRs, and that the Lynx family can be an effective pre-clinical therapeutic target for treating amblyopia. By combining cell-type specific gain/loss of gene expression through genetic and viral techniques with in vivo electrophysiology assisted by optogenetic tagging for cell-type specific measurements, and in vivo two- photon imaging of structural plasticity, we expect to identify the specific mechanisms of plasticity regulated by the Lynx family members, such as responsible cell-types, the nAChR subtypes, and neural circuits. Identification of such mechanisms would allow exploiting manipulations of the Lynx system in order to facilitate functional recovery after the end of the critical period, which would in turn lead to new opportunities to treat amblyopia and other disorders of cortical plasticity.

DESCRIPTION (provided by applicant): Aberrant brain development during critical periods of heightened vulnerability contributes to lifelong cognitive impairments underlying many psychiatric disorders. Mechanisms driving critical period circuit development are well described in sensory systems-but poorly characterized for complex cognitive behaviors. Identification of a critical period and underlying mechanisms for cognitive circuits and behavior would eventually improve diagnosis, prevention and treatment of psychiatric disorders. This study will test whether a mechanism critical for regulating the critical period for visual cortex development also modulates maturation of frontal cortex- dependent attentional functions. Lynx1 is an endogenous nicotinic acetylcholine receptor (nAchR) inhibitor and regulates the critical period of visual cortex plasticity by its increased expression across adolescence. In addition to its role in visual cortex, our preliminary data show that the Lynx1 knock-out mice with high nAChR signaling unexpectedly present with attention-deficits in adulthood. Strikingly, this deficit was prevented by transient suppression of nAChR signaling only during early adolescence but not acutely in adulthood. This study will test the overarching hypothesis that the excessive nAChR signaling during adolescence, normally limited by peri-adolescent increase in Lynx1 expression, causes long-lasting impairment in frontal cortical circuits and behaviors that support attention. Understanding how excess nAChR signaling disrupts normal circuit development could provide mechanistic insight into developmental neuropsychiatric disorders characterized by disrupted nAChR signaling, such as Autism, ADHD and schizophrenia. In Aim1, we will test the hypothesis that Lynx1 expression in anterior cingulate cortex during peri-adolescence is required for normal attentional function in the adult. We will precisely define the critical period and key brain regions mediating the long-lasting impact of excess nAChR signaling on adult attentional function by combining pharmacological, viral and genetic gene manipulations in vivo with 5-choice serial reaction time task (5- CCRTT) employing a translational touchscreen system. In Aim2, we will test the hypothesis that the fronto- posterior cortical circuits require nAChR signaling to be suppressed by Lynx1 during adolescence to establish attention in the adult. By introducing a novel viral system that allows circuit-specific labeling and gene manipulations at time points of interest, the impact of Lynx1 deletion to neurons projecting from anterior cingulate cortex to visual cortex, and the cell-autonomous contribution of Lynx1 in these circuits will be determined. At the completion of this study, we will have defined the developmental time window, anatomical region, and circuit that require optimal nAChR signaling for normal attentional function, which is identified by the NIMH Research Domain Criteria (RDoC) project as one major construct of cognitive systems impaired in a number of psychiatric conditions.

Experience-dependent brain plasticity is heightened during developmental critical periods but declines into adulthood, posing a major challenge to recovery of function following injury or disease later in life. A dominant model of critical period plasticity is the change in ocular dominance of neurons in primary visual cortex following monocular deprivation. Recent studies suggest that experience-dependent plasticity of Parvalbumin positive (PV-) GABA interneurons in visual cortex plays a key role in triggering ocular dominance plasticity during the critical period. Importantly, such rapid plasticity of PV cells is limited to the critical period and is not observed in adults. Our pilot study further showed that another major subtype of interneurons expressing Somatostatin (SST) can also modulate ocular dominance plasticity in the adult. These studies underscore the novel role of interneurons in rapidly gating cortical plasticity in adult visual cortex. However, the molecular and circuit mechanisms that limit the rapid plasticity triggered by interneurons in the adult are totally unknown. This proposal deals with this open question to provide new mechanisms of the initial phase of plasticity to effectively re-trigger plastic windows for recovery of cortical function in adults. We turn to proteolytic regulation because tissue plasminogen activator (tPA), a major protease in the brain, is known to rapidly elevate during only critical period but not in adulthood to mediate plasticity. Our preliminary study found that the removal of one endogenous tPA inhibitors called Neuroserpin, enriched in adult PV- and SST-cells, leads to a series of experience-dependent rapid changes to unmask plasticity in adult visual cortex. By combining in vivo extracellular recordings, patch-clamp electrophysiology with optogenetics, pharmacogenetics, and cell-type specific gain/loss of gene expression through genetic and viral techniques in vivo, we will test our hypothesis that proteolytic regulation by an endogenous serine inhibitor, Neuroserpin, gates the sensitivity of PV- and SST- interneurons to rapidly trigger visual cortex plasticity and recovery in adulthood. Proteolytic regulation of rapid plasticity associated with PV-interneurons and SST-interneurons will be examined in Aim1 and Aim2 respectively. In Aim3, therapeutic potential for recovery from amblyopia by targeting Neuroserpin in interneurons will be tested. Successful completion of this project will illuminate a new molecular mechanism that gates the initial cascade triggering cortical plasticity, which will have direct implications for Amblyopia, a condition with limited adult-applicable cure affecting 2?5% of the human population, but also for brain injury repair, sensory recovery.

Attention deficit symptoms are frequently observed in psychiatric disorders, yet finite understanding of the neural circuits mediating attentional behavior has limited pathophysiologic insight. Previous studies in humans and rodent demonstrate that the frontal cortex?especially the anterior cingulate cortex (ACC)? plays a key role in implementing a top-down control of attention. However, the precise neural circuit mechanisms mediating attention remain largely unknown. The goal of this study is to identify the specific frontal cortex projecting neural circuits that mediate top-down control of attentional behavior. Conserved long-range frontal cortico- cortical projections regulate visual cortex (VIS) responses to visual stimuli in attention based tasks in macaque, and optogenetic manipulation in mice shows that this circuit can enhance visual discrimination sensitivity. Improper frontal modulation of VIS activity in ADHD, schizophrenia, and autism accompanying visual attention deficits may be in part due to the dysfunction of direct long-range projections. However, activity in this circuit has never been directly and specifically examined during attentional behavior, nor has been selectively manipulated to causally improve attentional behavior. Here we will test the hypothesis that long-range top- down cortical circuits directly projecting from frontal cortex to sensory cortex coordinate temporal dynamics between these regions to effectively modulate attentional behavior by implementing an intersectional viral strategy to combinatorially (1) monitor and (2) manipulate neural activity within specific circuits of mice with a translation touchscreen system to assess attention during naturalistic freely moving behavior. In Aim1, we will identify when top-down circuits are activated during attentional behavior. In Aim2, we will manipulate top-down cortical circuit activity to modulate attentional behavior. Here, we will test the hypothesis that top-down cortical circuits coordinate temporal dynamics between ACC and VIS to effectively modulate attentional behavior. At the completion of this study, we will have established a strong basis for the causal role of top-down cortical circuit on attentional behavior.

The objective of this proposal is to determine the key functional properties at the single-spine level that are associated with experience-dependent gain and loss of spines. Spines are known to appear and disappear in an experience-dependent manner throughout life. Such changes have key implications for behaviors by physically rearranging the circuit connectivity. However, the specific functional properties of individual spines in vivo that are associated with gain and loss of spines are not known. Such information is important for a better understanding of and interventions into brain disorders accompanying spine deficits. A classical model of experience-dependent plasticity, ocular dominance plasticity in the primary visual cortex, accompanies gain and loss of spines during monocular deprivation in addition to the cellular changes in ocular preference. Recent advances in two-photon calcium imaging techniques have enabled direct observation of single-spine activity in vivo, and highly specific and diverse functional properties (e.g. orientation preference) of individual spines were revealed. However, little is known to what extent the functional properties of spines are related to the experience-dependent gain and loss of spines. By fluorescent visualization of local calcium transient using a green genetically encoded fluorescent indicator (GCaMP6) and visualization of spine structure with a red fluorescent protein, simultaneous time-lapse structural and functional two-photon imaging of ocular dominance at the single spine level in vivo will be achieved to test the overarching hypothesis that specific functional properties of individual spines are associated with their experience-dependent gain and loss which collectively impact the plasticity of parent neurons' output. In aim1, the functional signatures at single spines associated with the experience-dependent spine gain will be determined. In aim2, functional properties of single spines that predict experience-dependent loss of spines will be identified. At the completion of this study, we will have determined for the first time the key functional properties of spines associated with their experience-dependent gain and loss. Such findings will serve as novel targets for the development of therapeutic approaches for functional recovery in the adult brain disorders.

PROJECT SUMMARY Schizophrenia (SZ) is a common and debilitating neurodevelopmental disorder that affects nearly three million Americans. Despite more than fifty years of research, no cures exist and the standard of treatment remains unsatisfactory. Genome wide association studies (GWAS) indicate that SZ risk reflects both highly penetrant rare copy number variants as well as common single nucleotide polymorphisms with small effect sizes. By overlapping GWAS and post-mortem expression analyses, common variants with expression quantitative trait loci (eQTL) that may contribute to altered gene expression and liability in SZ have been identified; however, demonstrating which risk loci are the causal contributors to disease risk remains an intractable problem. Consequently, we propose to apply a human induced pluripotent stem cell (hiPSC)-based approach to manipulate the genotype and/or expression levels of putative causal SZ risk variants, focusing largely on genes implicated in synaptic formation, maturation and function. Through isogenic comparisons, we propose to examine the molecular and functional effects of perturbing five putative causal eQTLs and ten SZ GWAS- associated genes, testing the impact on cis-gene expression, global network expression patterns and synaptic function. Our hope is that this work may identify novel therapeutic points of intervention in order to improve the disease course in individuals with SZ.

Social experience during childhood is essential to establish proper function in the adult prefrontal cortex (PFC) and related behaviors including social behavior, a fundamental process across species. However, the specific circuits that undergo social experience-dependent maturation to regulate social behavior are poorly understood. Given that social process deficit is a common dimension of many neurodevelopmental and psychiatric disorders, identifying the specific circuits sensitive to experience-dependent modulation will point toward therapeutic targets that allow amelioration of social processing deficits shared across of range of disorders. The objective of this study is to elucidate the PFC circuit mechanisms underlying social experience- dependent maturation crucial to mediate proper social behavior. In mice, juvenile social isolation leads to adult social behavior deficits and accompanies deficits in sub-cortically projecting deep layer medial PFC (mPFC) pyramidal neurons. Among various sub-cortical targets, our preliminary study identified the limbic thalamus which receives and relays signals to various components of the classical reward circuitry, as the most prominent projection target from mPFC that is preferentially recruited by social interaction. Importantly, juvenile social isolation reduced excitability of this projection neuron and increased their inhibitory drive. Among various types of inhibitory neurons, a selective subclass of deep layer inhibitory neuron, known to be essential to oscillate subcortically projecting deep layer cortical neurons, were the only population that exhibited elevated excitability after juvenile social isolation. This project will test the hypothesis that juvenile social experience- dependent maturation of deep layer mPFC projection neurons to limbic thalamus and its modulation by deep layer mPFC inhibitory neurons drives coordinated activity in mPFC and limbic thalamus to effectively modulate social behavior. We will test this hypothesis by integrating techniques to measure (fiber photometry imaging, patch-clamp/ in vivo electrophysiology) and manipulate (optogenetics/chemogenetics) the activities of selective circuits during social behavior in mice.

Cognitive function relies on the cohesive activity of large-scale networks. Long-range cortico-cortical projections facilitate such network activities by integrating information from various inputs and then relaying it to other cortical regions. Of particular significance is the evolutionarily conserved top-down projection from prefrontal cortex (PFC) to sensory visual cortex (VIS) which integrates sensory, motor, and cognitive information to modulate sensory processing. Conserved PFC->VIS projections regulate VIS responses to visual stimuli in attention based tasks in macaque, and optogenetic manipulation in mice shows that this circuit can enhance visual discrimination sensitivity. Notably, deficits in PFC modulation of VIS activity are pervasively reported in neurodevelopmental and psychiatric disorders, and often emerge following childhood and adolescence. Yet limited knowledge of processes governing long-range cortical circuit development hinders further pathophysiological insight into these conditions. The goal of this study is to identify the developmental mechanisms of PFC circuitry that mediate top-down control of attentional network and behavior. Here we test the hypothesis that top-down PFC->VIS cortical circuits require a Lynx1, developmentally regulated nicotinic modulator,-dependent adolescent shift in the balance of local/long-range inputs to coordinate temporal dynamics between PFC and VIS modulate attentional behavior, and that Lynx1 modulation can ameliorate top- down circuit deficits in mice with a risk gene of neurodevelopmental disorders. We will test his hypothesis by integrating techniques to measure and manipulate neural activity and gene expression within specific circuits of mice tested in a translationally-relevant touchscreen system to assess attention during naturalistic freely- moving behavior.

Social experience during childhood is essential to establish proper function in the adult prefrontal cortex (PFC) and related behaviors including social behavior, a fundamental process across species. However, the specific circuits that undergo social experience-dependent maturation to regulate social behavior are poorly understood. Given that social process deficit is a common dimension of many neurodevelopmental and psychiatric disorders, identifying the specific circuits sensitive to experience-dependent modulation will point toward therapeutic targets that allow amelioration of social processing deficits shared across of range of disorders. The objective of this study is to elucidate the PFC circuit mechanisms underlying social experience- dependent maturation crucial to mediate proper social behavior. In mice, juvenile social isolation leads to adult social behavior deficits and accompanies deficits in sub-cortically projecting deep layer medial PFC (mPFC) pyramidal neurons. Among various sub-cortical targets, our preliminary study identified the limbic thalamus which receives and relays signals to various components of the classical reward circuitry, as the most prominent projection target from mPFC that is preferentially recruited by social interaction. Importantly, juvenile social isolation reduced excitability of this projection neuron and increased their inhibitory drive. Among various types of inhibitory neurons, a selective subclass of deep layer inhibitory neuron, known to be essential to oscillate subcortically projecting deep layer cortical neurons, were the only population that exhibited elevated excitability after juvenile social isolation. This project will test the hypothesis that juvenile social experience- dependent maturation of deep layer mPFC projection neurons to limbic thalamus and its modulation by deep layer mPFC inhibitory neurons drives coordinated activity in mPFC and limbic thalamus to effectively modulate social behavior. We will test this hypothesis by integrating techniques to measure (fiber photometry imaging, patch-clamp/ in vivo electrophysiology) and manipulate (optogenetics/chemogenetics) the activities of selective circuits during social behavior in mice.

Project Summary: The decline of cortical plasticity due to closure of the juvenile-specific critical period is the key impedance of recovery from neurodevelopmental disorders and brain trauma in later life. Neuromodulatory systems are abundant in the adult cortex and well-positioned to orchestrate experience-dependent physiological events to prompt robust plasticity. However, an increasingly recognized complexity of neuromodulatory circuits as well as the diversity of neuron subtypes pose a challenge to identify a specific neuromodulatory system and their cortical target to precisely restore plasticity. The goal of this study is to identify novel molecular and circuit targets for inducing neuromodulatory changes in the adult brain, to reactivate juvenile-like plasticity for treating brain disorders with enduring functional impairments. Using ocular dominance plasticity, a prevailing primary visual cortex critical period plasticity model, We will test the hypothesis that, among various possible combinations of neuromodulatory systems, and their cortical targets, nicotinic ACh modulation and somatostatin expressing interneurons in the deep layer of primary visual cortex expressing specific type of nicotinic ACh sub-type as a novel combination of neurmodulatory circuit elements to induce rapid local circuit modulation to restore juvenile-like visual cortex plasticity and recovery from Amblyopia in adulthood. We will test this hypothesis by taking full advantage of the recently developed genetically-engineered mouse lines to achieve sub-population and cortical-layer-specific circuit-selective manipulation and measurement of gene expression or neural activity beyond conventional cell-type level analysis in combination of in vivo extracellular and in vitro slice electrophysiology with optogenetics, chemogenetics, and behavior assay. In Aim1, we will examine the contribution of specific nAChR subunit?on inducing experience-dependent rapid change of deep layer interneurons to trigger ocular dominance plasticity. In Aim2, we will dissect the excitatory and inhibitory circuit mechanisms regulated by deep layer SST interneurons to trigger ocular dominance plasticity. In Aim3, we will examine the extent of recovery from Amblyopia by modulating nAChR in deep layer interneurons. Successful completion of this project will illuminate new molecular and circuit mechanisms that gate the initial cascade triggering cortical plasticity, which will have direct implications for Amblyopia, a condition with limited adult-applicable treatment affecting 2?5% of the human population, but also for brain injury repair, sensory recovery, and the treatment of neurodevelopmental disorders with sensory perceptual deficits.