Wen-jun Gao - US grants

Persistent neural activity in the absence of external stimuliin prefrontal neurons is the hallmark of the brain's working memory systems. This property is thought to arise from mutual excitation between and among pyramidal neurons to generate local reverberatory neuronal activity. The failure or deficiency of this Imemory-related activation in vivo is hypothesized to be the cellular basis of cognitive impairment in Ischizophrenia. It has been determined that this process is subject to different influences by specific receptors I responding to dopamine, serotonin, GABA and glutamate. Accordingly, in Project 3, we employ the methods l of single and multiple whole cell recording with synaptic stimulation, pharmacological intervention, and calcium imaging to examine the synaptic and signaling basis of these modulatory influences, focusing on the dopamine system. Specific Aim 1 is directed at examining the differential modulatory influences of D1 and D2 receptors on excitatory transmission between identified neurons in layers 3 and 5, amplifying preliminary evidence of selectivity in different elemental cortical microcircuits (ECMs). Specific Aim 2 carries out similar experiments on synaptic augmentation, a form of short-term synaptic plasticity which recruits neuronal ensembles in specific ECMs and resembles persistent activity in vivo. The objective of Specific Aim 3 is to examine if D1 and D2 receptor effects are mediated by the cAMP-PKA pathway or the PLC-IP3-PKC pathways by selective pharmacological inhibition of PKA and PKC in specific ECMs, as described in Specific Aim 1 and in genetically altered systems as they become available. The advantages of in vitro approaches in providingcell type specific, subcellularly localized, and temporally resolved information which is difficult or impossible to obtain by in vivo methods will therefore complement the findings in other subprojects as well as represent a new and challenging level of analysis of dopamine's involvement in neural function and potential neuropathology.

[unreadable] DESCRIPTION (provided by applicant): Schizophrenia is probably the most distressing and disabling mental disorder. It affects almost 1% of the human population. Although the pathological processes of this disease is still not clear, remarkable progresses have been made in the past decades to solve this mystery. According to the postmortem studies of the brains of schizophrenia patients, it is now clear that certain inhibitory neurons in the forebrain are selectively damaged. In addition, a group of dissociative anesthetics, such as phencyclidine (PCP, Sernyl, also called 'angel dust') and its related compounds MK-801 and ketamine, can produce a wide range of schizophrenia-like symptoms such as estrangement, thought disorder, hallucination, and psychosis in both surgical patients and health volunteers. These drugs could also cause similar damage of inhibitory interneurons in the forebrain of animal models. Since these drugs are also NMDA receptor antagonists, a hypofunction hypothesis of NMDA receptors and inhibitory interneuron has thus been proposed for the schizophrenia pathological process. This intriguing hypothesis has attracted a great deal of attention, but the direct evidence in support of this hypothesis is still lacking. In this proposal, we hypothesize that the receptor properties of a subpopulation of interneurons in the prefrontal cortex are uniquely organized and these properties make them highly vulnerable to environmental factors that cause schizophrenia. The experiments listed in this proposal are designed to provide a comprehensive assessment of the receptor properties on the inhibitory circuitry which are evidenced as one of the most vulnerable parts of the brain in the schizophrenia pathological process. The experiments will be conducted in both normal and MK-801-treated animal models. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): N-methyl-D-aspartate receptor (NMDAR)-mediated glutamate transmission, along with dopamine (DA) and 3-aminobutyric acid (GABA) systems, has long been linked to schizophrenia, but all commonly prescribed antipsychotic agents act on DA receptors. Recent studies indicate that metabotropic glutamate receptor (mGluR) agonists reverse the behavioral effects of the NMDAR antagonist phencyclidine (PCP) and dizocilpine (MK-801) in animal models and in patients with schizophrenia. These studies suggest that mGluR2/3 receptor agonists have antipsychotic properties and may provide a new treatment of schizophrenia. This finding is exciting, but it raises some fundamental questions: Why do mGluR2/3 agonists have the same therapeutic efficacy as D2 receptor antipsychotic agents and by what mechanisms do mGluR2/3 agonists ameliorate behavior? We hypothesize that mGluR2/3 agonists restore the disrupted NMDAR function induced by the MK- 801 blockade by directly regulating the expression and trafficking of NMDAR subunits in the prefrontal circuitry. An integrated approach of in vivo pharmacologic agents, in vitro patch clamp recording, and molecular techniques will be used to test our hypothesis in the MK-801 animal model of schizophrenia. The proposed experiments will provide insights into the underlying mechanisms of mGluR regulation of NMDAR-mediated transmission and will contribute to a better understanding of how mGluR agonists reverse behavioral effects of NMDAR antagonists in animal models and of the underlying molecular pathophysiological characteristics and treatment of schizophrenia.

DESCRIPTION (provided by applicant): The goal of this multi-investigator project is to develop an animal model of HIV neuropathology that can be used to assess: 1) cognitive function, 2) neuronal and non-neuronal degeneration in the prefrontal cortex and 3) electrophysiological properties of cells and circuits in prefrontal cortical networks. With the advent of improved combination antiretroviral therapy, HIV infection has been transformed from a fatal illness to a chronic manageable condition. This trend has resulted in an increasingly large population of aging individuals with prolonged exposure to HIV neurotoxins and to HIV therapeutic interventions. While there are excellent tissue culture models for studying the impact of HIV or HIV therapy on cellular processes, the options for in vivo investigation of the effects o HIV infection or chronic antiretroviral therapy are more limited, particularly as they relate to th aging brain. The ideal model for investigating such issues would provide the opportunity to examine and correlate cognitive performance with electrophysiological indices of neural function and neuropathology across the aging continuum with respect to onset of the HIV infection and progression of ensuing disease processes. The work outlined in this proposal will focus on CNS exposure to the HIV envelope protein gp120 in adult and aged rats and its impact on 1) performance of two prefrontal cortex-dependent behavioral tasks, 2) neuronal excitability and synaptic transmission in the prefrontal cortical circuitry and 3) the degree of neurotoxic insult t neuronal and non-neuronal cells in the prefrontal cortex. The most important aspect of this investigation is the development of an animal model that will have advantages for numerous additional in vivo studies focusing on the broad array of potential agents and mechanisms associated with HIV infection and its treatment, the time course of these events, and their impact on the aging brain. In particular this model will facilitate the identification and development of new targets and new compounds for therapeutic interventions in adult and aging HIV/AIDS patients. Across all inquiries, the model will validate the findings of in vitro tissue culture studies and their relevance to normative functions in the intact central nervous system. PUBLIC HEALTH RELEVANCE: The goal of this multi-investigator project is to develop a model of HIV neuropathology that can be used to assess: 1) executive function in behaving animals, 2) neuronal and non-neuronal degeneration in the prefrontal cortex (PFC) and 3) electrophysiological properties of cells and circuits in PFC networks. Studies will be conducted in adult and aging rats. Specific experiments will focus on CNS exposure to the HIV envelope protein gp120 and characterize its impact on: 1) performance of two PFC-dependent behavioral tasks, 2) neuronal excitability and synaptic transmission in the PFC circuitry and 3) the degree of neurotoxic insult to neuronal and non-neuronal cells in the PFC. The proposed model will have advantages for numerous additional in vivo studies focusing on the broad array of potential agents and mechanisms associated with HIV infection and its treatment, the time course of these events, and their impact on the aging brain.

DESCRIPTION (provided by applicant): The role of NMDAR in the pathophysiological process of schizophrenia Abstract Schizophrenia (SZ) is increasingly recognized as a neurodevelopmental disorder with cognitive impairments often preceding the onset of psychosis, while the N-methyl-D-aspartate receptor (NMDAR) has long been associated with learning and memory processes, neurodevelopment, and SZ. Yet, the cause of the cognitive deficits and what initiates the pathological process are incompletely understood. Given the importance of NMDARs for cognitive functions, it is likely that NMDAR mis-regulation/dysfunction plays a critical role in the pathological process of SZ. In the past two decades, a remarkably convergent observation across several animal models of SZ and human postmortem studies is the phenomenon of NMDAR hypofunction. However, the vast majority of SZ- related research has focused on NMDAR function in adults, leaving the role of NMDARs during brain development unexplored. An important next step is to identify the mechanisms that cause NMDAR dysfunction with different insults during development. To address this issue, we have conducted some pilot studies. Our preliminary data indicated that along with working memory and learning deficits, protein levels of NMDAR subunits are significantly reduced in the prefrontal cortex and hippocampus, starting from the juvenile period and becoming more prominent during the adolescent period. Furthermore, there is a clear alteration in NMDAR-mediated current in the prefrontal neurons in both methylazoxymethanol (MAM)-exposed rat and DISC1 mutant mouse models during the early stage of development. Based on these observations, we hypothesize that NMDAR hypofunction begins in the early stage of postnatal development and progresses until adulthood. This process is universal to different animal models. Correcting NMDAR hypofunction in the early stage (juvenile period) would be effective to restore glutamatergic synaptic transmission and thus to rescue cognitive deficits. Using a combination of molecular, biochemical, and physiological techniques, along with behavioral tests, in Aim 1 we will determine the time course of NMDAR mis-expression and dysfunction in the prefrontal cortex and hippocampus during postnatal development; as well as testing learning and memory functions in both MAM-exposed rats and inducible DISC1 mutant mice. In Aim 2 we will investigate the mechanisms underlying NMDAR dysfunction during postnatal development, focusing on transcriptional repression by epigenetic remodeling and signaling pathways involved in NMDAR downregulation. In Aim 3 we will determine whether pharmacologically correcting NMDAR hypofunction in the early stage (juvenile period) of development is able to restore NMDAR functions and thus rescue learning and memory deficits in MAM-exposed rats and DISC1 mutant mice. We believe that these experiments will elucidate the progression of NMDAR hypofunction, provide mechanistic insight into its cause, and generate possible new avenues for therapeutic intervention. Furthermore, the results would provide an interesting platform for exploring how early NMDAR hypofunction contributes to cognitive deficits in SZ and will address the very important conceptual question of whether early stage treatment is able to prevent the progression or reverse the cognitive deficits associated with this disease.

Role of prefrontal cortical dopamine in aggression Abstract Aggression is a complex social behavior that may impose a significant toll on society. Although the mechanisms remain elusive, the prefrontal cortex (PFC) exhibits top-down inhibitory control of aggressive behaviors. However, a fundamental question is how neurons in the PFC control aggression versus normal social interaction and how these neurons are regulated by dopamine (DA), a major neurotransmitter for emotional control. One mechanism through which this may occur is activation of neurons with differential DA receptor expression profiles. Recent studies have demonstrated a segregation of D1R- and D2R-expressing neurons in the PFC. Since D2R antagonists are effective in the treatment of aggressive behaviors, we predict that these divergent D1R- or D2R-expressing neurons are likely endowed with different synaptic connectivity and afferent inputs to enable the execution of distinct regulation in social and aggressive behaviors, respectively. Indeed, our preliminary data indicate that both aggression and normal social interaction induced significant increases in levels of c-fos expression compared with asocial groups in the PFC; but interestingly, episodes of attack significantly increased c-fos expression in D2R-expressing neurons, whereas basal social interaction clearly increased c-fos expression in D1R-expressing neurons in the PFC. Based on these observations, we hypothesize that prefrontal DA regulates social behaviors via differential effects on D1R- and D2R-expressing neurons within PFC circuitry. Specifically, DA promotes basal social interaction through activation of D1R-expressing neurons, but triggers aggressive behavior by activating D2R-expressing neurons. We will use Cre-dependent transgenic mice combined with pharmacogenetics to test this hypothesis. In Aim 1, we will identify the roles of D1R- and D2R-containing PFC neurons in social and aggressive behaviors, and then characterize their neuronal and synaptic properties that are differentially affected by social interaction and aggression, respectively. In Aim 2, we will determine whether pharmacogenetically manipulating the functionality of D1R- and D2R-expressing cells within the PFC is capable of promoting social interaction or controlling aggressive behavior. This proposal will determine the role of the PFC in aggression control and dissect the specific effects of DA receptor subtypes on social behaviors in a cell-type specific manner. Our study will bridge a gap in the literature given that we aim to offer causative evidence of the ?social neural circuit? containing prefrontal D1R- and D2R-expressing neurons in regular social interaction and escalated aggression.

Targeting parvalbumin neurons in the PFC for cognitive deficits Abstract: Treatment-resistant cognitive deficits in schizophrenia (SZ) represent a significant clinical burden and are the primary predictor of functional outcome in individual patients. Although the mechanisms associated with cognitive deficits remain unclear, pathological GABAergic signaling likely plays an essential role. Deficits in parvalbumin (PV)-containing fast-spiking (FS) interneurons in the prefrontal cortex (PFC) are consistently found in post-mortem tissue of patients with SZ. In addition, the observation of decreased PV cells has been replicated across a wealth of SZ animal models, suggesting this pathology may represent a convergence point in this disorder. Specifically, our studies have shown a significant decrease of synaptic inhibition and PV interneurons in the PFC, along with working memory and learning deficits in both rat methylazoxymethanol acetate (MAM) exposure and NMDA receptor antagonist MK-801 models for SZ, analogous to endophenotypes seen in clinical populations. These findings not only highlight the importance of PV neurons in the pathological processes of SZ, but also a potential causal link between the impaired PV cells and cognitive deficits. However, it remains unclear whether directly targeting PV neurons in the PFC is capable of ameliorating cognitive deficits. We hypothesize that decreased inhibitory neurotransmission in the PFC underlies cognitive deficits in these two animal models of SZ, whereas augmenting the activity of the remaining PV interneurons will alleviate the cognitive impairments by adjusting excitation/inhibition balance in the PFC circuitry. Thus, the goal of this study is to improve cognitive performance by utilizing a targeted pharmacogenetic upregulation of PV interneuron activity, with a novel excitatory DREADD. This will follow electrophysiological tests to elucidate the mechanisms through which the DREADD affects prefrontal cortical function in both neurodevelopmental and pharmacological models for SZ. This study will have translational implications for clarifying how disrupted inhibitory circuitry plays a role in cognitive deficits in SZ and will help to answer a fundamental question of whether cognitive impairment can be improved by enhancing the neuronal activity of a specific subtype of GABAergic interneurons in the PFC. Further, it will provide important insight into treating other disorders with cognitive symptoms.

Behavioral Effect of IgSF9b in the Prefrontal Cortex Abstract IgSF9b was recently identified as a high-risk gene strongly associated with negative symptoms of schizophrenia (SZ) and major depressive disorders (MDD). IgSF9b gene codes for adhesion protein IgSF9b, which is specifically expressed in the brain, especially in the forebrain region of the neocortex. In vitro studies indicate IgSF9b is primarily expressed in GABAergic interneurons and subset of pyramidal neurons, with coupling to neuroligin 2 to promote inhibitory signaling. However, whether and how IgSF9b deficiency affects cognitive and social behaviors, especially those related to social withdrawal and anhedonia, remains untested. We endeavored to explore the functional roles of protein IgSF9b in the medial prefrontal cortex (mPFC) in the regulation of motivational and social behaviors as this brain region is highly associated with executive function, cognition, and social behavior control, as well as deficits related to depression and SZ. Our studies aim to elucidate the fundamental physiological role of protein IgSF9b, at a cellular, circuit, and behavioral level. In our pilot study, we performed successful knockdown (KD) of protein IgSF9b in the young adult mouse mPFC, and interestingly, we found that IgSF9b KD significantly impairs both social preference and social memory. We hypothesize that selective KD of IgSF9b impairs behaviors associated with social withdrawal and anhedonia by reducing inhibition and affecting neurons projecting to subcortical areas related to social behavior such as nucleus accumbens (NAc). We will first evaluate the effects of IgSF9b KD in the mPFC on motivational and social withdrawal behaviors and then determine whether IgSF9b KD in the mPFC selectively affects the mPFC-NAc pathway to regulate social behaviors. IgSF9b is mainly expressed in the forebrain of the neocortex and is proposed to be highly associated with negative symptoms. Therefore, examining the effects of IgSF9b on mPFC-associated behaviors has the potential to elucidate unidentified mechanisms of behavioral deficits seen in neuropsychiatric diseases such as SZ.