Sachin Patel, M.D., Ph.D. - US grants

Psychosocial stress is a major risk factor for the precipitation and exacerbation of mental illness in susceptible individuals. Understanding the neuroadaptations induced by chronic stress could afford new opportunities for therapeutic intervention for stress-related psychiatric disorders. The candidate has shown that levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) exhibit a progressive increase in response to repeated stress exposure in limbic brain regions including the amygdala, and that this increase contributes stress-response habituation. In aim 1 of this proposal the candidate will determine the temporal dynamics of the 2-AG response to stress and the molecular mechanisms subserving these effects. The candidate will test the hypothesis that the stress hormone corticosterone is required for the adaptations in endogenous cannabinoid signaling to occur in response to repeated stress exposure. Although stress increases 2-AG in the amygdala, it is not known if this increase is associated with enhanced endocannabinoid-mediated synaptic signaling. In aim 2 the candidate will test the hypothesis that this stress-induced increase in 2-AG in the amygdala is associated with enhanced capacity of amygdala neurons to participate in endocannabinoid-mediated synaptic signaling. Finally, in aim 3, the candidate will test the hypothesis that stress-induced increases in 2-AG levels contribute to the behavioral dysregulation induced by chronic stress. Elucidating the stress-induced adaptations in endocannabinoids signaling could provide novel molecular targets for drug development for the treatment of affective disorders.

DESCRIPTION (provided by applicant): Mood and anxiety disorders exert substantial societal cost and personal burden for patients and families. Current treatment approaches for these disorders are primarily focused on increasing brain neurotransmitters like serotonin, i.e. via selective serotonin-reuptake inhibitors (SSRIs). However, large-scale effectiveness studies have demonstrated this class of drugs is only partially effective. Recent studies have suggested that elevating levels of endogenous brain cannabinoids could exert therapeutic benefit in mood and anxiety disorders including PTSD. We have recently developed novel inhibitors of cyclooxygenase-2 (COX-2) that selectively prevent inactivation of brain endocannabinoids without preventing synthesis of prostaglandins; which are inflammatory mediators required for normal immune and vascular function. Given that long-term inhibition of prostaglandin synthesis is associated with gastrointestinal and cardiovascular toxicity, development, validation, and preclinical evaluation of novel pharmacological strategies to modulate COX-2 activity to enhance endocannabinoid signaling without affecting prostaglandin synthesis is a high research priority. We will test the hypothesis that substrate-selective inhibitors of COX-2 selectively increase brain endocannabinoid levels without affecting prostaglandin levels and that they exert preclinical efficacy in models of mood and anxiety disorders. Completion of these studies will provide preclinical evidence for the efficacy of a novel class of antidepressant and anxiolytic drugs, and could validate COX-2 as a viable molecular target for future drug discovery directed at the treatment of affective disorders.

DESCRIPTION (provided by applicant): The central amygdala (CeA) is a key nodal brain structure that integrates autonomic, arousal, and sensory information to initiate fear and anxiety responses to appropriate salient stimuli. Amygdalar activity links environmental stress to the development of psychopathological states in susceptible individuals, and dysregulation of amygdala function has been demonstrated in numerous affective disorders including major depression, and anxiety disorders such as posttraumatic stress disorder. Thus, understanding the cellular and synaptic organization of the amygdala, and the mechanisms regulating information processing in this region could provide important insights into the mechanisms regulating fear and anxiety generation and stress response physiology. Moreover, understanding the synaptic adaptations induced by stress exposure could reveal novel mechanisms contributing to the development of stress-related neuropsychiatric disorders. Here we aim to elucidate the synaptic organization of the CeA using optogenetic projection-targeting approaches in order to uncover synaptic and functional anatomical mechanisms by which different excitatory inputs to the CeA could exert differential control of anxiety states via cell type-specific targeting. We will determine the role of endogenous cannabinoid signaling in the cell-type- and afferent-specific modulation of excitatory drive to the CeA. Lastly, we will test th hypothesis that cell-type specific adaptations in endogenous cannabinoid signaling counteract stress-induced synaptic remodeling associated with anxiety states. These studies will provide an unprecedented understanding of the synaptic organization of the CeA and elucidate endogenous cannabinoid signaling mechanisms regulating glutamatergic drive to CeA neurons in an afferent and cell type-specific manner. These studies could also reveal novel synaptic adaptations in endocannabinoid signaling that could serve a homeostatic function aimed at normalizing stress-induced anxiety states. Understanding the synaptic and molecular mechanisms regulating CeA function could ultimately advance our understanding of the pathophysiological mechanisms subserving mood and anxiety disorders in humans.

DESCRIPTION (provided by applicant): A significant number of individuals who start smoking cannabis develop a cannabis use disorder (CUD) and seek treatment on their own initiative, yet only a small percentage achieves sustained abstinence. Efficacious treatments for CUD are critically needed. Here we propose a proof-of-concept, human laboratory study to test a novel and innovative pharmacological strategy to reduce cannabis withdrawal and facilitate cannabis abstinence in chronic cannabis smokers. In recent preclinical studies, we have identified cyclooxygenase-2 (COX-2) as a key enzyme that inactivates brain endocannabinoids, and have demonstrated that non-steroidal anti-inflammatory drugs (NSAIDS) that block COX-2 activity can increase central endocannabinoid levels and subsequent endocannabinoid activity at the CB1 receptor. Importantly, another recent study showed that THC administration increases brain COX-2 expression via CB1 activation. Based on these two recent studies, we propose the novel hypothesis that chronic THC exposure up-regulates COX-2 expression, which in turn inactivates endogenous cannabinoids, resulting in an endocannabinoid-deficient state. We hypothesize that this endocannabinoid deficiency contributes to the negative reinforcement driving continued cannabis use, and that blocking COX-2 activity will normalize this endocannabinoid deficiency, reduce withdrawal symptoms, and reduce relapse to drug taking. The primary objective is of this application is to test the efficacy of the FDA- approved COX-2 selective inhibitor, celecoxib (200 mg BID), on discrete features of cannabis use in the human laboratory: positive subjective effects ('good drug effect', cannabis 'liking'), withdrawal (mood, sleep, food intake), and relapse (cannabis self-administration after a period of abstinence). Participants will be non- treatment-seeking daily cannabis smokers who will complete two inpatient study phases, with one 11-day phase testing celecoxib and the other 11-day phase testing placebo maintenance, with treatment condition counter-balanced across participants. A secondary objective is to measure plasma levels of endogenous cannabinoids following acute cannabis administration and withdrawal as a function of celecoxib treatment, to determine if the behavioral effects of celecoxib are related to increased plasma levels of endogenous cannabinoids. We hypothesize that celecoxib will reduce the severity of cannabis withdrawal symptoms and will reduce our laboratory measure of cannabis relapse while producing few adverse events. We also hypothesize that plasma endocannabinoid levels will be increased by celecoxib treatment relative to placebo and will be negatively correlated with withdrawal severity. If successful, thes studies will ultimately introduce COX-2 inhibition as a novel pharmacological strategy for the treatment of CUD. Since celecoxib is an FDA- approved drug and well-tolerated, rapid translation of these data to treat CUD can be easily envisioned.

PROJECT SUMMARY Stress is a major risk factor for the development of mood and anxiety disorders and the causative agent in posttraumatic stress disorder (PTSD). Development of stress-related psychopathology is variable among individuals and involves complex interactions between susceptibility mechanisms favoring development of psychopathology and resiliency mechanisms protecting against the development of mental illness in the face of adversity. Understanding the molecular, cellular, and circuit-level mechanisms by which stress exposure is translated into affective pathology could have broad implications for understanding the pathophysiology of stress-related psychiatric disorders and for the development of novel treatment approaches. We propose to test the overarching hypothesis that the endogenous cannabinoid 2-Arachidonoylglycerol (2-AG) is a critical regulator of stress adaptation and propose to elucidate the synaptic and cellular mechanisms by which 2-AG signaling serves to mitigate pathological responses to stress exposure. We will first test the hypothesis that 2-AG signaling inhibits bi-directional excitatory coupling between the amygdala and ventral hippocampus, a neural circuit critical for mediating innate danger avoidance and generalization of learned fear responses. We will test the hypothesis that stress causes a functional collapse of 2-AG signaling within this circuit leading to synaptic strengthening, the generation of increased avoidance, and fear generalization. We will also test the hypothesis that stress exposure leads to increases in 2-AG release within distinct amygdala circuits in a neural activity-dependent manner using a novel virally encoded GPCR-based endocannabinoid biosensor. These studies will reveal, for the first time, the temporal dynamics and activity-dependent mechanisms mediating stress-induced endocannabinoid mobilization within amygdala circuits. Lastly, we will use in vivo single-cell calcium imaging approaches to test the hypothesis that 2-AG is required for the dynamic changes in neuronal ensemble representations to threat predictive cues that occur during fear learning, expression, generalization, and extinction. Taken together, these data will provide new mechanistic insight into how 2-AG signaling regulates stress-related biobehavioral processes relevant to neuropsychiatric disorders including PTSD and could reveal novel pathophysiological mechanisms contributing to the translation of stress exposure into affective pathology.

PROJECT SUMMARY  Alcohol  use  disorders  (AUDs)  manifest  from  a  convergence  of  characteristics  of  the  individual,  the  environment,  and  the  alcohol  itself.    The  affective  disturbances  associated  with  alcohol  withdrawal  are  examples  of  this  convergence  and  represent  a  critical  barrier  to  successful  treatment.  Reduction  of  these  affective disturbances has been suggested to represent an important conceptual approach to reduce negative  reinforcement-­based  alcohol  intake  in  dependent  individuals.  However,  treatment  of  these  affective  disturbances is complicated by data indicating reduced efficacy of antidepressants such as selective-­serotonin  reuptake  inhibitors  (SSRIs)  in  patients  with  AUDs,  and  that  these  traditional  treatments  can  actually  increase  alcohol  intake  in  some  people;?  thus,  alternate  non-­monoamine-­based  treatment  approaches  for  affective  symptoms  associated  with  AUDs  are  critically  needed.    Here  we  will  test  the  novel  hypothesis  that  pharmacological augmentation of endogenous cannabinoid signaling could represent an effective treatment for  negative  affective  states  associated  with  alcohol  withdrawal  including  anxiety  and  depression,  and  could  thereby  facilitate  abstinence  in  patients  with  AUDs.  We  will  test  the  overall  hypothesis  that  2-­ arachidonoylglycerol  (2-­AG)-­mediated  endocannabinoid  signaling  reduces  anxiety  and  depressive-­like  behaviors  associated  with  acute  and  protracted  alcohol  withdrawal  in  a  mouse  model  of  voluntary  alcohol  consumption.  To  interrogate  the  underlying  mechanism  of  this  effect,  we  will  test  the  hypothesis  that  insula  cortical-­extended  amygdala  circuits  are  hyperactive  during  alcohol  withdrawal  and  that  over  activation  of  this  circuit is causally linked to the affective phenotypes observed during alcohol withdrawal. Finally, we will test the  hypothesis  that  2-­AG-­mediated  inhibition  of  insula-­extended  amygdala  circuit  activity  represents  a  key  mechanism by which endocannabinoid signaling reduces alcohol withdrawal-­induced anxiety and depressive-­ like  behaviors.    These  data  could  provide  new  insight  into  the  neural  circuit  mechanisms  responsible  for  generating  negative  affective  states  associated  with  alcohol  withdrawal,  and  reveal  novel  neuromodulatory  mechanisms  capable  of  counteracting  these  processes.  If  successful,  these  studies  could  support  advancement of 2-­AG-­based pharmacological treatments for AUDs and co-­morbid affective disorders.       

? DESCRIPTION: Since initial funding of this grant in May 2000, research literature cited by PubMed on cannabinoid and endocannabinoid physiology and pharmacology has expanded by two orders of magnitude, reflecting the expansion of basic science in this field. Findings have potential clinical applications in drug abuse and addictions, metabolic disorders, mental health and neurodegenerative diseases, and pathologies in other organ systems of the body. The International Cannabinoid Research Society's (ICRS) annual Symposia on the Cannabinoids is the single comprehensive meeting in which leading international laboratories disclose critical research findings on cannabinoid and endocannabinoid physiology and pharmacology well in advance of publication of the data. Participation of trainees as well as established research investigators ensures future progress. The goal of this grant is to facilitate trainee attendance a the symposia and their retention in the field by providing travel awards, student presentation awards and career development programs. In addition, the ICRS proposes to host an annual translational symposium with invited speakers. Symposia abstracts are archived on the ICRS website and as a citable document available to the public.

PROJECT SUMMARY Acquisition and extinction of learned fear responses are critical for survival and require modification of conserved neural circuits that promote or suppress fear expression, respectively. Disruption of these important physiological processes are thought to underlie the development of stressor and trauma- related disorders including posttraumatic stress disorders. Understanding the neuronal circuits and synaptic mechanisms regulating fear memory formation and extinction could have important implications for elucidating pathophysiological mechanisms of stress-related disorders and provide insight into fundamental mechanisms subserving learning and memory processes. The central nucleus of the amygdala (CeA) is a striatal-like subcortical brain structure that sits functionally at the limbic-motor interface. Recent studies have identified distinct cell-types within this region that are sufficient to generate diverse survival-oriented behavioral responses including freezing, flight, hunting, and feeding. Relevant to the current proposal, very recent work has identified the CeA as a novel locus of fear-learning and identified distinct cell-types responsible for generating learned fear responses in the form of freezing and flight-like escape behavior. Despite these advances, how top-down cortical signals are able to select distinct behavioral outputs via targeted activation of different CeA cell-types is not well understood. Here we will utilize a combination of cutting-edge cell-type- specific electrophysiological, optogenetic, chemogenetic and viral reporter approaches combined with a Pavlovian fear-conditioning and extinction paradigm to gain insight into this critical open question. Aim 1 of this proposal will test the hypothesis that acquisition and extinction of conditioned fear responses is associated with dynamic shifts in the relative glutamatergic drive from the basolateral amygdala (BLA) to CeA corticotrophin releasing factor expressing (CRF+) and somatostatin-expressing (SOM+) neurons. We hypothesize that fear acquisition shifts BLA excitatory drive to favor SOM+ neurons, which have been shown to drive conditioned freezing behavior, while extinction learning reverses the relative input bias from the BLA to favor CRF+ neurons, which we show facilitate extinction of conditioned freezing behavior. Aim 2 will test the requirement for neuronal activity in the induction of this form of experience-dependent plasticity and its necessity for the expression of conditioned fear and extinction using circuit-specific chemogenetic and behavioral approaches. Aim 3 will test the hypothesis that retrograde endocannabinoid signaling is an important mediator of experience-dependent changes in synaptic strength between the BLA and CeA CRF+ and SOM+ neurons, and that modulation of eCB signaling within the BLA-CeA-SOM+ circuit promotes fear extinction. Completion of these studies will provide novel insight into the circuit and cell-type-specific mechanisms regulating fear acquisition and extinction and enhance our understanding of the pathophysiology of stressor and trauma- related psychiatric disorders.

PROJECT SUMMARY Cannabinoid signaling is composed of Cannabinoid receptors CB1 and CB2 and their cognate exogenous and endogenous ligands. Cannabinoid signaling is widely distributed throughout the CNS and periphery and is implicated in a wide variety of important neurobiological processes including regulation of energy homeostasis, learning and memory, motor function, reward, anxiety, and social function. Moreover, use of cannabis has increases over the past decade and is expected to continue to increase in the face of the expanding anti-prohibition movement in the United States. Cannabis use by vulnerable populations and during critical periods, such as adolescence and pregnancy, carry unclear risk and are the focus of intense ongoing scientific investigation. Commensurate with these growing potential public health concerns, research into the fundamental biology of cannabinoids and endogenous cannabinoids related to brain development, function, and pathology are becoming of greater interest to the public, policymakers, researchers and healthcare professionals. Therefore, understanding the neurobiology of cannabinoid signaling could have broad implications for physiological function and the pathophysiology of somatic and neuropsychiatric disorders. The International Cannabinoid Research Society (ICRS) is the only annual scientific meeting devoted to dissemination of scientific information on all aspects of cannabinoid sciences. The ICRS will hold its 31st annual symposium in 2021 and has been an unparalleled success in the field of cannabinoid research. During the previous funding cycle, NIH support has allowed the ICRS to provide substantial travel support to increase trainee attendance at our annual meeting. The student travel grant program will be used to increase the participation of early stage cannabinoid researchers in the ICRS Symposia on the Cannabinoids. Special emphasis has been, and will continue to be, placed on support for individuals traditionally underrepresented in science, including women and U.S. racial and ethnic minorities. In addition to the travel award program, we propose to provide a career development program at each annual ICRS symposium, recognize outstanding predoctoral and postdoctoral student presentations with awards given during each symposium, and publish an abstract book in printed form that will be made available to the public on the ICRS website after the annual symposium. Completion of these aims will continue to invigorate the next generation of cannabinoid scientists by facilitating attendance and presentation of their original research in an open, inclusive, and egalitarian forum and provide networking and mentorship opportunities to young scientists in all areas of cannabinoid research.