While most of the general population, and particularly combat veterans, experience traumatic events, only a relatively small proportion go on to develop post-traumatic stress disorder (PTSD), suggesting there are individual differences in susceptibility to the long-term consequences of traumatic stress. PTSD patients also show impairments in fear extinction, extinction recall, and safety learning, so extinction learning processes form the basis of exposure-based therapies. We have observed individual variation in the behavioral (freezing) and cardiovascular responses during the extinction of cued fear memories in outbred rats, providing a model to study PTSD-like resistance to fear extinction. Our overarching goal is to define the mechanistic role of cholinergic system, and specifically muscarinic receptors (mACHRs), in differentially regulating the cortico- amygdalar circuit to induce individual differences in extinguishing fear memories. Cholinergic inputs from the basal forebrain (BF) provide strong inputs to the prefrontal (PFC)-amygdalar circuit which is important for fear extinction. Preliminary data in our rat model suggests that individual differences in cued fear extinction are related to differences in cholinergic neurotransmission and function, but particularly mACHR regulation in the basolateral amygdala (BLA). Our hypothesis is that muscarinic receptor activation in the amygdala decreases excitatory inputs from the PFC to diminish fear extinction learning in the resistant (high freezing) phenotype, and thus extinction learning can be enhanced specifically in this extinction resistant population with mACHR antagonists. All experiments compare individual differences in male and female rats with distinct extinction phenotypes. Using a multidisciplinary approach, three Aims explore the ability of pharmacological approaches to enhance extinction learning or recall in the extinction resistant phenotype, and provide a mechanistic view of the neurobiological effects underlying these effects.
Aim 1 tests the therapeutic potential of mACHR antagonists, agonists, and positive allosteric modulators in shifting fear extinction, with a focus on M1 and M4 selective compounds. We will examine extinction of conditioned freezing, ultrasonic vocalizations, and cardiovascular responses during extinction learning and recall with both systemic and intra-amygdalar injections of mACHR compounds.
Aim 2 examines if the differential release of acetylcholine from the basal forebrain and/or acetylcholinesterase activity mediates differences in fear extinction. In vivo microdialysis will be used to assess acetylcholine and glutamate efflux, and a transgenic rat model will be used for optogenetically stimulating or inhibiting basal forebrain cholinergic inputs to examine effects in our extinction phenotypes.
Aim 3 examines muscarinic modulation of excitatory responses and synaptic plasticity evoked by stimulation of PFC inputs using optogenetic and electrophysiological approaches ex vivo in amygdalar brain slices. Pharmacological manipulations of mACHRs will be used in conjunction with optogenetic stimulation of the infralimbic inputs critical for fear extinction, while recording from BLA pyramidal neurons. These studies will focus on amygdalar responses associated with PFC inputs, due to the strong cholinergic innervation to the BLA from the basal forebrain, as well as being downstream from PFC for mediating behavioral outputs. These studies will provide evidence for mACHR based pharmacological interventions that might enhance extinction- based approaches such as exposure therapy, plus provide a mechanistic profile of how muscarinic agents may be acting within the neural circuitry underlying fear learning and extinction in both male and female rats.

Public Health Relevance

to VA: The proposed studies use innovative technologies and a translational animal model to help establish evidence-based medical practices for treatment of PTSD & stress disorders in men and women veterans. The VA?s ORD supports a strong program of research toward understanding, treating, and preventing the neurobiological responses to a traumatic event during combat duty. While most combat veterans experience traumatic events, only a relatively small proportion go on to develop PTSD, suggesting some individuals are more susceptible to these stressors. The proposed studies use an animal model showing similar individual differences in traumatic stress responses, to better understand the neurobiological mechanisms underlying inability to reduce fear memories during repeated exposure to trauma cues, even in a safe environment. Further, we use this model to test pharmacologic approaches that could augment the effectiveness of treatment approaches, such as exposure therapy, for veterans.

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Veterans Affairs (VA)
Non-HHS Research Projects (I01)
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