Elucidating the mechanisms underlying ketamine-induced protection against fear overgeneralization

PROJECT SUMMARY / ABSTRACT Though stress is necessary for the adaptive survival of a species, stress exposure can also elicit maladaptive physiological and behavioral responses. Chronic stress in particular often leads to maladaptive responses, and subsequent development of psychiatric disorders such as post-traumatic stress disorder (PTSD) and major depressive disorder (MDD). A core symptom observed in these disorders is increased fear generalization, as defined by the overgeneralization of fear from a conditioned, fear-inducing stimulus to novel, neutral stimuli. We have previously discovered that a single injection of (R,S)-ketamine, a rapid-acting antidepressant, protects mice against stress-induced depressive-like behavior (Brachman et al., 2016) and attenuates learned fear following contextual fear conditioning (CFC) (McGowan et al., 2017). We have also found that, using a contextual fear discrimination (CFD) protocol, (R,S)-ketamine decreases fear generalization (Mastrodonato et al., 2018). The Denny laboratory has reported that the ventral hippocampus (vHPC), specifically ventral CA3 (vCA3), mediates (R,S)-ketamine's effects on attenuating learned fear in the CFC paradigm. However, how exactly (R,S)-ketamine modulates the ensembles in vCA3 to decrease fear generalization is yet to be explored. This research plan will lay the groundwork to uncover the effects of (R,S)-ketamine administration on fear generalization throughout CFD using a combination of in vivo microdialysis and in vivo Ca2+ imaging in vCA3. Both of these in vivo techniques will allow for chronic assessment of underlying changes throughout (R,S)- ketamine administration and during behavioral expression (e.g., fear overgeneralization). In Aim 1, I will test the hypothesis that neurochemical changes in glutamate, gamma-aminobutyric acid (GABA), and serotonin (5- HT) in vCA3 potentially mediate (R,S)-ketamine's effects on fear generalization in male and female mice. By using in vivo microdialysis, I will determine how neurotransmitters relevant to learning and memory are contributing to fear generalization in both saline (control)-injected and (R,S)-ketamine-injected mice. I will test the hypothesis that glutamate and 5-HT in vCA3 are persistently increased, while GABA is decreased, during expression of fear in saline-treated mice, but that this persistent increase is mitigated by (R,S)-ketamine treatment. In Aim 2, I will utilize nVoke minimicroscopes developed by Inscopix to perform in vivo Ca2+ imaging and will test the hypothesis that (R,S)-ketamine prevents the heightened excitatory Ca2+ activity in vCA3 induced by stress, and thus, decreases fear generalization in male and female mice. For Aim 2A, I will visualize Ca2+ transients in excitatory cells of vCA3. For Aim 2B, I will visualize Ca2+ transients in inhibitory cells of vCA3. To date, no longitudinal studies utilizing in vivo Ca2+ imaging or in vivo microdialysis studies have yet been performed for fear generalization and/or following (R,S)-ketamine administration. Overall, my goal for this proposal is to better understand how (R,S)-ketamine may alter neurotransmitters and neural ensembles, resulting in decreased fear generalization, a core symptom in PTSD and MDD patients.