Analysis of cFos expression and microglial branch density in a mouse model of trauma-induced hyperarousal
Post-traumatic stress disorder (PTSD) is a severe psychiatric condition that is characterized by a failure in recovery following a traumatic event. A key feature of PSTD is persistent symptoms of hyperarousal, including an exaggerated startle responses and increased sympathetic tone. Current treatment options for PTSD have been largely unsuccessful in treating these symptoms and this could likely be attributed to a gap in the knowledge of the biological underpinnings of these more physiological symptoms. Previous research has supported the idea that dysregulation of noradrenergic signaling contributes to the presentation of the hyperarousal symptoms of PTSD. I hypothesized the Nucleus of the Solitary Tract (NTS), a major source of norepinephrine in the brainstem, may experience a change in neurotransmission or glial activation in response to trauma, contributing to the pathophysiology of hyperarousal in PTSD. These changes in noradernergic signaling may also be reflected downstream in the projection targets of the NTS. Two candidate targets include the central nucleus of the amygdala (CeA) and paraventricular nucleus (PVN) of the hypothalamus, two nuclei involved in the output of the fear response and regulation of the Hypothalamic-Pituitary-Adrenal (HPA) axis, respectively. Hyperarousal symptoms were induced in mice using the Traumatic Experience with Reminders of Stress (TERS) paradigm. Male and female C57Bl/6J mice were randomly assigned to control (N = 27; 14 Male, 13 Female) or TERS (N = 53; 27 Male, 26 Female). TERS mice experienced a single exposure to a traumatic foot shock (10s, 2 mA) followed by six, one-minute, contextual reminders over 24 days. Mice that underwent the TERS paradigm demonstrated enhanced acoustic startle (p=0.0120, Mann-Whitney test) and elevated heart rate (p=0.0271, Mann-Whitney test). No sex differences were observed in either measure. TERS and control mice were transcardially perfused ninety minutes after a final acoustic startle measurement. Using immunohistochemistry, brain sections containing the NTS were stained for tyrosine hydroxylase (TH) as a biomarker for noradrenergic neurons, and the immediate early gene cFos as an indicator of neuronal activation. PVN and CeA sections underwent cFos staining. cFos expression was quantified in the PVN and CeA, as well as the basolateral amygdala (BLA) since previous research has established a central role of the BLA in fear circuitry. Different trends were shown between control and shocked mice in neuronal activation of the BLA and PVN. However, further experimentation will be required with additional animals to determine the potential significance of these trends. In additional sections from the NTS, CeA, and BLA staining for Iba-1 was performed. Iba-1 is a calcium binding protein specific to macrophages, including microglia. High resolution laser scanning microscope images were taken at 40x magnification to quantify microglial somas and branch densities with ImageJ. Regional differences were observed in the average number of microglial somas, but no significant variation was present between TERS groups. A main finding was susceptible TERS mice displayed reduced microglial branch density in the NTS compared to the control and resilient mice, indicating a higher percentage of microglia may be activated. In the BLA, susceptible mice exhibited hyper-ramified microglia, indicated by higher branch densities compared to controls. The microglial adaptations indicate persistent effects of trauma on the neuroimmune system and point towards the presence of regional heterogeneity in microglial function.