Investigating the Neural Circuitry of Learned Safety Expression in Rats

The prevalence of clinical conditions rooted in anxiety and trauma, such as posttraumatic stress disorder (PTSD), underscores the need for better understanding of the neural mechanisms involved with fear/threat and safety expression. The neural circuitry of fear learning and memory expression is well understood. However, investigations of the neural mechanisms of safety learning and its expression are limited. The paraventricular nucleus of the thalamus (PVT) and the lateral septum (LS) both contribute to resolving motivational conflict. There are robust, bidirectional projections between the PVT and the LS and both regions are implicated in the expression of learned safety. In the present project, we first demonstrated safety learning in a large sample of rats using a discrete safety stimulus in the presence of a threatening context. Male and female rats showed reduced freezing in the threatening context in the presence of the safety stimulus. Next, we selected a subset of males and females from threat and safety training conditions based on their performance. “Threat” animals were not trained using the safety stimulus and we selected a subset of animals that showed no change in freezing during the safety stimulus presentation in the test session. The “high safety” subset of animals were those safety-trained animals that showed the greatest decrease in freezing during the presence of the safety stimulus, compared to the context-alone period. The “low safety” subset of animals were those safety-trained animals that showed the least change in freezing during the safety stimulus, compared to the context-alone period. We evaluated c-Fos immunoreactivity in specific subregions of the PVT and LS. In the anterior and posterior PVT and dorsal/intermediate regions of LS, safety-trained animals showed reduced c-Fos expression compared to threat-trained animals, regardless of whether they demonstrated “high-safety” or “low-safety” in their freezing behavior during the test.

123 adult Long-Evans rats (male and female) underwent surgery to infuse retrobead tracers (not used in this study). One month following surgery, each rat was exposed to two sessions of light/tone stimulus and context in the absence of footshock (habituation). Following habituation, rats underwent safety training across two consecutive days. Rats were presented with 5 light/tone stimuli and 5 footshocks per session. Footshocks were explicitly unpaired from the light/tone presentations.  On the next day, each rat underwent testing. During the test, rats were either presented with 5 light/tone stimuli in the absence of footshock or in absence of all stimuli. 90 minutes after completion of the testing, the rats were perfused and brains harvested for subsequent c-Fos analysis.To assess safety expression, a difference score was calculated (safety stimulus freezing minus pre-stimulus freezing). Immunohistochemistry was used to stain tissue samples, which were then Brightfield imaged using a Leica microscope at 10x magnification. A subset of brains were used and categorized into “threat,” “low safety,” and “high safety” based on freezing performance. Image analysis was conducted through ImageJ. C-Fos+ cells were quantified in the anterior and posterior PVT (aPVT, pPVT) as well as the dorsal, intermediate and ventral LS (dLS, iLS, vLS) both rostrally and caudally.

There are four main conclusions of this study. First, inhibition of the PVT and the LS appear to support the expression of learned safety. Second, activation of the PVT appears comparable across males and females. Third, activation of the LS in “threat” animals may be sex-dependent, with males showing greater activation of the rostral LS and females showing greater activation of the caudal LS. Lastly, these data suggest that both the PVT and the LS may be important in regulating learned safety. Future studies will aim to selectively manipulate these regions to determine whether inhibition of PVT or LS would reduce the expression of learned fear in “threat” tested animals. In addition, it is important to determine whether stimulation of PVT or LS will impair the expression of learned safety. Future analysis of tissue samples will indicate whether these trends hold over time with larger sample sizes.

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This research was approved by the Miami University IACUC: Protocol 1083_2029_Mar.