GABA receptors type A (GABAAR) are ligand-gated ion channels permeable for chloride anions. In the mammalian brain they mediate most of the inhibitory transmission. Moreover, the dysfunctions of the GABA-mediated system result in many neurological disorders, including epilepsy, anxiety and depression. Neurosteroids are cholesterol metabolites interacting with a variety of membrane receptors and have a direct effect on neuronal excitability. The neurosteroids allo-pregnanolone (allo-PA) and pregnanolone (PA) are potent positive modulators of the GABAAR. The goal of this work is to establish a newly constructed application system and a calcium imaging method using the GCaMP sensor to examine the effects of PA on the activity of primary hippocampal cultures. In this work we validate the application system and test the GCaMP calcium sensor in vitro. Application of PA inhibited the spontaneous calcium peaks, which agrees with its known actions on the GABAAR. We discovered that the neurosteroid inhibitory effect on the neuronal network activity changes after repeated applications. The results suggest that there might be some compensatory actions on the GABAAR level during prolonged or repeated exposition to PA. Key words: GABAAR; neurosteroid; pregnanolone; allopregnanolone; calcium; GCaMP; inhibition

The ability of animals to sense, interpret, and respond appropriately to social stimuli in their environment is essential for identifying and distinguishing between members of their own species. In mammals, social interactions both within and across species play a key role in determining if an animal will live to pass on its genes to the next generation or else be removed from the gene pool. The result of this selection pressure can be observed in specialized neural circuits that respond to social stimuli and orchestrate appropriate behavioral responses. This highly conserved network of brain structures is often referred to as the Social Behavior Network (SBN). The medial amygdala (MeA) is a central node in the SBN and has been shown to be involved in transforming information from olfactory sensory systems into social and defensive behavioral responses. Previous research has shown that individual neurons in the MeA of anesthetized mice respond selectively to different chemosensory social cues, a characteristic not observed in its upstream relay, the accessory olfactory bulb (AOB). However, the cause of this stimulus selectivity in the MeA is not yet understood. Here, I hypothesize that a subpopulation of neurons in the MeA that express the enzyme aromatase are involved in the sensory representation of social stimuli in awake, behaving animals. To test this hypothesis, I designed and built a novel behavioral apparatus that allows for discrete presentations of social stimuli in a highly controllable and reproducible environment. I then injected the adeno-associated virus (AAV) AAV-Syn-Flex-GCAMP6s into the MeA of Aromatase:Cre transgenic mice and implanted a fiber optic cannula slightly above the injection site. The combination of this transgenic mouse line and conditional AAV caused GCaMP6s expression to be exclusive to aromatase-expressing neurons. By coupling my novel behavioral apparatus to a fiber photometry system, I successfully recorded the moment-to-moment activity of aromatase neurons in the MeA of awake, behaving animals as they investigated various social stimuli. Aromatase neurons in the MeA of adult male mice respond strongly to conspecific social stimuli, including live adult mice, mouse pups, and mouse urine samples. Sniffing and investigative behaviors correlated strongly with increased GCaMP6s signal in aromatase neurons, reflecting increases in their neural activity. Interestingly, after repeated investigations of the same stimuli the activity of aromatase neurons gradually diminished. Presenting a novel stimulus following repeated investigations of a familiar stimulus reinstated some, but not all of the initial GCaMP6s signal. This points to the potential role that aromatase neurons may play in the habituation to social stimuli that are consistently present in their environment. Investigations of predator stimuli did not evoke significant responses from aromatase neurons, nor did investigations of non-social stimuli. These results demonstrate that aromatase expressing neurons in the MeA of awake, behaving animals encode the sensory representation of conspecific social stimuli, and their responses are highly selective to the type of stimulus presented.