I first ensured that this expression was indeed selective by using immunohistochemistry. This involved visualising the “tag” along with staining for tryptophan hydroxylase (TPH2), a marker for serotonin neurons. Once selective targeting of serotonin and GABA neurons was established (with more than 95% specificity), I tested the functionality of this system by measuring the effect of the DREADD activator, clozapine-N-oxide (CNO), on the expression of the immediate early gene cFos, a marker for recent neuronal activity. I found that CNO significantly increased cFos expression in the DRN in both the SERT-Cre and vGAT-Cre mice in the correct neuronal populations. In addition, CNO increased cFos expression in the basolateral amygdala (BLA) of SERT-Cre mice, suggesting serotonin activation of downstream anxiety-related circuits.
Once serotonin-selective and functional expression of these receptors was confirmed, I tested the effect of manipulating neuronal activity within the DRN in various anxiety models. On the elevated plus maze, I found that increasing the excitability of serotonin neurons through expression and activation of these receptors in the SERT-Cre mice caused an increase in time spent in the open arms. This suggests that it makes them less anxious. vGAT-Cre mice did not show this effect. In two other tests of anxiety (the light and dark box and novelty supressed feeding), however, I saw no changes in the SERT-Cre mice. Surprisingly, vGAT-Cre mice spent a significantly reduced time in the light compartment of the light and dark box while at the same time exhibiting increased exploration of the central area of an open field, suggesting increased and decreased anxiety, respectively. These findings suggest that chemogenetic activation of serotonin neurons reduces anxiety, while chemogenetic activation of GABA neurons in the DRN increases anxiety, at least under certain conditions, and might be doing this independently of serotonin.
I then set out to characterise whether chemogenetically activating serotonin neurons in the raphe would have an impact on the acquisition and retrieval of fear memories. Surprisingly, I found no effects using this system in several variations of fear conditioning, a well-used test to investigate negative emotional memory formation and retrieval. These results suggested that chemogenetically activating serotonin neurons in the DRN is not sufficient to enhance nor disrupt the acquisition and retrieval of emotional fear memories.
