Pharmacological and Chemogenetic Characterization and Modulation of the Brain Neuropeptide S and Oxytocin

Anxiety is a natural response to a real or perceived threat that has been conserved throughout evolution. From this perspective, sensation of anxiety is viewed as an adaptive behavioral state, which occurs in response to signals of danger. However, excessive or inappropriate anxiety can become pathological. Anxiety disorders represent one major burden of our modern society, and the available treatment options are limited by adverse side effects. During the last decades, research centered on anxiety disorders has focused on neuropeptides. Neuropeptide S (NPS) and oxytocin (OXT) represent potential neuropeptide candidates for the treatment of various psychopathologies, including anxiety disorders due to their potent anxiolytic profile. Both signal through G protein-coupled receptors and thereby regulate complex neuronal signaling pathways. How these pathways contribute to behavioral and physiological effects of NPS and OXT remains to be elucidated. Therefore, in the present thesis I aimed to reveal, (i) whether NPS acts on the brain OXT system to exert its anxiolytic properties, (ii) whether the anxiolytic profile of NPS is mediated in a Gq pathway-dependent manner, and (iii) to characterize the spatiotemporal characteristics of chemogenetically induced OXT neurons activation. NPS receptors (NPSR) are found within the hypothalamic paraventricular nucleus (PVN), where OXT is synthesized besides the supraoptic and accessory nuclei. Besides anxiolysis, NPS and OXT share other effects, such as the reversal of social fear, the attenuation of aggressive-like behavior as well as the inhibition of food-intake and anti-nociceptive properties as demonstrated in rodents. These behavioral similarities, together with the neuroanatomical overlapping of the NPSR and OXT expression within the PVN, led me to hypothesize that NPS effects are mediated by acting on the OXT system within the PVN. Herein, I present a chain of evidence that the effects of NPS within the PVN are mediated via actions on local OXT neurons in male Wistar rats. Retrograde tracing revealed that NPS-immunoreactive neurons originating within the Locus coeruleus innervate the PVN. Moreover, fluorescence-activated cell sorting identified NPSR expression in PVN-OXT neurons. NPS reliably induced transient Ca2+-influx in a subpopulation of OXT neurons - an effect mediated via the NPSR. Moreover, intracerebroventricular (icv) NPS evoked a significant release of OXT within the PVN as assessed by microdialysis in combination with a highly sensitive radioimmunoassay. Both, chemogenetic silencing of the endogenous OXT system using designer receptors exclusively activated by designer drugs (DREADD) as well as pharmacological blockade of brain OXT receptors (OXTR) abolished the anxiolytic effect of NPS infused icv. These findings provide the first evidence for an intra-hypothalamic mechanism involving NPSR-expressing OXT neurons in the potent anxiolytic profile of NPS, and fill an important gap in our neurophysiological understanding of brain neuropeptide interactions in the context of regulation of emotional behaviour within the hypothalamus. Next, I performed studies in order to extend our knowledge on the intraneuronal mechanisms underlying the anxiolytic profile of NPS by studying signaling pathways downstream of NPSR activation. Here, bilateral microinfusion of NPS into the medial amygdala (MeA) of male adult Wistar rats reduced anxiety-related behavior on the elevated plus maze and the open field. Moreover, icv infusion of NPS evoked expression as well as increased phosphorylation of Ca2+/calmodulin-dependent kinase II in the amygdala, and subsequent phosphorylation of the mitogen-activated protein kinase ERK1/2. Importantly, NPS-induced anxiolysis in the MeA was prevented by local inhibition of phospholipase C using the specific inhibitor U73122. In contrast, local pharmacological blockade of adenylyl cyclase signaling using the specific inhibitor 2’,5’-dideoxyadenosine failed to inhibit the anxiolytic effect of NPS infused into the MeA. Hence, NPS promotes acute anxiolysis within the MeA dependent on NPSR-mediated phospholipase C signaling. Finally, in order to validate an innovative and highly specific tool for activating OXT neurons within the PVN in a behavioral context, I performed a series of experiment using the DREADD technique. Acute chemogenetic activation of PVN-OXT neurons resulted in enhanced release of OXT within the PVN within one hour as assessed using intracerebral microdialysis. Further, DREADD activation reduced anxiety-related behavior in the light/dark box, and increased self-grooming behavior in the home cage. In addition, chemogenetic activation of PVN-OXT neurons marginally improved ethanol-induced locomotor deficits. In summary, my experimental data advance our understanding of the mechanisms by which NPS promotes anxiolysis and the temporal release patterns of OXT following chemogenetic activation and its behavioral relevance.