Role of Kiss1 in serotonergic modulation in the zebrafish

KISS1/Kiss1 gene produces a neuropeptide, kisspeptin with the ability to activate its G-protein coupled receptor, GPR54 (Kiss-R), which is involved in the control of vertebrate reproduction. Previously, two forms of genes encoding kisspeptin (kiss1 and kiss2) and its respective receptor (kissr1 and kissr2) were identified in the zebrafish (Danio rerio). Kiss1 neurons are observed in the ventral habenula (vHb) and project down to the median raphe (MR) while Kiss2 neurons express in the hypothalamus, implicating the independent function of two kisspeptin systems. Previous studies suggest the function of Kiss2 in reproduction, while the physiological significance of Kiss1 remains unknown. This thesis aims to determine the role of habenular Kiss1 in serotonin (5-HT)-dependent outcomes in the zebrafish, particularly odorant, alarm substance (AS)-induced fear responses. Chapter 2 initially tested the hypothesis that Kiss1 modulates 5-HT-dependent anxiety and fear outcomes in mature adult zebrafish. Although Kiss1 showed no effects on anxiety with an exception of increased exploratory behaviour, 7 days exposure to the AS resulted in significant decreases in the expression of kiss1 and the 5-HT-related genes. Additionally, central administration of the Kiss1 peptide supressed AS-induced fear outcomes in a neuromodulatory manner. Furthermore, this effect was successfully blocked by selective antagonists for 5-HT receptors (5-HT1AR and 5-HT2R). The findings in Chapter 2 thus indicate that in the zebrafish, Kiss1 modulates AS-induced unconditioned fear outcomes mediated at least in part by 5-HT1A and 5-HT2 receptors. In Chapter 3, the neuronal mechanism underlying the action of Kiss1 on the 5-HT system was determined. Immunohistochemistry utilising a specific antibody to zebrafish Kiss1 and Kiss-R1 showed Kiss1 and Kiss-R1 cells sending fiber projections down to the MR through the fasciculus retroflexus (FR), adjacent to the serotonergic raphe region (superior raphe, SR) with no direct contact observed in 5-HT neuronal cell bodies, implicating an indirect mechanism of modulation. Dual-fluorescence labelling showed majority of Kiss1 neurons are glutamatergic and in the raphe region, the Kiss1 fibers were closely associated with glutamatergic and GABAergic cells and a few with 5-HT. Hence, these morphological findings in Chapter 3 implicate that Kiss1 neurons modulate the 5-HT system in the SR via neurotransmitter assistance involving mainly glutamate and GABA. Finally in Chapter 4, to further investigate the endogenous role of Kiss1 in AS-induced fear outcomes, Kiss1-SAP (zebrafish Kiss1 peptide conjugated to saporin), to selectively inactivate Kiss-R1-expressing neurons, as well as Kiss1 mutant (Kiss1-/-) zebrafish that showed complete absence of the peptide in the brain were utilised. Kiss1-SAP significantly decreased the neuronal activity in the vHb and MR 3 days post-administration alongside reduced Kiss1-immunoreactivity in these regions 12 days post-administration. The mutants lacked Kiss1-immunoreactivity in the vHb-MR pathway with no differences observed in the baseline activity levels compared to controls. Upon exposure to the AS, Kiss1-SAP-adminsistered and Kiss1-/- zebrafish showed no fear responses suggesting the AS-triggered signals were inhibited from being relayed downstream. The findings in Chapter 4 implicate the importance of the Kiss1/Kiss-R1 system in modulating AS-induced fear responses. Taken together, the work presented in this thesis provides evidences on the role of Kiss1 in modulating the 5-HT system to regulate olfactory-driven fear (specifically AS-induced) that is mediated via the interaction of the 5-HT1A and 5-HT2 receptors. The effects brought on involve the pre-synaptic action of Kiss1 and the interaction with glutamatergic and GABAergic neurotransmitters. These findings provide understanding into one of the functional implications of Kiss1 in the innate fear pathway that extends beyond reproductive control, providing a therapeutic resolution and link in understanding psychiatric disorder development such as depression and phobias.