Role of Tachykinin-1 peptides in the regulation of habenular kisspeptin neurons in the zebrafish

Fear is a conserved emotion which serves as the root cause of psychiatric disorders such as phobias and depression. Hence understanding the neural pathway underlying fear is important to find a cure. Previously it was reported that the odorant-evoked fear response is regulated by habenular Kiss1 neurons through the serotonergic system. However the afferent signalling pathway upon exposure to the odorant cue remains unknown. Substance P (SP), a member of the tachykinin family of neuropeptides, is known to be involved in fear-related functions. Mammalian studies have demonstrated the role of SP in the habenula in depression-related behaviours. <i>Tac1</i> gene, encodes for SP and neurokinin A (NKA). SP and NKA are co-localized and co-released from the same neurons and nerves. The role of these peptides depends on its receptor activation and binding selectivity. The present study was designed to investigate the role of tachykinin1 (Tac1) peptides in the habenular Kiss1-regulating fear pathway in the zebrafish. <br>        In chapter 2, zebrafish-specific Tac1 antibody was generated and Tac1 cells were localized. The distribution of Tac1 fibers and their association to habenular Kiss1 neurons were examined. Immonohistochemical labelling showed Tac1 cell distribution in several regions of the brain such as in the anterior part of the parvocellular preoptic nucleus, the ventral region of the periventricular hypothalamus, the nucleus of medial longitudinal fascicle, the oculomotor nucleus and the central gray while the fiber distributions were noted throughout the brain. Alternate section labelling of Tac1 immunoreactivity and Kiss1 showed Tac1-immunoreactive fibers innervating the Kiss1-expressing region. Detection of SP receptor <i>NK1a</i> mRNA expression in the Kiss1 neurons suggests the presence of a possible direct regulation of Kiss1 by Tac1 through its cognate receptor. <br>        In chapter 3, the potential brain regions involved in the zebrafish AS-evoked fear pathway and the connectivity of AS with Tac1 was examined. Mapping of neural activity upon AS exposure revealed that several brain regions such as telencephalon, diencephalic region, cerebellum and spinal cord are involved in the fear pathway. Double labelling of Tac1 cells with a neuronal activity marker, <i>npas4a</i> showed increased activity in Tac1 cells, suggesting that the AS activates the Tac1 cells in the preoptic area. <br>     In chapter 4, to identify which Tac1-encoding peptides (SP or NKA) is involved in the habenular kisspeptin-modulating fear pathway, we examined the effect of exogenous SP and NKA on <i>kiss1</i> expression. Administration of NKA peptide had no effect on <i>kiss1 </i>mRNA expression, whereas administration of SP significantly reduced kiss1 mRNA expression. Blockade of SP receptor using a SP receptor antagonist (sendide), eliminated the effect of SP on <i>kiss1</i> and also blocked the effect of AS on <i>kiss1</i> mRNA expression, suggesting that SP but not NKA may regulate Kiss1 in AS-evoked fear response. <br>        In conclusion, the present study presents the g of a zebrafish-specific Tac1 antibody and the possible role of tachykinin peptides in the regulation of habenular Kiss1 neurons in the zebrafish. Innervation of Tac1 fibers into the Kiss1-expressing ventral habenula and the expression of a SP receptor, NK1a, in Kiss1 neurons suggest the presence of a possible direct action of Tac1 on Kiss1 neurons. Neural activity in the POA of Tac1 cells suggests that the AS-driven olfactory signalling may involve the activation of Tac1 cells in the preoptic area followed by transmission of signals to habenula Kiss1 neurons. Finally, the effects of SP and NKA on Kiss1 neurons suggest that SP exclusively regulate Kiss1 in AS-evoked fear response.