Serotonergic regulation of clock genes and kisspeptin in the anteroventral periventricular nucleus of male mice

Abstract

Kisspeptin neurons localized in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC) ensure normal reproductive functions. Neuroendocrine and behavioural aspects of reproduction are regulated by both serotonin (5-HT) and the circadian system such that 5-HT disruptions trigger sexual irregularities. The circadian system coordinates reproductive mechanisms with rhythms depicted in kisspeptin encoded by the Kiss1 gene; pre-requisite for temporal hormonal secretion. However, the serotonergic control of circadian processes within the AVPV and kisspeptin neurons is unknown. Therefore, this thesis investigated the presence and modulation of an endogenous clock in the AVPV and kisspeptin neurons in a chronic citalopram (CIT, 5-HT enhancing drug) model; and validated a potential serotonergic-kisspeptin pathway. In chapter 2, examination of a slave oscillator in the AVPV revealed the time-dependent expression (Zeitgeber Time (ZT) 4 vs ZT14) of Kiss1 and clock genes (Circadian locomotor output cycle kaput, Clock; Brain and muscle ARNT-like, Bmal1; Period1, Per1; and Period2, Per2) with higher levels of Kiss1, Per1 and Per2 at ZT14, opposing Bmal1 peaking at ZT4. Additionally, clock proteins, CLOCK and BMAL1 co-localized with kisspeptin neurons, providing evidence of an endogenous oscillatory mechanism. Clock mRNA was up-regulated with a decrease in CLOCK-ir and BMAL1-ir kisspeptin neurons in the chronic CIT model, implying a serotonergic-mediated regulation of circadian components in the AVPV. In chapter 3, the pathways transmitting the serotonergic regulatory cues to the AVPV were assessed. Connectivity from the suprachiasmatic nucleus (SCN) was examined as a result of the synchronous kisspeptin activity to the masterclock. Circadian synchronizers from the SCN, i.e. arginine vasopressin (AVP) and vasoactive intestinal peptide (VIP) as well as their receptors (V1a and VPAC2) in the AVPV were unaltered in the chronic CIT model. Alternately, a direct route from the raphe to the AVPV was validated through the expression of 6 out of 7 5-HT receptor families and the presence of 5-HT-ir fibres. Furthermore, ~60% of kisspeptin neurons were innervated by 5-HT consolidating the 5-HT-AVPV connectivity. 5-HT1A receptor mRNA was up-regulated in the chronic CIT model, implying that clock-related changes were potentially mediated by a modified serotonergic system. Chapter 4 addressed the functional 5-HT-AVPV connectivity with emphasis on Kiss1 and clock genes. Manipulation of endogenous 5-HT levels using p-chlorophenylalanine (PCPA) reduced 5-HT by 60% in the dorsal raphe (quantified by HPLC-MS/MS). PCPA increased Clock with no effects on Kiss1 in the AVPV. Contrastingly, specific 5-HT1A antagonism (WAY100635) potently affected clock, 5-HT-related and Kiss1 genes at specific timepoints. Bmal1 upregulation 12hrs post-injection was accompanied by a substantial decrease in Kiss1, Rev-erbα, Per1 and Per2; while 5-HT1A and 5-HT2A were higher 6hrs post-treatment. These findings endorsed the regulatory role of 5-HT on clock and Kiss1 genes potentially mediated by 5-HT1A receptors; with decreased sensitivity to low 5-HT levels. Overall, the AVPV incorporates an endogeneous oscillator, regulated in part by a direct pathway mediated by 5-HT. On the basis of these findings, chronic CIT may target the circadian components of the kisspeptin neurons while Kiss1 and clock genes in the AVPV are functionally regulated by the serotonergic system and mediated by the 5-HT1A or 5-HT2A receptors depending on the status of 5-HT levels.