Habenular kisspeptin modulates fear in the zebrafish

Abstract

Kisspeptin, a neuropeptide encoded by the KISS1/Kiss1, and its cognate G protein-coupled receptor, GPR54 (kisspeptin receptor, Kiss-R), are critical for the control of reproduction in vertebrates. We have previously identified two kisspeptin genes (kiss1 and kiss2) in the zebrafish, of which kiss1 neurons are located in the habenula, which project to the median raphe. kiss2 neurons are located in the hypothalamic nucleus and send axonal projections to gonadotropin-releasing hormone neurons and regulate reproductive functions. However, the physiological significance of the Kiss1 expressed in the habenula remains unknown. Here we demonstrate the role of habenular Kiss1 in alarm substance (AS)-induced fear response in the zebrafish. We found that AS-evoked fear experience significantly reduces kiss1 and serotonin-related genes (plasmacytoma expressed transcript 1 and solute carrier family 6, member 4) in the zebrafish. Furthermore, Kiss1 administration suppressed the AS-evoked fear response. To further evaluate the role of Kiss1 in fear response, zebrafish Kiss1 peptide was conjugated to saporin (SAP) to selectively inactivate Kiss-R1-expressing neurons. The Kiss1-SAP injection significantly reduced Kiss1 immunoreactivity and c-fos mRNA in the habenula and the raphe compared with control. Furthermore, 3 d after Kiss1-SAP injection, the fish had a significantly reduced AS-evoked fear response. These findings provide an insight into the role of the habenular kisspeptin system in inhibiting fear.5-HT | interpeduncular | neuroendocrine | anxiolytic K isspeptin, a hypothalamic neuropeptide derived from the KISS1/Kiss1, with the ability to activate the kisspeptin receptor (Kiss-R), has proven to play a key role in vertebrate reproduction (1). Kisspeptin neurons are present in the hypothalamic region, but their neural targets are not restricted to the hypothalamic region (2, 3). Furthermore, recent studies in mammals have revealed the expression of Kiss1 in several brain regions, including the medial amygdala (4). However, the knowledge of the potential role of kisspeptin-Kiss-R in nonhypothalamic regions remains limited. Using the teleost fish, we have previously identified two homologous genes (kiss1 and kiss2) encoding kisspeptin (5), of which kiss1 is a conserved ortholog of mammalian KISS1/Kiss1, whereas kiss2 has been found in hypothalamic nuclei of only nonmammalian vertebrates, which include amphibians and teleosts (6). In the zebrafish, kiss1 and kissr1 mRNAs are predominantly expressed in the ventral habenula (vHb) (5, 7). In nonmammalian vertebrates, the dorsal habenula (dHb) and the vHb are homologous to the medial (mHb) and lateral (lHb) habenula in mammals (8, 9). The lHb in primates regulates punishment avoidance behavior (10) and in rodents, it controls anxiety and fear (11), which suggests that nonmammalian vHb, homologous to the mammalian lHb, could modulate fear response. Furthermore, the vHb projects Kiss1 neuronal fibers to the median raphe (MR) (7, 12), a site adjacent to serotonergic (5-hydroxytrypt...

Emerging Roles of Kisspeptin in Sexual and Emotional Brain Processing

“…By contrast, a more recent study observed that DREADD activation of medial amygdala kisspeptin neurones in male rodents increased time in the open arms of a maze suggesting a decrease in anxiety [31]. This latter study is in agreement with studies in zebrafish demonstrating anxiolytic effects of kisspeptin administration in a novel tank diving test designed to assess anxiety (increased top-to-bottom transitions), associated with enhanced 5-HT-related gene expression [36,43]. Therefore, these contrasting data propose a differential effect between global activation of central kisspeptin receptors via central kisspeptin administration in rodents and zebrafish, versus pure activation of medial amygdala kisspeptin neurones which may release kisspeptin as well as additional neurotransmitters.…”

“…This included the anterior and posterior cingulate, amygdala, thalamus, and globus pallidus, which are all implicated in romantic, maternal, and unconditional love [53][54][55][56] and consistent with structures known to be involved in kisspeptin signalling in humans [19,20] and rodents [10,15,21,23,24]. Interestingly, kisspeptins enhanced activation of the amygdala in response to bonding images correlated with improved positive mood [40], a Sexual behaviours -Enhances limbic and paralimbic brain activity with correlations to reward, sexual aversion, and positive mood (H) [40] -Triggers erections (R) [51] Fear -Reduces fear responses (Z) [36][37][38]43] Social behaviours -MeA kisspeptin neurone apposition with dopaminergic and vasopressinergic neurones (R) [24] Audition -Male rodent ultrasonic vocalisations increase female ARC kisspeptin activity which correlates to the duration of female searching for male (R) [34] Mood -Antidepressant-like effects (R, H) [40,44] -Anxiolytic effects (R, Z) [31,36] -Anxiogenic effects (R) [42] Olfaction -Kisspeptin anatomical framework (R, H) [24] -Kiss1r and MeA Kiss1 roles in male olfactory partner preference (R) [30,31] -Opposite-sex urinary odours stimulate RP3V and limbic kisspeptin neurones and enhance LH surge (R) [32,33] Kisspeptin signalling potential mechanism that may contribute to the desire to bond with a partner.…”

“…In this way, AS can also be used to evoke fear in fish 198 behavioural studies [35]. In zebrafish, AS-evoked fear stimuli significantly reduces Kiss1 and serotonin-related gene transcription (pet1 and slc6a4a), while intracranial kisspeptin administration suppresses AS-evoked fear responses (erratic movement and freezing) [36]. In subsequent studies, the authors observed that pharmacological blockade of serotonin receptors (5-HT 1A and 5-HT 2 ) removed this anxiolytic effect of kisspeptin [37] and that kisspeptin modulated the serotonergic system here primarily via glutamatergic neurotransmission [38].…”

“…In subsequent studies, the authors observed that pharmacological blockade of serotonin receptors (5-HT 1A and 5-HT 2 ) removed this anxiolytic effect of kisspeptin [37] and that kisspeptin modulated the serotonergic system here primarily via glutamatergic neurotransmission [38]. Furthermore, selective inactivation of Kiss1 neurones (using kisspeptin conjugated to saporin) resulted in reduced Kiss1 immunoreactivity and Fos expression within the habenula and raphe (key structures in the fear response), which resulted in attenuated AS-evoked fear responses [36]. The role of kisspeptin in fear has as yet not been explored in other species, but these studies in zebrafish provide evidence that kisspeptin can attenuate fear responses via serotonergic pathways, a finding with implications for successful reproduction and conservation of the species.…”

Anatomical and functional analysis of non-retinal tectal afferents in larval zebrafish

“…At 6dpf, zebrafish are able to track and capture prey (Borla et al, 2002), can respond to predatory visual cues (Temizer et al, 2015;Dunn et al, 2016), perform phototaxis (Burgess et al, 2010) and can display optomotor and optokinetic responses (Roeser and Baier, 2003). Beyond visual behaviours, zebrafish have been shown to be capable of exhibiting complex social behaviours such as shoaling and aggression (Engeszer et al, 2004;Engeszer et al, 2007;Saverino and Gerlai, 2008;Dreosti et al, 2015), and are capable of responding appropriately to chemical stimuli (Speedie and Gerlai, 2008;Ogawa et al, 2014). They have a diverse motor repertoire which includes both turning and normal beat and glide swimming (Budick and O'Malley, 2000), undergoing exploratory behaviour (Ahmad and Richardson, 2013) and performance of the escape behaviour (Eaton et al, 1977;Eaton et al, 2001).…”