**1. Introduction**

The *KISS1* gene was discovered as a novel metastasis-suppressor in human melanoma cells in 1996 in Hershey, named after the famous chocolate of the city, Hershey's Kisses [1].

*KISS1* encodes a 145-amino-acid propeptide, from which kisspeptin-54 (Kp-54) is cleaved. The proteolytical cleavage of this 54-amino-acid peptide results in shorter biologically active products, designated kisspeptin-14 (Kp-14), kisspeptin-13 (Kp-13) and kisspeptin-10 (Kp-10) [2]. Mammalian kisspeptins belong to the family of RF-amide peptides, as they carry the characteristic, conserved carboxyl-terminal Arg–Phe–NH2 sequence [3].

The canonical receptor of kisspeptins is a G protein-coupled receptor, Gpr54, that is fully activated by all biologically active products of the *Kiss1* gene [4]. Although Gpr54 was initially described in 1999 as an orphan receptor similar to galanin receptors [5], after being deorphanized in 2001, it was designated kisspeptin-1 receptor (Kiss1r) [6].

**Citation:** Ibos, K.E.; Bodnár, É.; Bagosi, Z.; Bozsó, Z.; Tóth, G.; Szabó, G.; Csabafi, K. Kisspeptin-8 Induces Anxiety-Like Behavior and Hypolocomotion by Activating the HPA Axis and Increasing GABA Release in the Nucleus Accumbens in Rats. *Biomedicines* **2021**, *9*, 112. https://doi.org/10.3390/ biomedicines9020112

Academic Editor: Insop Shim Received: 24 December 2020 Accepted: 22 January 2021 Published: 25 January 2021

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Upon activation of the Gαq/11-coupled Kiss1r, phospholipase C (PLC) is activated, leading to inositol 1,4,5-trisphosphate (IP3)-mediated intracellular Ca2+ mobilization. Moreover, the activation of protein kinase C (PKC) and the Gαq-independent recruitment of β-arrestins result in the phosphorylation of several mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 [4]. MAPKs in turn regulate gene expression and induce long-term alterations in a wide range of biological processes [7], including progesterone secretion [8], trophoblast adhesion [9], and glucose-induced insulin secretion [10].

Kisspeptins also bind and activate both neuropeptide FF receptors (NPFFR1 and NPFFR2) [11]. As NPFF receptors are coupled with Gαi/o, their activation inhibits cAMP production. The Gβγ heterodimer released from Gi/o proteins was found to inhibit voltagegated Ca2+ channels. Moreover, it is capable of potentiating Gq signaling via physical interaction with PLC [3].

Kisspeptin is expressed in several regions of the rat central nervous system, including hypothalamic nuclei [e.g., arcuate nucleus, anteroventral paraventricular nucleus (AVPV)], thalamic nuclei, the amygdala, hippocampus, lateral septum, the bed nucleus of stria terminalis, striatum, nucleus accumbens (NAc), periaqueductal grey, and locus coeruleus [12,13]. Likewise, Kiss1r has been localized in rats in the hypothalamus (e.g., paraventricular, arcuate and supraoptic nucleus), thalamus, hippocampus, amygdala, septum, striatum, raphe nuclei, and cortex [5,14].

The expression of NPFF1 receptor mRNA has been detected in the lateral septum, in thalamic and brainstem nuclei, as well as in the ventral tegmental area (VTA), NAc, the bed nucleus of the stria terminalis, the amygdala and hippocampus. NPFF2 receptor mRNA expression has been reported in thalamic nuclei, in the hypothalamus, hippocampus, VTA, the A5 noradrenergic cell group and also in the dorsal horn of the spinal cord [15,16].

Following the original discovery of its metastasis suppressor role in melanoma [1], the anti-metastatic activity of kisspeptin has been found in a variety of tumors, including bladder, ovary, colorectal, pancreas, pituitary, prostate and thyroid cancer [17].

The involvement of kisspeptin in reproduction has been a topic of extensive research since it was discovered in 2003 that kisspeptin is a potent stimulator of gonadotropin secretion [18]. The role of kisspeptin in the regulation of puberty is underlined by the finding that various loss-of-function mutations of *KISS1R* and *KISS1* are associated with isolated hypogonadotropic hypogonadism, whereas activating mutations result in central precocious puberty [19]. Hypothalamic *Kiss1* neuron populations are responsible for the regulation of the estrous cycle by mediating positive and negative feedback of gonadal steroids on gonadotropin secretion [20]. The sexually dimorphic *Kiss1* neuron population of the AVPV is responsible for the positive feedback of estrogen, thus it contributes to the surge-like secretion of GnRH. However, pulsatile GnRH secretion is regulated by the KNDy neurons (coexpressing kisspeptin, neurokinin B, and dynorphin) of the arcuate nucleus that mediate the negative feedback of estrogen [21]. Compelling evidence has suggested that KNDy neurons in the arcuate nucleus function as a major integrator of various modifiers of the reproductive axis, including metabolic signals, olfactory clues, and circadian rhythm [22–25].

Similarly to other members of the RF-amide family [26], kisspeptin has also been implicated in the regulation of nociception [27]. In a recent study, Kp-13 lowered the nociceptive threshold in mice, decreased the analgesic effect of morphine, diminished morphine tolerance and caused mechanical hypersensitivity [28].

Based on the expression of *Kiss1* and *Kiss1r* in limbic brain structures [29,30], several studies have investigated the behavioral effects of kisspeptin.

An antidepressant-like effect of kisspeptin has been reported in rats [31], and intravenous kisspeptin has also decreased negative mood in human subjects [32].

Kisspeptin neurons in the rostral periventricular area of the 3rd ventricle (RP3V) seem to regulate sexual behavior in rodents, as they are activated by male urinary odors in female mice and facilitate copulatory behavior in a NO-dependent pathway [33,34].

An interplay between kisspeptin and the hypothalamic-pituitary-adrenal (HPA) axis was suggested in 2009, when Kinsey-Jones et al. discovered that stress-induced elevation of plasma corticosterone suppresses hypothalamic kisspeptin signaling in rodents [35]. Since that time, several studies have been conducted with controversial results.

In paraventricular nucleus-derived cell lines, Kp-10 increased the gene expression of arginine vasopressin (AVP) and oxytocin, while suppressing the expression of corticotropin releasing hormone (CRH). However, it failed to influence the activity of the HPA axis in vivo, as intraperitoneally (ip.) administered Kp-10 had no effect on plasma corticosterone and adrenocorticotropic hormone (ACTH) levels in rats [36]. Likewise, kisspeptin administration had no effect on anxiety in human subjects [32].

In 2013, our group reported an anxiogenic effect of intracerebroventricularly (icv.) administered Kp-13 in rats. Kp-13 not only induced a significant increase in plasma corticosterone level, but also decreased the number of entries into the open arms and the time spent in them in the elevated plus maze test. Moreover, it has stimulated spontaneous locomotion and it also had a hyperthermic effect lasting for several hours after treatment [37].

An anxiogenic property of kisspeptin signaling has also been proposed by the experiments of Delmas et al., in which *Kiss1r* KO mice have spent more time in the open arms in the elevated plus maze test, indicating a suppression of anxiety. The most pronounced anxiolytic effect was observed when kisspeptin signaling in GnRH neurons was selectively rescued in *Kiss1r* KO animals, suggesting a modulatory role of gonadal steroids. Interestingly, no significant effect of *Kiss1r* KO was detected on the behavioral parameters of the open field test [38].

In zebrafish, however, the central administration of kisspeptin has been associated with an anxiolytic tendency in the novel-tank diving test and a significantly reduced fear response to alarm substance [39].

In a recent study, a Cre-dependent, stimulatory DREADD (Designer Receptors Exclusively Activated by Designer Drugs) viral construct has been targeted to the *Kiss1* neurons of the posterodorsal medial amygdala (MePD) in mice. Upon selective activation of MePD *Kiss1* neurons by clozapine-N-oxide, a significant increase in open arm exploration has been observed in the elevated plus maze, suggesting an anxiolytic role of this neuron population [40].

There are several possible explanations for the ambiguous results reported in the literature. On one hand, the route of administration could be a determining factor. Peripheral administration of Kp has failed to influence the activity of the HPA axis in rats (0.13 μg/μL Kp-54 ip.) [36] and the activity of the limbic system in human subjects (1 nmol/kg/h Kp-54 iv. over 75 min) [32]. In contrast, central Kp-13 (1 or 2 μg icv.) had a pronounced anxiogenic effect in rats [37]. It is likely that the doses applied by Rao et al. [36] and Comninos et al. [32] were too low to exert an anxiogenic effect. In their investigation into the effect of peripheral or central Kp administration on the reproductive axis in rats, Thomson et al. have found that 1 nmol of icv. Kp-10 was sufficient to significantly raise plasma luteinizing hormone (LH) concentration, but a 100-fold dose was required for the same effect in case of ip. treatment [41]. Likewise, the selective activation of MePD *Kiss1* neurons [40] points to the function of a distinct neuron population, whereas central kisspeptin treatment [37] reflects a general central effect by activating neurons bearing Kiss1r throughout the brain.

On the other hand, the differences could also be attributed to the variety of species involved in these experiments. The kisspeptin system of zebrafish is strikingly different from the mammalian one, both in terms of anatomy and function [39,42], thus the results of studies on zebrafish should be interpreted with caution.

Some studies have also reported that kisspeptin might play a role in the regulation of locomotor activity. Icv. Kp-13 has induced an increase in not only spontaneous, but also in exploratory locomotion in male Sprague-Dawley rats [37]. In line with these results, Tolson et al. have found that *Kiss1r* KO female mice exhibit decreased locomotor activity and energy expenditure, leading to obesity [43].

It has been discovered that kisspeptin attenuates morphine effect [28], and is expressed in the NAc [44], pointing to its possible involvement in the regulation of mesocorticolimbic dopaminergic activity. Interestingly, the centers of reward and addiction have also been implicated in the regulation of locomotion. First, quinpirole (a D2 receptor agonist) injected into the NAc has suppressed exploratory locomotion in rats [45], whereas bicuculline (a GABAA receptor antagonist) administration into the nucleus induced hyperactivity with prolonged exploratory behavior in rats [46]. Second, the selective activation of dopaminergic neurons in the VTA by DREADD has induced a pronounced and sustained hyperactivity in rats, which effect could be reproduced by activating selectively activating the dopaminergic pathway between the VTA and NAc [47]. Thus, it is possible that kisspeptin might influence locomotion by modulating the activity of the VTA or NAc.

Nowadays kisspeptin analogs and antagonists are attracting considerable attention due to their potential therapeutic use in various gynecological conditions, including infertility, polycystic ovary syndrome and precocious puberty [48]. The shortest natural bioactive form of kisspeptin is the 10 amino acid long Kp-10, which has higher affinity to Kiss1r than Kp-54 [49]. According to molecular docking studies, ASN4, SER5, GLY7, ARG9 and PHE10 of Kp-10 are involved in the formation of hydrogen bonds between the peptide and Kiss1r [50]. Consequently, shorter kisspeptin fragments containing these amino acids might be able to bind and possibly activate the receptor.

The aim of the current study was to investigate whether the 8 amino acid long fragment of kisspeptin is capable of influencing the behavior of rats similarly to kisspeptin. Following icv. treatment with Kp-8, elevated plus maze (EPM), computerized open field (OF), and marble burying (MB) tests were performed. Serum corticosterone and luteinizing hormone concentrations were measured to assess the activation of the HPA axis and Kiss1 receptors, respectively. Moreover, dopamine release from the VTA and NAc and GABA release from the NAc were measured using ex vivo superfusion to further characterize the mechanism of action.
