The habenula, located on the dorsal thalamic surface, is an emotional and reward processing center. As in the mammalian brain, the zebrafish habenula is divided into dorsal (dHb) and ventral (vHb) subdivisions that project to the interpeduncular nucleus and median raphe (MR) respectively. Previously, we have shown that kisspeptin 1 (Kiss1) expressing in the vHb, regulates the serotonin (5-HT) system in the MR. However, the connectivity between the Kiss1 neurons and the 5-HT system remains unknown. To resolve this issue, we generated a specific antibody against zebrafish Kiss1 receptor (Kiss-R1); using this primary antibody we found intense immunohistochemical labeling in the ventro-anterior corner of the MR (vaMR) but not in 5-HT neurons, suggesting the potential involvement of interneurons in 5-HT modulation by Kiss1. Double-fluorescence labeling showed that the majority of habenular Kiss1 neurons are glutamatergic. In the MR region, Kiss1 fibers were mainly seen in close association with glutamatergic neurons and only scarcely within GABAergic and 5-HT neurons. Our findings indicate that the habenular Kiss1 neurons potentially modulate the 5-HT system primarily through glutamatergic neurotransmission via as yet uncharacterized interneurons. The neuropeptide kisspeptin (Kiss1) play a key role in vertebrate reproduction. We have previously shown modulatory role of habenular Kiss1 in the raphe serotonin (5-HT) systems. This study proposed that the habenular Kiss1 neurons modulate the 5-HT system primarily through glutamatergic neurotransmission, which provides an important insight for understanding of the modulation of 5-HT system by the habenula-raphe pathway.
Kiss1, a neuropeptide predominantly expressed in the habenula, modulates the serotonin (5-HT) system to decrease odorant cue [alarm substance (AS)]-evoked fear behaviour in the zebrafish. The purpose of this study was to assess the interaction of Kiss1 with the 5-HT system as well as to determine the involvement of the 5-HT receptor subtypes in AS-evoked fear. We utilized 0. 28 mg/kg WAY 100635 (WAY), a selective 5-HT1A receptor antagonist, to observe the effects of Kiss1 administration on AS-evoked fear. We found WAY significantly inhibited the anxiolytic effects of Kiss1 (p < 0.001) with an exception of freezing behaviour. Based on this, we utilized 92.79 mg/kg methysergide, a 5-HT1 and 5-HT2 receptor antagonist, and found that methysergide significantly blocked the anxiolytic effects of Kiss1 in the presence of the AS (p < 0.001). From this, we conclude that Kiss1 modulates AS-evoked fear responses mediated by the 5-HT1A and 5-HT2 receptors. Kiss1 peptide intracranially (IC) administrated has been shown to decrease olfactory, alarm substance (AS)-evoked fear response. Blockade of the 5-HT1A receptor utilizing WAY 100635 (0.28 mg/kg) and the 5-HT1 and 5-HT2 receptor utilizing methysergide (92.79 mg/kg) produced increased AS-evoked fear responses that were unable to be overcome even during the recovery period. Blockade of this 5-HT system followed by Kiss1 administration showed that the peptide was unable to recover the anxiolytic effects upon 5-HT1A blocking using WAY 100635 with the exception of freezing behaviour while methysergide significantly blocked all the anxiolytic effects of Kiss1. These findings implicate that Kiss1 could modulate AS-evoked fear responses mediated by 5-HT1A and 5-HT2 receptors.
Sprouty-related protein-2 (Spred-2) is a negative regulator of extracellular signal-regulated kinases (ERK) pathway, which is important for cell proliferation, neuronal differentiation, plasticity and survival. Nevertheless, its general molecular characteristics such as gene expression patterns and potential role in neural repair in the brain remain unknown. Thus, this study aimed to characterise the expression of spred-2 in the zebrafish brain. Digoxigenin-in situ hybridization showed spred-2 mRNA-expressing cells were mainly seen in the proliferative zones such as the olfactory bulb, telencephalon, optic tectum, cerebellum, and the dorsal and ventral hypothalamus, and most of which were neuronal cells. To evaluate the potential role of spred-2 in neuro-regeneration, spred-2 gene expression was examined in the dorsal telencephalon followed by mechanical-lesion. Real-time PCR showed a significant reduction of spred-2 mRNA levels in the telencephalon on 1-day till 2-days post-lesion and gradually increased to normal levels as compared with intact. Furthermore, to confirm involvement of Spred-2 signalling in the cell proliferation after brain injury, double-labelling of spred-2 in-situ hybridization with immunofluorescence of BrdU and phosphorylated-ERK1/2 (p-ERK1/2), a downstream of Spred-2 was performed. Increase of BrdU and p-ERK1/2 immunoreactive cells suggest that a decrease in spred-2 after injury might associated with activation of the ERK pathway to stimulate cell proliferation in the adult zebrafish brain. The present study demonstrates the possible role of Spred-2 signalling in cell proliferative phase during the neural repair in the injured zebrafish brain.
Morphine is a potent analgesic opiate commonly used in treating pain, and it is also a substance of abuse and highly addictive. Hence, it is vital to discover the action sites of morphine in the brain to increase its efficacy of treatment. In the present study, we aimed at identifying comprehensive neuroanatomical locations that are sensitive to morphine in the adult zebrafish (Danio rerio). We performed in situ hybridization to localize the mu opioid receptor (oprm1) gene and to map the morphine sensitive brain areas using neuronal PAS domain-containing protein 4a (npas4a), an early gene marker. Real-time PCR was used to detect changes in mRNA levels of oprm1 and npas4a in control and acute morphine treated fish (2 mg/L; 20 min). Intense positive oprm1 signals were seen in the telencephalon, preoptic area, habenula, hypothalamic area and periventricular gray zone of the optic tectum. Acute morphine exposure significantly increased oprm1 and npas4a mRNA levels in the medial zone of dorsal telencephalon (Dm), ventral region of the ventral telencephalon (Vv), preoptic area, and in the hypothalamus but a decrease in oprm1 and npas4a signals in the dorsal habenula. This study provides a detailed map of oprm1 localization in the brain, which includes previously unreported oprm1 in the habenula of teleost. Presence of oprm1 in multiple brain sites implies multiple action targets of morphine and potential brain functions which could include reward, cognitive and negative emotions.
Mechanisms for fish social behaviours involve a social brain network (SBN) which is evolutionarily conserved among vertebrates. However, considerable diversity is observed in the actual behaviour patterns amongst nearly 30000 fish species. The huge variation found in socio-sexual behaviours and strategies is likely generated by a morphologically and genetically well-conserved small forebrain system. Hence, teleost fish provide a useful model to study the fundamental mechanisms underlying social brain functions. Herein we review the foundations underlying fish social behaviours including sensory, hormonal, molecular and neuroanatomical features. Gonadotropin-releasing hormone neurons clearly play important roles, but the participation of vasotocin and isotocin is also highlighted. Genetic investigations of developing fish brain have revealed the molecular complexity of neural development of the SBN. In addition to straightforward social behaviours such as sex and aggression, new experiments have revealed higher order and unique phenomena such as social eavesdropping and social buffering in fish. Finally, observations interpreted as 'collective cognition' in fish can likely be explained by careful observation of sensory determinants and analyses using the dynamics of quantitative scaling. Understanding of the functions of the SBN in fish provide clues for understanding the origin and evolution of higher social functions in vertebrates.
G-protein coupled receptor 139 (GPR139) is an evolutionarily conserved orphan receptor, predominantly expressing in the habenula of vertebrate species. The habenula has recently been implicated in aversive response and its associated learning. Here, we tested the hypothesis that GPR139 signalling in the habenula may play a role in fear learning in the zebrafish. We examined the effect of intraperitoneal injections of a human GPR139-selective agonist (JNJ-63533054) on alarm substance-induced fear learning using conditioned place avoidance paradigm, where an aversive stimulus is paired with one compartment, while its absence is associated with the other compartment of the apparatus. The results indicate that fish treated with 1 µg/g body weight of GPR139 agonist displayed no difference in locomotor activity and alarm substance-induced fear response. However, avoidance to fear-conditioned compartment was diminished, which suggests that the agonist blocks the consolidation of contextual fear memory. On the other hand, fish treated with 0.1 µg/g body weight of GPR139 agonist spent a significantly longer time in the unconditioned neutral compartment as compared to the conditioned (punished and unpunished) compartments. These results suggest that activation of GPR139 signalling in the habenula may be involved in fear learning and the decision-making process in the zebrafish.
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.
Sleep deprivation or sleep loss, a prevalent issue in modern society, is linked to cognitive impairment, leading to heightened risks of errors and accidents. Chronic sleep deprivation affects various cognitive functions, including memory, attention, and decision-making, and is associated with an increased risk of neurodegenerative diseases, cardiovascular issues, and metabolic disorders. This review examines the potential of β-caryophyllene, a dietary non-psychotropic cannabinoid, and FDA-approved flavoring agent, as a therapeutic solution for sleep loss-induced cognitive impairment. It highlights β-caryophyllene's ability to mitigate key contributors to sleep loss-induced cognitive impairment, such as inflammation, oxidative stress, neuronal death, and reduced neuroplasticity, by modulating various signaling pathways, including TLR4/NF-κB/NLRP3, MAPK, Nrf2/HO-1, PI3K/Akt, and cAMP/PKA/CREB. As a naturally occurring, non-psychotropic compound with low toxicity, β-caryophyllene emerges as a promising candidate for further investigation. The review underscores the therapeutic potential of β-caryophyllene for sleep loss-induced cognitive impairment and provides mechanistic insights into its action on crucial pathways, suggesting that β-caryophyllene could be a valuable addition to strategies aimed at combating cognitive impairment and other health issues due to sleep loss.
The role of steroid/thyroid hormones in the regulation of endocrine cells at the level of the pituitary has remained unclear. Therefore, using single-cell quantitative real-time PCR, we examined absolute amounts of transcripts for nuclear receptors [estrogen receptors (ERs) alpha, beta, and gamma; androgen receptors (ARs) a and b; glucocorticoid receptors (GRs) 1, 2a, and 2b; and thyroid hormone receptors (TRs) alpha1, alpha2, and beta] in pituitary cells of immature (IM) and mature (M) male tilapia, Oreochromis niloticus. In the two reproductive stages, ACTH cells expressed only ERbeta, whereas all other pituitary cell types expressed ERalpha + beta, and a subpopulation coexpressed ARa, ARb, GR1, GR2b, and TRbeta but lacked ERgamma, GR2a, TRalpha1, and TRalpha2. IM males had high percentages of LH cells (IM 46.0% vs. M 10.0%), GH cells (IM 23.3% vs. M 7.9%), and prolactin cells (IM 68.8% vs. M 6.0%) with ERbeta, and TSH cells (IM 19.2% vs. M 0.0%) and MSH cells (IM 25.6% vs. M 0.0%) with ERalpha + TRbeta. A high percentage of FSH cells in IM males expressed ERbeta (IM 46.9% vs. M 18.8%), and FSH cells in M males showed significantly high GR1 transcripts (IM 76.0 +/- 5.0 vs. M 195.0 +/- 10.7 copies per cell; P < 0.05), suggesting that FSH cells are regulated differently in the two reproductive stages. Coexpression of ERalpha + beta in high percentages of cells of the GH family (GH, IM 43.8% vs. M 14.3%; prolactin, IM 8.3% vs. M 59.7%; somatolactin, IM 22.2% vs. M 42.2%) suggests that the expression of both ERs is important for functionality. Thus, differential coexpression of genes for nuclear receptors in subpopulations of pituitary cell types suggests multiple steroid/thyroid hormone regulatory pathways at the level of the pituitary during the two reproductive stages.
Kisspeptin is a hypothalamic neuropeptide, which acts directly on gonadotropin-releasing hormone (GnRH)-secreting neurons via its cognate receptor (GPR54 or Kiss-R) to stimulate GnRH secretion in mammals. In non-mammalian vertebrates, there are multiple kisspeptins (Kiss1 and Kiss2) and Kiss-R types. Recent gene knockout studies have demonstrated that fish kisspeptin systems are not essential in the regulation of reproduction. Studying the detailed distribution of kisspeptin receptor in the brain and pituitary is important for understanding the multiple action sites and potential functions of the kisspeptin system. In the present study, we generated a specific antibody against zebrafish Kiss2-R (=Kiss1Ra/GPR54-1/Kiss-R2/KissR3) and examined its distribution in the brain and pituitary. Kiss2-R-immunoreactive cell bodies are widely distributed in the brain including in the dorsal telencephalon, preoptic area, hypothalamus, optic tectum, and in the hindbrain regions. Double-labeling showed that not all but a subset of preoptic GnRH3 neurons expresses Kiss2-R, while Kiss2-R is expressed in most of the olfactory GnRH3 neurons. In the posterior preoptic region, Kiss2-R immunoreactivity was seen in vasotocin cells. In the pituitary, Kiss2-R immunoreactivity was seen in corticotropes, but not in gonadotropes. The results in this study suggest that Kiss2 and Kiss2-R signaling directly serve non-reproductive functions and indirectly subserve reproductive functions in teleosts.
Substance P (SP) and neurokinin A (NKA), encoded by TAC1/Tac1 gene are members of the tachykinin family, which exert their neuromodulatory roles in vertebrate reproduction. In mammals, SP and NKA have been shown to regulate gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion via kisspeptin neurons. On the other hand, the role of SP/NKA in the regulation of reproduction in non-mammalian vertebrates is not well known. In the present study, we first localized expression of tac1 mRNA in the brain of male and female zebrafish, Danio rerio. Next, using an antibody against zebrafish tachykinin1 (Tac1), we examined the neural association of SP/NKA neural processes with GnRH3 neurons, and with kisspeptin (kiss2) neurons, in the brains of male and female zebrafish. In situ hybridization showed an apparent male-dominant tac1 expression in the ventral telencephalic area, the anterior and posterior parts of the parvocellular preoptic nucleus, and the suprachiasmatic nucleus. On the other hand, there was female-dominant tac1 expression in the ventral periventricular hypothalamus. Confocal images of double-labeled zebrafish Tac1 and GnRH3 showed associations between Tac1-immunoreactive processes and GnRH3 neurons in the ventral telencephalic area. In contrast, there was no apparent proximity of Tac1 processes to kiss2 mRNA-expressing neurons in the hypothalamus. Lastly, to elucidate possible direct action of SP/NKA on GnRH3 or Kiss2 neurons, expression of SP/NKA receptor, tacr1a mRNA was examined in regions containing GnRH3 or Kiss2 neurons by in situ hybridization. Expression of tacr1a mRNA was seen in several brain regions including the olfactory bulb, preoptic area and hypothalamus, where GnRH3 and Kiss2 cells are present. These results suggest that unlike in mammals, Tac1 may be involved in male reproductive functions via direct action on GnRH3 neurons but independent of kisspeptin in the zebrafish.
Ghrelin, a gut-brain peptide hormone, is implicated in a multiplicity of biological functions, including energy homeostasis and reproduction. Neuronal systems that are involved in energy homeostasis as well as reproduction traverse the hypothalamus; however, the mechanism by which they control energy homeostasis is not fully understood. The present study analyzes the anatomical relationship of neurons expressing gonadotropin-releasing hormone (GnRH), neuropeptide Y (NPY) and growth hormone-releasing hormone (GHRH) in a cichlid, tilapia (Oreochromis niloticus). Additionally, we examine in vivo effects of ghrelin on these hypothalamic neurons and plasma growth hormone (GH) and insulin-like growth factor-1 (IGF-1) levels. Double-immunofluorescence showed neuronal fiber associations between GnRH, NPY and GHRH in the brain and pituitary. Intracerebroventricular injection of ghrelin had no effect on numbers, soma size, or optical density of GnRH and NPY neurons, whereas the number of GHRH neurons was significantly decreased in the animals injected with ghrelin when compared to controls, which may indicate administered ghrelin promoted GHRH release. Plasma GH and pituitary GH mRNA levels were significantly increased in the animals injected with ghrelin. These results suggest that central administration of ghrelin primarily act on hypothalamic GHRH neurons to stimulate GH release from the pituitary in the tilapia.
Non-rod non-cone photopigments in the eyes and the brain can directly mediate non-visual functions of light in non-mammals. This was supported by our recent findings on vertebrate ancient long (VAL)-opsin photopigments encoded by the val-opsinA (valopa) and val-opsinB (valopb) genes in zebrafish. However, the physiological functions of valop isoforms remain unknown. Here, we generated valop-mutant zebrafish using CRISPR/Cas genome editing, and examined the phenotypes of loss-of-function mutants. F0 mosaic mutations and germline transmission were confirmed via targeted insertions and/or deletions in the valopa or valopb gene in F1 mutants. Based on in silico analysis, frameshift mutations converted VAL-opsin proteins to non-functional truncated forms with pre-mature stop codons. Most F1 eggs or embryos from F0 female valopa/b mutants showed either no or only partial chorion elevation, and the eggs or embryos died within 26 hour-post-fertilization. However, most F1 embryos from F0 male valopa mutant developed but hatched late compared to wild-type embryos, which hatched at 4 day-post-fertilization. Late-hatched F1 offspring included wild-type and mutants, indicating the parental effects of valop knockout. This study shows valop gene knockout affects chorion formation and embryonic hatching in the zebrafish.
A cichlid fish, the Nile tilapia (Oreochromis niloticus), is a maternal mouthbrooder, which exhibits minimum energy expenditure and slower ovarian cycles during mouthbrooding. The objective of this study was to observe changes in the gene expression of key neuropeptides involved in the control of appetite and reproduction, including neuropeptide Y a (NPYa), reproductive neuropeptides: gonadotropin-releasing hormone (GnRH1, GnRH2 and GnRH3) and kisspeptin (Kiss2) during mouthbrooding (4- and 12-days), 12-days of food restriction and 12-days of food restriction followed by refeeding. The food restriction regime showed a significant increase in npya mRNA levels in the telencephalon. However, there were no significant alterations in npya mRNA levels during mouthbrooding. gnrh1 mRNA levels were significantly lower in mouthbrooding female as compared with females with food restriction. gnrh3 mRNA levels were also significantly lower in female with 12-days of mouthbrooding, 12-days of food restriction followed by 12-days of refeeding when compared with controls. There were no significant differences in gnrh2 and kiss2 mRNA levels between groups under different feeding regimes. No significant changes were observed in mRNA levels of receptors for peripheral metabolic signaling molecules: ghrelin (GHS-R1a and GHS-R1b) and leptin (Lep-R). These results suggested that unaffected npya mRNA levels in the telencephalon might contribute to suppression of appetite in mouthbrooding female tilapia. Furthermore, lower gnrh1 and gnrh3 mRNA levels may influence the suppression of reproductive functions such as progression of ovarian cycle and reproductive behaviours, while GnRH2 and Kiss2 may not play a significant roles in reproduction under food restriction condition.
Three paralogous genes for gonadotropin-releasing hormone (GnRH; gnrh1, gnrh2, and gnrh3) and GnRH receptors exist in non-mammalian vertebrates. However, there are some vertebrate species in which one or two of these paralogous genes have become non-functional during evolution. The developmental migration of GnRH neurons in the brain is evolutionarily conserved in mammals, reptiles, birds, amphibians, and jawed teleost fish. The three GnRH paralogs have specific expression patterns in the brain and originate from multiple sites. In acanthopterygian teleosts (medaka, cichlid, etc.), the preoptic area (POA)-GnRH1 and terminal nerve (TN)-GnRH3 neuronal types originate from the olfactory regions. In other fish species (zebrafish, goldfish and salmon) with only two GnRH paralogs (GnRH2 and GnRH3), the TN- and POA-GnRH3 neuronal types share the same olfactory origin. However, the developmental origin of midbrain (MB)-GnRH2 neurons is debatable between mesencephalic or neural crest site. Each GnRH system has distinctive anatomical and physiological characteristics, and functions differently. The POA-GnRH1 neurons are hypophysiotropic in nature and function in the neuroendocrine control of reproduction. The non-hypophysiotropic GnRH2/GnRH3 neurons probably play neuromodulatory roles in metabolism (MB-GnRH2) and the control of motivational state for sexual behavior (TN-GnRH3).
Hypothalamic kisspeptin encoded by KISS1/Kiss1 gene emerged as a regulator of the reproductive axis in mammals following the discovery of the kisspeptin receptor (Kissr) and its role in reproduction. Kisspeptin-Kissr systems have been investigated in various vertebrates, and a conserved sequence of kisspeptin-Kissr has been identified in most vertebrate species except in the avian linage. In addition, multiple paralogs of kisspeptin sequences have been identified in the non-mammalian vertebrates. The allegedly conserved role of kisspeptin-Kissr in reproduction became debatable when kiss/kissr genes-deficient zebrafish and medaka showed no apparent effect on the onset of puberty, sexual development, maturation and reproductive capacity. Therefore, it is questionable whether the role of kisspeptin in reproduction is conserved among vertebrate species. Here we discuss from a comparative and evolutional aspect the diverse functions of kisspeptin and its receptor in vertebrates. Primarily this review focuses on the role of hypothalamic kisspeptin in reproductive and non-reproductive functions that are conserved in vertebrate species.
Kisspeptins encoded by the kiss1 and kiss2 genes play an important role in reproduction through the stimulation of gonadotropin-releasing hormone (GnRH) secretion by activating their receptors (KissR1 EU047918 and KissR2 EU047917). To understand the mechanism through which temperature affects reproduction, we examined kiss1 and kiss2 and their respective receptor (kissr1 and kissr2) gene expression in the brain of male zebrafish exposed to a low temperature (15°C), normal temperature (27°C), and high temperature (35°C) for 7-days. kiss1 mRNA levels in the brain were significantly increased (2.9-fold) in the low temperature compared to the control (27°C), while no noticeable change was observed in the high temperature conditions. Similarly, kissr1 mRNA levels were significantly increased (1.5-2.2-folds) in the low temperature conditions in the habenula, the nucleus of the medial longitudinal fascicle, oculomotor nucleus, and the interpeduncular nucleus. kiss2 mRNA levels were significantly decreased (0.5-fold) in the low and high temperature conditions, concomitant with kissr2 mRNA levels (0.5-fold) in the caudal zone of the periventricular hypothalamus and the posterior tuberal nucleus. gnrh3 but not gnrh2 mRNA levels were also decreased (0.5-fold) in the low and high temperature conditions. These findings suggest that while the kiss1/kissr1 system is sensitive to low temperature, the kiss2/kissr2 system is sensitive to both extremes of temperature, which leads to failure in reproduction.
The central nervous system (CNS) of the non-mammalian vertebrates has better neuroregenerative capability as compared with the mammalian CNS. Regeneration of habenula was observed 40 days after damage in zebrafish. During the early stage of regeneration, we found a significant increase of apoptotic cells on day-1 post-damage and of proliferative cells on day-3 post-damage. To identify the molecular factor(s) involved in the early stages of neuroregeneration, differentially expressed proteins during sham, 20- and 40-h post-habenula damage were investigated by proteomic approach by using two-dimensional differential gel electrophoresis (2D-DIGE) coupled with Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-ToF) and tandem mass spectrometry. Protein profiles revealed 17 differentially (>1.5-fold) expressed proteins: 10 upregulated, 4 downregulated, 2 proteins were found to be downregulated at the early stage but upregulated at a later stage, and 1 protein was found to be upregulated at 2 different time points. All proteins identified can be summarized under few molecular processes involved in the early stages of neuroregeneration in zebrafish CNS: apoptosis regulation (Wnt inhibitory factor 1 [WIF1]), neuroprotection (metallothionein), cell proliferation (Spred2, ependymin, Lhx1, and Wnts), differentiation (Spred2, Lhx9, and Wnts), and morphogenesis (cytoplasmic actins and draculin). These protein profiling results suggest that drastic molecular changes occur in the neuroregenerative process during this period, which includes cell proliferation, differentiation, and protection.
G protein-coupled receptor 139 (GPR139), a highly conserved orphan receptor, is predominantly expressed in the habenula of vertebrate species. Habenula is an ancient epithalamic structure, which is critical to comprehending adaptive behaviors in vertebrates. We have previously demonstrated the role of GPR139 agonists in fear-associated decision-making processes in zebrafish. However, how GPR139 signaling in the habenula modulates such adaptive behavioral responses remains unsolved. Fish centrally administered with a synthetic antagonist for human GPR139 (NCRW0005-F05) exhibited significant suppression of odorant cue (alarm substance, AS)-induced fear learning in the conditioned place avoidance paradigm. On the other hand, co-treatment with a GPR139 antagonist and a synthetic agonist for human GPR139 (JNJ-63533054) interrupted the fear conditioning process by significantly reducing locomotion during post-conditioning. Calcium imaging of acute brain slices showed a significant increase in peak amplitude of calcium transients in the habenula upon bath application of either a GPR139 antagonist or agonist. Furthermore, KCl-evoked calcium transients were reduced by the GPR139 antagonist and co-treatment of the GPR139 antagonist-agonist. These results suggest that the GPR139 antagonist did not block the inhibitory action of the GPR139 agonist in the decision-making process during the fear-retrieval phase; however, solitarily, it functions in governing the fear consolidation process via activation of the ventral habenula neurons in zebrafish.
Neurokinin B (NKB) was recently identified as a key regulator of reproduction in mammals and fish. Fish were found to possess a specific novel neurokinin termed NKF. To study the role of NKB/NKF in the regulation of fish reproduction and to investigate the role of NKB/NKF and their receptors in the piscine pituitary, we have identified the NKB/tachikinin 3 receptor (tac3r) system in tilapia. Bioinformatics and phylogenetic analyses have demonstrated that the tilapia holds 1 putative tac3 gene and 2 NKB receptor genes (tac3ra and tac3rb) that clustered with other piscine Tac3 and NKB receptor lineages. Furthermore, we found that in African cichlids, NKB peptides differ from other vertebrate NKBs in their C-terminal sequence, possessing isoleucine instead of valine as the X in the NKB FXGLM-NH2-terminal consensus sequence. Signal transduction analysis demonstrated that tilapia NKB (tiNKB), tiNKF, and human NKB activated both CRE-luc and SRE-luc transcriptional activity of both tilapia and human NKB receptors. Two hours after ip injection of tiNKB, the plasma levels of both FSH and LH were increased, whereas tiNKF was more effective in increasing LH levels. However, tiNKB was more effective than tiNKF in increasing both FSH and LH from tilapia pituitary dispersed cells. Using in situ hybridization and fluorescent immunohistochemistry, we have shown that LH cells possess tac3, tac3ra, and tac3rb mRNAs, whereas FSH cells possess mainly tac3rb and tac3ra and tac3 to a much lesser extent. These results suggest that the members of the NKB/tac3r system may serve as paracrine/autocrine regulators of gonadotropin release in fish pituitary.