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 bactericidal/permeability-increasing (BPI) fold-containing (BPIF) superfamily of genes expressed in the brain are purportedly involved in modulating brain function in response to stress, such as inflammation. Kisspeptin, encoded by kiss, is affected by inflammation in the brain; therefore, BPIF family genes might be involved in the modulation of kisspeptin in the brain. In this study, we investigated the expression of BPIF family C, like (bpifcl) in zebrafish brain and its involvement in kiss2 regulation. The identified, full-length sequence of a bpifcl isoform expressed in the zebrafish brain contained the BPI fold shared by BPIF family members. bpifcl mRNA expression in female zebrafish brains was significantly higher than that in males. Exposure of female zebrafish to 11-ketotestosterone decreased bpifcl and kiss2 mRNA expression. bpifcl knockdown by bpifcl-specific small interfering RNA administration to female zebrafish brain decreased kiss2 mRNA expression. bpifcl expression was widely distributed in the brain, including in the dorsal zone of the periventricular hypothalamus (Hd). Furthermore, bpifcl was also expressed in KISS2 neurons in the Hd. These results suggest that the Bpifcl modulates kiss2 mRNA expression under the influence of testosterone in the Hd of female zebrafish.
Conventional mammalian models of neurodegeneration are often limited by futile axonogenesis with minimal functional recuperation of severed neurons. The emergence of zebrafish, a non-mammalian model with excellent neuroregenerative properties, may address these limitations. This study aimed to establish an adult zebrafish-based, neurotoxin-induced Parkinson's disease (PD) model and subsequently validate the regenerative capability of dopaminergic neurons (DpN). The DpN of adult male zebrafish (Danio rerio) were lesioned by microinjecting 6-hydroxydopamine (6-OHDA) neurotoxin (6.25, 12.5, 18.75, 25, 37.5, 50 and 100 mg/kg) into the ventral diencephalon (Dn). This was facilitated by an optimised protocol that utilised 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanineperchlorate (DiI) dye to precisely identify the injection site. Immunostaining was utilised to identify the number of tyrosine hydroxylase immunoreactive (TH-ir) DpN in brain regions of interest (i.e. olfactory bulb, telencephalon, preoptic area, posterior tuberculum and hypothalamus). Open tank video recordings were performed for locomotor studies. The Dn was accessed by setting the injection angle of the microinjection capillary to 60° and injection depth to 1200 μm (from the exposed brain surface). 6-OHDA (25 mg/kg) successfully ablated >85% of the Dn DpN (preoptic area, posterior tuberculum and hypothalamus) whilst maintaining a 100% survival. Locomotor analysis of 5-min recordings revealed that 6-OHDA-lesioned adult zebrafish were significantly (p zebrafish showed full recovery of Dn DpN 30 days post-lesion. This study had successfully developed a stable 6-OHDA-induced PD zebrafish model using a straightforward and reproducible approach. Thus, this developed teleost model poses exceptional potentials to study DpN regeneration.
Despite the known importance of long-chained polyunsaturated fatty acids (LC-PUFA) during development, very little is known about their utilization and biosynthesis during embryogenesis. Combining the advantages of the existence of a complete range of enzymes required for LC-PUFA biosynthesis and the well established developmental biology tools in zebrafish, we examined the expression patterns of three LC-PUFA biosynthesis genes, Elovl2-like elongase (elovl2), Elovl5-like elongase (elovl5) and fatty acyl desaturase (fad) in different zebrafish developmental stages. The presence of all three genes in the brain as early as 24 hours post fertilization (hpf) implies LC-PUFA synthesis activity in the embryonic brain. This expression eventually subsides from 72 hpf onwards, coinciding with the initiation of elovl2 and fad expression in the liver and intestine, 2 organs known to be involved in adult fish LC-PUFA biosynthesis. Collectively, these patterns strongly suggest the necessity for localized production of LC-PUFA in the brain during in early stage embryos prior to the maturation of the liver and intestine. Interestingly, we also showed a specific expression of elovl5 in the proximal convoluted tubule (PCT) of the zebrafish pronephros, suggesting a possible new role for LC-PUFA in kidney development and function.
pbx1, a TALE (three-amino acid loop extension) homeodomain transcription factor, is involved in a diverse range of developmental processes. We examined the expression of pbx1 during zebrafish development by in situ hybridization. pbx1 transcripts could be detected in the central nervous system and pharyngeal arches from 24 hpf onwards. In the swim bladder anlage, pbx1 was detected as early as 28 hpf, making it the earliest known marker for this organ. Morpholino-mediated gene knockdown of pbx1 revealed that the swim bladder failed to inflate, with eventual lethality occurring by 8 dpf. The knockdown of pbx1 did not perturb the expression of prdc and foxA3, with both early swim bladder markers appearing normally at 36 and 48 hpf, respectively. However, the expression of anxa5 was completely abolished by pbx1 knockdown at 60 hpf suggesting that pbx1 may be required during the late stage of swim bladder development.
In Parkinson's disease (PD), several genes have been identified as the PD-related genes, however, the regulatory mechanisms of these gene expressions have not been fully identified. In this study, we investigated the effect of inflammation, one of the major risk factors in PD on expressions of the PD-related genes. Lipopolysaccharide (LPS) was intraperitoneally administered to mature male zebrafish and gene expressions in the brains were examined by real-time PCR. In the inflammation-related genes, expressions of tnfb, il1b and il6 were increased at 2 days post administration in the 10 μg group, and tnfb expression was also increased at 4 days post administration in the 1 μg and 10 μg group. In the PD-related genes, pink1 expression was significantly decreased at 4 days, atp13a2 expression was significantly increased at 7 days, and uchl1 expression was significantly decreased at 7 days. This suggests that pink1, atp13a2 and uchl1 expressions are regulated by inflammation, and this regulatory mechanism might be involved in the progress of PD.
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.
Excessive production of melanin implicates hyperpigmentation disorders. Flavokawain A (FLA) and flavokawain B (FLB) have been reported with anti-melanogenic activity, but their melanogenic inhibition and toxicity effects on the vertebrate model of zebrafish are still unknown. In the present study, cytotoxic as well as melanogenic effects of FLA and FLB on cellular melanin content and tyrosinase activity were evaluated in α-MSH-induced B16/F10 cells. Master regulator of microphthalmia-associated transcription factor (Mitf) and the other downstream melanogenic-related genes were verified via quantitative real time PCR (qPCR). Toxicity assessment and melanogenesis inhibition on zebrafish model was further observed. FLA and FLB significantly reduced the specific cellular melanin content by 4.3-fold and 9.6-fold decrement, respectively in α-MSH-induced B16/F10 cells. Concomitantly, FLA significantly reduced the specific cellular tyrosinase activity by 7-fold whilst FLB by 9-fold. The decrement of melanin production and tyrosinase activity were correlated with the mRNA suppression of Mitf which in turn down-regulate Tyr, Trp-1 and Trp-2. FLA and FLB exhibited non-toxic effects on the zebrafish model at 25 and 6.25 µM, respectively. Further experiments on the zebrafish model demonstrated successful phenotype-based depigmenting activity of FLA and FLB under induced melanogenesis. To sum up, our findings provide an important first key step for both of the chalcone derivatives to be further studied and developed as potent depigmenting agents.
The habenula is a phylogenetically conserved epithalamic structure, which conveys negative information via inhibition of mesolimbic dopamine neurons. We have previously shown the expression of kisspeptin (Kiss1) in the habenula and its role in the modulation of fear responses in the zebrafish. In this study, to investigate whether habenular Kiss1 regulates fear responses via dopamine neurons in the zebrafish, Kiss1 peptides were intracranially administered close to the habenula, and the expression of dopamine-related genes (th1, th2 and dat) were examined in the brain using real-time PCR and dopamine levels using LC-MS/MS. th1 mRNA levels and dopamine levels were significantly increased in the telencephalon 24-h and 30-min after Kiss1 administration, respectively. In fish administered with Kiss1, expression of neural activity marker gene, npas4a and kiss1 gene were significantly decreased in the ventral habenula. Application of neural tracer into the median raphe, site of habenular Kiss1 neural terminal projections showed tracer-labelled projections in the medial forebrain bundle towards the telencephalon where dopamine neurons reside. These results suggest that Kiss1 negatively regulates its own neuronal activity in the ventral habenula via autocrine action. This, in turn affects neurons of the median raphe via interneurons, which project to the telencephalic dopaminergic neurons.
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.
Early-life stress can cause long-term effects in the adulthood such as alterations in behaviour, brain functions and reproduction. DNA methylation is a mechanism of epigenetic change caused by early-life stress. Dexamethasone (DEX) was administered to zebrafish larvae to study its effect on reproductive dysfunction. The level of GnRH2, GnRH3, Kiss1 and Kiss2 mRNAs were measured between different doses of DEX treatment groups in adult zebrafish. Kiss1 and GnRH2 expression were increased in the 200mg/L DEX treated while Kiss2 and GnRH3 mRNA levels were up-regulated in the 2mg/L DEX-treated zebrafish. The up-regulation may be related to programming effect of DEX in the zebrafish larvae, causing overcompensation mechanism to increase the mRNA levels. Furthermore, DEX treatment caused negative impact on the development and maturation of the testes, in particular spermatogenesis. Therefore, immature gonadal development may cause positive feedback by increasing GnRH and Kiss. This indicates that DEX can alter the regulation of GnRH2, GnRH3, Kiss1 and Kiss2 in adult zebrafish, which affects maturation of gonads. Computer analysis of 1.5 kb region upstream of the 5' UTR of Kiss1, Kiss2, GnRH2 and GnRH3 promoter showed that there are putative binding sites of glucocorticoid response element and transcription factors involved in stress response. GnRH3 promoter analysed from pre-optic area, ventral telencephalon and ventral olfactory bulb showed higher methylation at CpG residues located on -1410, -1377 and -1355 between control and 2mg/L DEX-treated groups. Hence, early-life DEX treatment can alter methylation of GnRH3 gene promoter, which subsequently affects gene regulation and reproductive functions.
The kiss1/gpr54 signaling system is considered to be a critical regulator of reproduction in most vertebrates. However, this presumption has not been tested vigorously in nonmammalian vertebrates. Distinct from mammals, multiple kiss1/gpr54 paralogous genes (kiss/kissr) have been identified in nonmammalian vertebrates, raising the possibility of functional redundancy among these genes. In this study, we have systematically generated the zebrafish kiss1(-/-), kiss2(-/-), and kiss1(-/-);kiss2(-/-) mutant lines as well as the kissr1(-/-), kissr2(-/-), and kissr1(-/-);kissr2(-/-) mutant lines using transcription activator-like effector nucleases. We have demonstrated that spermatogenesis and folliculogenesis as well as reproductive capability are not impaired in all of these 6 mutant lines. Collectively, our results indicate that kiss/kissr signaling is not absolutely required for zebrafish reproduction, suggesting that the kiss/kissr systems play nonessential roles for reproduction in certain nonmammalian vertebrates. These findings also demonstrated that fish and mammals have evolved different strategies for neuroendocrine control of reproduction.
In addition to vision, light information is used to regulate a range of animal physiology. Such nonimage-forming functions of light are mediated by nonvisual photoreceptors expressed in distinct neurons in the retina and the brain in most vertebrates. A nonvisual photoreceptor vertebrate ancient long opsin (VAL-opsin) possesses two functional isoforms in the zebrafish, encoded by valopa and valopb, which has received little attention. To delineate the neurochemical identities of valop cells and to test for colocalization of the valop isoforms, we used in situ hybridization to characterize the expression of the valop genes along with that of neurotransmitters and a neuropeptide known to be present at the sites of valop expression. Double labeling showed that the thalamic valop population coexpresses valopa and valopb. All the thalamic valop cells overlapped with a GABAergic cell mass that continues from the anterior nucleus to the intercalated thalamic nucleus. A novel valopa cell population found in the superior raphe was serotonergic in nature. A valopb cell population in the Edinger-Westphal nucleus was identified as containing thyrotropin-releasing hormone. Valopb cells localized in the hindbrain intermediate reticular formation were noncholinergic in nature (nonmotorneurons). Thus, the presence of valop cell populations in different brain regions with coexpression of neurotransmitters and neuropeptides and the colocalization of valop isoforms in the thalamic cell population indicate regulatory and functional complexity of VAL-opsin in the brain of the zebrafish.
The Kiss1/KISS1 gene has recently been implicated as a potent hypothalamic regulator of reproductive functions, in particular, the onset of puberty in mammals. In zebrafish (Danio rerio), there are two kiss1 homologues (kiss1 and kiss2) expressed in the brain: Kiss2-expressing neurons in the hypothalamic nuclei are considered potent regulators of reproduction, whereas the role of Kiss1-expressing neurons in the habenula remains unknown. We first analyzed the expression of kiss1 mRNA in a transgenic zebrafish, in which the habenula-interpeduncular nucleus (IPN) pathway is labelled with green fluorescent protein, and our application of a biocytin neural tracer into the habenula showed the presence of neuronal projections of Kiss1 neurons to the ventral IPN. Therefore, we speculated that kiss1 neurons might regulate the serotonergic system in the raphe. However, laser microdissection followed by real-time PCR revealed the expression of Kiss1 receptor (kissr1) mRNA in the habenula and the ventral IPN but not in the dorsal IPN or the serotonergic neurons in the raphe nuclei. Dual-fluorescent in situ hybridization revealed the coexpression of kiss1 and kissr1 mRNA in the habenula. Administration of Kiss1 significantly decreased the level of kiss1 mRNA (0.3- to 0.5-fold, P < 0.001), but the level of c-fos mRNA was increased (≈ 3-fold, P < 0.05) in the ventral habenula, suggesting that there is autocrine regulation of the kiss1 gene. Kiss1 administration significantly increased the c-fos mRNA levels in the raphe nuclei (2.5-fold, P < 0.001) and genes involved in the regulation of serotonin levels (pet1 and slc6a4a; 3.3- and 2.2-fold, P < 0.01). These findings suggest that the autocrine-regulated habenular Kiss1 neurons indirectly regulate the serotonergic system in the raphe nuclei through the IPN in the zebrafish.
Newly discovered kisspeptin (metastin), encoded by the Kiss1/KISS1 gene, is considered as a major gatekeeper of puberty through the regulation of GnRH. In the present study, we cloned a novel kisspeptin gene (kiss2) in the zebrafish Danio rerio and the medaka Oryzias latipes, which encodes a sequence of 125 and 115 amino acids, respectively, and its core sequence (FNLNPFGLRF, F-F form) is different from the previously characterized kiss1 (YNLNSFGLRY, Y-Y form). Our in silico data mining shows kiss1 and kiss2 are highly conserved across nonmammalian vertebrate species, and we have identified two putative kisspeptins in the platypus and three forms in Xenopus. In the brain of zebrafish and medaka, in situ hybridization and laser capture microdissection coupled with real-time PCR showed kiss1 mRNA expression in the ventromedial habenula and the periventricular hypothalamic nucleus. The kiss2 mRNA expression was observed in the posterior tuberal nucleus and the periventricular hypothalamic nucleus. Quantitative real-time PCR analysis during zebrafish development showed a significant increase in zebrafish kiss1, kiss2 (P < 0.002), gnrh2, and gnrh3 (P < 0.001) mRNA levels at the start of the pubertal phase and remained high in adulthood. In sexually mature female zebrafish, Kiss2 but not Kiss1 administration significantly increased FSH-beta (2.7-fold, P < 0.05) and LH-beta (8-fold, P < 0.01) mRNA levels in the pituitary. These results suggest that the habenular Kiss1 and the hypothalamic Kiss2 are potential regulators of reproduction including puberty and that Kiss2 is the predominant regulator of gonadotropin synthesis in fish.
Epilepsy is a devastating neurological condition exhibited by repeated spontaneous and unpredictable seizures afflicting around 70 million people globally. The basic pathophysiology of epileptic seizures is still elusive, reflecting an extensive need for further research. Developing a novel animal model is crucial in understanding disease mechanisms as well as in assessing the therapeutic target. Most of the pre-clinical epilepsy research has been focused on rodents. Nevertheless, zebrafish disease models are relevant to human disease pathophysiology hence are gaining increased attention nowadays. The current study for the very first time developed a pilocarpine-induced chronic seizure-like condition in adult zebrafish and investigated the modulation in several neuroinflammatory genes and neurotransmitters after pilocarpine exposures. Seizure score analysis suggests that compared to a single dose, repeated dose pilocarpine produces chronic seizure-like effects maintaining an average seizure score of above 2 each day for a minimum of 10 days. Compared to the single dose pilocarpine treated group, there was increased mRNA expression of HMGB1, TLR4, TNF-α, IL-1, BDNF, CREB-1, and NPY; whereas decreased expression of NF-κB was upon the repeated dose of pilocarpine administration. In addition, the epileptic group demonstrates modulation in neurotransmitters levels such as GABA, Glutamate, and Acetylcholine. Moreover, proteomic profiling of the zebrafish brain from the normal and epileptic groups from LCMS/MS quantification detected 77 and 13 proteins in the normal and epileptic group respectively. Summing up, the current investigation depicted that chemically induced seizures in zebrafish demonstrated behavioral and molecular alterations similar to classical rodent seizure models suggesting the usability of adult zebrafish as a robust model to investigate epileptic seizures.
The habenula is an evolutionarily conserved brain structure, which has recently been implicated in fear memory. In the zebrafish, kisspeptin (Kiss1) is predominantly expressed in the habenula, which has been implicated as a modulator of fear response. Hence, in the present study, we questioned whether Kiss1 has a role in fear memory and morphine-induced fear memory impairment using an odorant cue (alarm substances, AS)-induced fear avoidance paradigm in adult zebrafish, whereby the fear-conditioned memory can be assessed by a change of basal place preference (= avoidance) of fish due to AS-induced fear experience. Subsequently, to examine the possible role of Kiss1 neurons-serotonergic pathway, kiss1 mRNA and serotonin levels were measured. AS exposure triggered fear episodes and fear-conditioned place avoidance. Morphine treatment followed by AS exposure, significantly impaired fear memory with increased time-spent in AS-paired compartment. However, fish administered with Kiss1 (10-21 mol/fish) after morphine treatment had significantly lower kiss1 mRNA levels but retained fear memory. In addition, the total brain serotonin levels were significantly increased in AS- and Kiss1-treated groups as compared to control and morphine treated group. These results suggest that habenular Kiss1 might be involved in consolidation or retrieval of fear memory through the serotonin system.
Large doses of ionizing radiation can damage human tissues. Therefore, there is a need to investigate the radiation effects as well as identify effective and non-toxic radioprotectors. This study evaluated the radioprotective effects of Kelulut honey (KH) from stingless bee (Trigona sp.) on zebrafish (Danio rerio) embryos. Viable zebrafish embryos at 24 hpf were dechorionated and divided into four groups, namely untreated and non-irradiated, untreated and irradiated, KH pre-treatment and amifostine pre-treatment. The embryos were first treated with KH (8 mg/mL) or amifostine (4 mM) before irradiation at doses of 11 Gy to 20 Gy using gamma ray source, caesium-137 (137Cs). Lethality and abnormality analysis were performed on all of the embryos in the study. Immunohistochemistry assay was also performed using selected proteins, namely γ-H2AX and caspase-3, to investigate DNA damages and incidences of apoptosis. KH was found to reduce coagulation effects at up to 20 Gy in the lethality analysis. The embryos developed combinations of abnormality, namely microphthalmia (M), body curvature and microphthalmia (BM), body curvature with microphthalmia and microcephaly (BMC), microphthalmia and pericardial oedema (MO), pericardial oedema (O), microphthalmia with microcephaly and pericardial oedema (MCO) and all of the abnormalities (AA). There were more abnormalities developed from 24 to 72 h (h) post-irradiation in all groups. At 96 h post-irradiation, KH was identified to reduce body curvature effect in the irradiated embryos (up to 16 Gy). γ-H2AX and caspase-3 intensities in the embryos pre-treated with KH were also found to be lower than the untreated group at gamma irradiation doses of 11 Gy to 20 Gy and 11 Gy to 19 Gy, respectively. KH was proven to increase the survival rate of zebrafish embryos and exhibited protection against organ-specific abnormality. KH was also found to possess cellular protective mechanism by reducing DNA damage and apoptosis proteins expression.
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.
This study assessed the cholesterol lowering effect of Pediococcus acidilactici LAB4 and Lactobacillus plantarum LAB12 using adult zebrafish. Animals were fed with a high cholesterol diet (HCD) with/without LAB for seven weeks. Serum and liver cholesterol was quantified using colorimetric and dye staining methods. Expressions of npc1l1 and abca1 in the liver and intestine and appa in the brain were quantified using RT-PCR. Serum and liver cholesterol was significantly lowered in LAB4- and LAB12-fed zebrafish (≤64% and ≤71%, respectively), with reduced liver cholesterol deposition. The cholesterol lowering effect was accompanied by down-regulation of npc1l1 in intestines (≤28.7%), up-regulation of abca1 in the liver (≥30.5%) and down-regulation of appa in the brain (≤24.5%). A moderately strong positive Pearson correlation (r = 0.617, p < 0.01) was found between appa and serum cholesterol. LAB-fed zebrafish exhibited improved spatial learning and memory. LAB4 and LAB12 can be potentially used in preventing hypercholesterolaemia and Alzheimer's diseases.