The decrease in serotonergic neurotransmission during aging can increase the risk of neuropsychiatric diseases such as depression in elderly population and decline the reproductive system. Therefore, it is important to understand the age-associated molecular mechanisms of brain aging. In this study, the effect of aging and chronic escitalopram (antidepressant) treatment to admit mice was investigated by comparing transcriptomes in the preoptic area (POA) which is a key nucleus for reproduction. In the mid-aged brain, the immune system-related genes were increased and hormone response-related genes were decreased. In the escitalopram treated brains, transcription-, granule cell proliferation- and vasoconstriction-related genes were increased and olfactory receptors were decreased. Since homeostasis and neuroprotection-related genes were altered in both of mid-age and escitalopram treatment, these genes could be important for serotonin related physiologies in the POA.
Kisspeptin is a neuropeptide, encoded by kisspeptin 1 (KISS1)/Kiss1 gene, which primarily acts as the regulator of reproductive functions via its receptor, kisspeptin receptor (KissR) in vertebrates. In the brain, Kiss1 gene is mainly expressed in the hypothalamic region, but KissR gene is widely distributed throughout the brain, suggesting that kisspeptin-KissR system may be involved in not only reproductive, but also non-reproductive functions. In non-mammalian vertebrates, there are two or more kisspeptin and KissR types. The zebrafish (Danio rerio) possess two kisspeptin (Kiss1 and Kiss2) and their respective receptors [Kiss1 receptor (KissR1) and KissR2]. In the brain of zebrafish, while Kiss2 is expressed in the preoptic-hypothalamic area, Kiss1 is predominantly expressed in the habenula, an evolutionarily conserved epithalamic structure. Similarly, KissR1 is expressed only in the habenula, while KissR2 is widely distributed in the brain, suggesting that the two kisspeptin systems play specific roles in the brain. The habenular Kiss1 is involved in the modulation of the raphe nuclei and serotonin-related behaviors such as fear response in the zebrafish. This review summarizes the roles of multiple kisspeptin-KissR systems in reproductive and non-reproductive functions and neuronal mechanism, and debates the biological and evolutional significance of habenular kisspeptin-KissR systems in teleost species.
Gonadotropin-releasing hormone (GnRH) is a reproductive neuropeptide, which controls vertebrate reproduction. In most vertebrates, there are more than two GnRH orthologs in the brain. In cichlid fish, the Nile tilapia (Oreochromis niloticus), GnRH1 is the primary hypophysiotropic hormone, while GnRH2 and GnRH3 are non-hypophysiotropic but neuromodulatory in function. Hypophysiotropic GnRH neurons are thought to inter-communicate, while it remains unknown if hypophysiotropic and non-hypophysiotropic GnRH systems communicate with each other. In the present study, we examined interrelationship between three GnRH types using specific antibodies raised against their respective GnRH associated peptide (GAP) sequence. Double-immunofluorescence labeling coupled with confocal microscopy revealed that in sexually mature males, GnRH-GAP1-immunoreactive (-ir) processes are in proximities of GnRH-GAP3-ir cell somata in the terminal nerve, while GnRH-GAP1-ir cell somata were also accompanied by GnRH-GAP3-ir processes in the preoptic area. However, such interaction was not seen in immature males. Further, there was no interaction between GnRH-GAP2 and GnRH-GAP1 or GnRH-GAP3 neurons. Single cell gene expression analysis revealed co-expression of multiple GnRH receptor genes (gnrhr1 and gnrhr2) in three GnRH-GAP cell types. In mature males, high levels of gnrhr2 mRNA were expressed in GnRH-GAP1-ir cells. In immature males, gnrhr1 and gnrhr2 mRNAs are highly expressed in GnRH-GAP3-ir cells. These results suggest heterologous interactions between the three GnRH-GAP cell types and their potential functional interaction during different reproductive stages.
Reproduction is associated with the circadian system, primarily as a result of the connectivity between the biological clock in the suprachiasmatic nucleus (SCN) and reproduction-regulating brain regions, such as preoptic area (POA), anteroventral periventricular nucleus (AVPV), and arcuate nucleus (ARC). Networking of the central pacemaker to these hypothalamic brain regions is partly represented by close fiber appositions to specialized neurons, such as kisspeptin and gonadotropin-releasing hormone (GnRH) neurons; accounting for rhythmic release of gonadotropins and sex steroids. Numerous studies have attempted to dissect the neurochemical properties of GnRH neurons, which possess intrinsic oscillatory features through the presence of clock genes to regulate the pulsatile and circadian secretion. However, less attention has been given to kisspeptin, the upstream regulator of GnRH and a potent mediator of reproductive functions including puberty. Kisspeptin exerts its stimulatory effects on GnRH secretion via its cognate Kiss-1R receptor that is co-expressed on GnRH neurons. Emerging studies have found that kisspeptin neurons oscillate on a circadian basis and that these neurons also express clock genes that are thought to regulate its rhythmic activities. Based on the fiber networks between the SCN and reproductive nuclei such as the POA, AVPV, and ARC, it is suggested that interactions among the central biological clock and reproductive neurons ensure optimal reproductive functionality. Within this neuronal circuitry, kisspeptin neuronal system is likely to "time" reproduction in a long term during development and aging, in a medium term to regulate circadian or estrus cycle, and in a short term to regulate pulsatile GnRH secretion.
In addition to reproduction, gonadotropin-releasing hormone (GnRH) has been postulated to control cholesterol metabolism via cholesterol transport, which is carried out partly by the members of ATP-binding cassette (ABC) transporters G1 (ABCG1) and G4 (ABCG4). However, there is yet to be evidence demonstrating the relationship between these transporters with reference to GnRH neurons. In the present study, we cloned two ABCG1 messenger RNA (mRNA) variants and one ABCG4 mRNA and examined their expression in the brain including GnRH neurons (GnRH1, GnRH2, and GnRH3) in the cichlid tilapia (Oreochromis niloticus). Comparison of nucleotide sequences of the tilapia ABCG1 and ABCG4 with that of other fish species showed that both of these genes are evolutionarily conserved among fishes. ABCG1 and ABCG4 were shown to have high mRNA expressions in the CNS, pituitary, and gonads. In the brain, real-time polymerase chain reaction (PCR) showed that ABCG4 mRNA was higher than ABCG1a in all brain regions including the olfactory bulb (ABCG1=13.34, ABCG4=6796.35; P<0.001), dorsal telencephalon (ABCG1=8.64, ABCG4=10149.13; P=0.001), optic tectum (ABCG1=22.12, ABCG4=13931.04; P<0.01), cerebellum (ABCG1=8.68, ABCG4=12382.90; P<0.01), and preoptic area-midbrain-hypothalamus (ABCG1=21.36, ABCG4=13255.41; P=0.001). Similarly, although ABCG1 mRNA level is much higher in the pituitary compared with the brain, it was still significantly lower compared with ABCG4 (ABCG1=337.73, ABCG4=1157.87; P=0.01). The differential pattern of expression of ABCG1 and ABCG4 in the brain versus pituitary suggests that the two transporters are regulated by different mechanisms. Furthermore, ABCG1 and ABCG4 mRNA expressions were found in all three types of laser-captured GnRH neurons with highly similar percentage of expressions, suggesting that cholesterol efflux from GnRH neurons may require heterodimerization of both ABCG1 and ABCG4.
Migration and final positioning of gonadotropin-releasing hormone (GnRH) neurons in the preoptic area (POA) is critical for reproduction. It is known that maternal dexamethasone (DEX) exposure impairs reproductive function and behaviour in the offspring. However, it is still not known whether maternal DEX exposure affects the postnatal GnRH neurons in the offspring. This study determined the neuronal movement of enhanced green fluorescent protein (EGFP)-tagged GnRH neurons in slice culture of postnatal day 0 (P0), P5 and P50-60 transgenic male rats. Effect of maternal DEX treatment on EGFP-GnRH neuronal movement and F-actin distribution on GnRH neurons at P0 stage were studied. Time-lapse analysis of P0 and P5 EGFP-GnRH neurons displayed active cellular movement within the POA compared to young adult P50-60 stages, suggesting possible fine-tuning movement for positioning of early postnatal GnRH neurons. The DEX-treated EGFP-GnRH neurons demonstrated decreased motility in the POA and reduced F-actin distribution in the GnRH neurons at 60 h culture compared to the vehicle-treated. These results suggest that the P0 GnRH neuronal movement in the POA is altered by maternal DEX exposure, which possibly disrupts the fine-tuning process for positioning and development of early postnatal GnRH neurons in the brain, potentially linked to reproductive dysfunction in adulthood.
Water deprivation (WD) induces changes in plasma volume and osmolality, which in turn activate several responses, including thirst, the activation of the renin-angiotensin system (RAS) and vasopressin (AVP) and oxytocin (OT) secretion. These systems seem to be influenced by oestradiol, as evidenced by the expression of its receptor in brain areas that control fluid balance. Thus, we investigated the effects of oestradiol treatment on behavioural and neuroendocrine changes of ovariectomized rats in response to WD. We observed that in response to WD, oestradiol treatment attenuated water intake, plasma osmolality and haematocrit but did not change urinary volume or osmolality. Moreover, oestradiol potentiated WD-induced AVP secretion, but did not alter the plasma OT or angiotensin II (Ang II) concentrations. Immunohistochemical data showed that oestradiol potentiated vasopressinergic neuronal activation in the lateral magnocellular PVN (PaLM) and supraoptic (SON) nuclei but did not induce further changes in Fos expression in the median preoptic nucleus (MnPO) or subfornical organ (SFO) or in oxytocinergic neuronal activation in the SON and PVN of WD rats. Regarding mRNA expression, oestradiol increased OT mRNA expression in the SON and PVN under basal conditions and after WD, but did not induce additional changes in the mRNA expression for AVP in the SON or PVN. It also did not affect the mRNA expression of RAS components in the PVN. In conclusion, our results show that oestradiol acts mainly on the vasopressinergic system in response to WD, potentiating vasopressinergic neuronal activation and AVP secretion without altering AVP mRNA expression.
Glial cell line-derived neurotrophic factor (GDNF) was initially described as important for dopaminergic neuronal survival and is involved in many other essential functions in the central nervous system. Characterization of GDNF phenotype in mammals is well described; however, studies in non-mammalian vertebrate models are scarce. Here, we characterized the anatomical distribution of gdnf-expressing cells in adult zebrafish brain by means of combined in situ hybridization (ISH) and immunohistochemistry. Our results revealed that gdnf was widely dispersed in the brain. gdnf transcripts were co-localized with radial glial cells along the ventricular area of the telencephalon and in the hypothalamus. Interestingly, Sox2 positive cells expressed gdnf in the neuronal layer but not in the ventricular zone of the telencephalon. A subset of GABAergic precursor cells labeled with dlx6a-1.4kbdlx5a/6a: green fluorescence protein (GFP) in the pallium, parvocellular preoptic nucleus, and the anterior and dorsal zones of the periventricular hypothalamus also showed expression with gdnf mRNA. In addition, gdnf signals were detected in subsets of dopaminergic neurons, including those in the ventral diencephalon, similar to what is seen in mammalian brain. Our work extends our knowledge of gdnf action sites and suggests a potential role for gdnf in adult brain neurogenesis and regeneration.
Sexual dysfunction and cognitive deficits are markers of the aging process. Mammalian sirtuins (SIRT), encoded by sirt 1-7 genes, are known as aging molecules which are sensitive to serotonin (5-hydroxytryptamine, 5-HT). Whether the 5-HT system regulates SIRT in the preoptic area (POA), which could affect reproduction and cognition has not been examined. Therefore, this study was designed to examine the effects of citalopram (CIT, 10 mg/kg for 4 weeks), a potent selective-serotonin reuptake inhibitor and aging on SIRT expression in the POA of male mice using real-time PCR and immunocytochemistry. Age-related increases of sirt1, sirt4, sirt5, and sirt7 mRNA levels were observed in the POA of 52 weeks old mice. Furthermore, 4 weeks of chronic CIT treatment started at 8 weeks of age also increased sirt2 and sirt4 mRNA expression in the POA. Moreover, the number of SIRT4 immuno-reactive neurons increased with aging in the medial septum area (12 weeks = 1.00 ± 0.15 vs. 36 weeks = 1.68 ± 0.14 vs. 52 weeks = 1.54 ± 0.11, p < 0.05). In contrast, the number of sirt4-immunopositive cells did not show a statistically significant change with CIT treatment, suggesting that the increase in sirt4 mRNA levels may occur in cells in which sirt4 is already being expressed. Taken together, these studies suggest that CIT treatment and the process of aging utilize the serotonergic system to up-regulate SIRT4 in the POA as a common pathway to deregulate social cognitive and reproductive functions.
Postnatal treatment with selective serotonin reuptake inhibitors (SSRIs) has been found to affect brain development and the regulation of reproduction in rodent models. The normal masculinization process in the brain requires a transient decrease in serotonin (5-HT) levels in the brain during the second postnatal week. Strict regulation of androgen receptor (AR) and gonadotropin-releasing hormone (GnRH) expression is important to control male reproductive activity. Therefore, this study was designed to examine the effects of a potent SSRI (citalopram) on male sexual behavior and expression levels of AR and GnRH in adult male mice receiving either vehicle or citalopram (10mg/kg) daily during postnatal days 8-21. The citalopram-treated male mice showed altered sexual behavior, specifically a significant reduction in the number of intromissions preceding ejaculation compared with the vehicle-treated mice. The citalopram-treated male mice displayed elevated anxiety-like behavior in an open field test and lower locomotor activity in their home cage during the subjective night. Although there was no change in GnRH and AR mRNA levels in the preoptic area (POA), quantified by real-time polymerase chain reaction, immunostained AR cell numbers in the medial POA were decreased in the citalopram-treated male mice. These results suggest that the early-life inhibition of 5-HT transporters alters the regulation of AR expression in the medial POA, likely causing decreased sexual behavior and altered home cage activity in the subjective night.