Kisspeptin regulates gonadotropin-releasing hormone secretion in gonadotropin-releasing hormone/enhanced green fluorescent protein transgenic rats

Kisspeptin is essential for activation of the hypothalamo-pituitary-gonadal axis. In this study, we established gonadotropin-releasing hormone/enhanced green fluorescent protein transgenic rats. Rats were injected with 1, 10, or 100 pM kisspeptin-10, a peptide derived from full-length kisspeptin, into the arcuate nucleus and medial preoptic area, and with the kisspeptin antagonist peptide 234 into the lateral cerebral ventricle. The results of immunohistochemical staining revealed that pulsatile luteinizing hormone secretion was suppressed after injection of antagonist peptide 234 into the lateral cerebral ventricle, and a significant increase in luteinizing hormone level was observed after kisspeptin-10 injection into the arcuate nucleus and medial preoptic area. The results of an enzyme-linked immunosorbent assay showed that luteinizing hormone levels during the first hour of kisspeptin-10 infusion into the arcuate nucleus were significantly greater in the 100 pM kisspeptin-10 group than in the 10 pM kisspeptin-10 group. These findings indicate that kisspeptin directly promotes gonadotropin-releasing hormone secretion and luteinizing hormone release in gonadotropin-releasing hormone/enhanced green fluorescent protein transgenic rats. The arcuate nucleus is a key component of the kisspeptin-G protein-coupled receptor 54 signaling pathway underlying regulating luteinizing hormone pulse secretion.     

Glycinergic neurons expressing enhanced green fluorescent protein in bacterial artificial chromosome transgenic mice

Although glycine is a major inhibitory transmitter in the mammalian CNS, the role of glycinergic neurons in defined neuronal circuits remains ill defined. This is due in part to difficulties in identifying these cells in living slice preparations for electrophysiological recordings and visualizing their axonal projections. To facilitate the morphological and functional analysis of glycinergic neurons, we generated bacterial artificial chromosome (BAC) transgenic mice, which specifically express enhanced green fluorescent protein (EGFP) under the control of the promotor of the glycine transporter (GlyT) 2 gene, which is a reliable marker for glycinergic neurons. Neurons expressing GlyT2-EGFP were intensely fluorescent, and their dendrites and axons could be visualized in great detail. Numerous positive neurons were detected in the spinal cord, brainstem, and cerebellum. The hypothalamus, intralaminar nuclei of the thalamus, and basal forebrain also received a dense GlyT2-EGFP innervation, whereas in the olfactory bulb, striatum, neocortex, hippocampus, and amygdala positive fibers were much less abundant. No GlyT2-EGFP-positive cell bodies were seen in the forebrain. On the subcellular level, GlyT2-EGFP fluorescence was colocalized extensively with glycine immunoreactivity in somata and dendrites and with both glycine and GlyT2 immunoreactivity in axon terminals, as shown by triple staining at all levels of the neuraxis, confirming the selective expression of the transgene in glycinergic neurons. In slice preparations of the spinal cord, no difference between the functional properties of EGFP-positive and negative neurons could be detected, confirming the utility of visually identifying glycinergic neurons to investigate their functional role in electrophysiological studies.     

Social signals regulate gonadotropin-releasing hormone neurons in the green treefrog

Animals coordinate their physiological state with external cues to appropriately time reproduction. These external cues exert effects through influences on the gonadotropin-releasing hormone neurons (GnRH), at the apex of the hypothalamus-pituitary-gonad (HPG) axis. In green treefrogs, mating calls are important regulators of reproductive behavior and physiology. Reception of mating calls causes an increase in androgen levels, and androgens promote the production of mating calls, demonstrating a mutual influence between the communication and endocrine systems. In order to investigate the central nervous system correlates of social regulation of the HPG axis in green treefrogs, we exposed males to a mating chorus or a control stimulus (tones), counted the resulting number of septo-preoptic GnRH-immunoreactive cells (GnRH-ir), and measured changes in plasma androgens. We found that reception of the mating chorus caused an increase in the number of GnRH-ir cells. As previously shown, we also found that the reception of the mating chorus resulted in higher androgen levels, suggesting that the higher GnRH-ir cell number represents increased GnRH production and release. We suggest that mating calls are an important supplementary cue that promotes GnRH production and release within the context of GnRH regulation by seasonal cues. Previous studies have proposed a neuroanatomical link between the anuran auditory system and GnRH neurons. Our results demonstrate a functional role for this proposed sensory-endocrine circuit, and show for the first time an influence of acoustic signals on GnRH neurons.     

GABA- and glutamate-activated currents in glial cells of the mouse corpus callosum slice.

Whole-cell transmitter-activated currents were recorded with the patch-clamp technique from glial cells in thin frontal brain slices of the corpus callosum. In slices from 6- to 8-day-old mice, glioblasts were predominantly found, while oligodendrocytes were predominant in slices from 10- to 13-day-old mice. These developmental stages could be readily distinguished by their K+ channel pattern and their morphology and ultrastructural features. Both cell types expressed GABA and glutamate receptors in this in situ preparation. GABA responses showed similarities to those described for GABAA receptors, i.e., they were mimicked by muscimol, blocked by bicuculline, and enhanced by pentobarbital. Glutamate responses showed similarities to those of the kainate/quisqualate receptor subtype. The amplitude of GABA-activated currents recorded in oligodendrocytes was significantly smaller than that from glioblasts, while glutamate responses did not show marked differences in either cell type.     

Simulated GABA synaptic input and L-type calcium channels form functional microdomains in hypothalamic gonadotropin-releasing hormone neurons

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons integrate the multiple internal and external cues that regulate sexual reproduction. In contrast to other neurons that exhibit extensive dendritic arbors, GnRH neurons usually have a single dendrite with relatively little branching. This largely precludes the integration strategy in which a single dendritic branch serves as a unit of integration. In the present study, we identify a gradient in L-type calcium channels in dendrites of mouse GnRH neurons and its interaction with GABAergic and glutamatergic inputs. Higher levels of L-type calcium channels are in somata/proximal dendrites (i.e., 0-26 μm) and distal dendrites (～130 μm dendrite length), but intervening midlengths of dendrite (～27-130 μm) have reduced L-type calcium channels. Using uncaging of GABA, there is a decreasing GABAergic influence along the dendrite and the impact of GABA(A) receptors is dependent on activation of L-type calcium channels. This results in amplification of proximal GABAergic signals and attenuation of distal dendritic signals. Most interestingly, the intervening dendritic regions create a filter through which only relatively high-amplitude, low-frequency GABAergic signaling to dendrites elicits action potentials. The findings of the present study suggest that GnRH dendrites adopt an integration strategy whereby segments of single nonbranching GnRH dendrites create functional microdomains and thus serve as units of integration.     

Real-time imaging of green fluorescent protein-tagged beta 2-adrenergic receptor distribution in living cells

In an attempt to investigate the subcellular trafficking of beta(2)-adrenergic receptor (beta(2)AR) in living cells, we performed real-time imaging of beta(2)AR tagged with green fluorescent protein (GFP). We transiently transfected a chimera construct of beta(2)AR and GFP (beta(2)AR-GFP) into HEK 293 cells, primary cultured rat hippocampal neurons and cortical neuronal cells, and then compared the dynamic changes in subcellular localization of beta(2)AR-GFP in these live cells. In the absence of ligands, beta(2)AR-GFP fluorescence was detected predominantly on the plasma membrane in HEK 293 cells as well as on the surface of cell somata and dendrites in cortical neuronal cells. In contrast, in hippocampal neurons, beta(2)AR-GFP was diffusely distributed not only on the surface of cells but in the whole cell somata and dendrites. In HEK 293 cells, cortical neuronal cells and cortical glial cells, time-lapse images showed the rapid appearance of a punctate distribution pattern that became more numerous over the 15-min course of agonist exposure. Semiquantitative analysis revealed the time-course internalization of beta(2)AR-GFP in a single living cell. In hippocampal neurons, beta(2)AR-GFP distribution became scattered both in cell somata and dendrites following agonist exposure. Three-dimensional analysis of time-lapse images revealed a significant portion of beta(2)AR-GFP was distributed in endosomal compartments, along with Alexa 546-labeled transferrin, in all types of cells. Our results demonstrate spatial and temporal redistribution pattern of beta(2)AR in living non-neuronal cells and neuronal cells.     

Expression of galanin immunoreactivity in gonadotropin-releasing hormone neurons in mice: a confocal microscopic study

The expression of galanin immunoreactivity (galanin-IR) in gonadotropin-releasing hormone (GnRH) neurons was investigated in mice using double label immunohistochemistry combined with confocal laser scanning microscopy. A large proportion of GnRH cells in proestrous mice and very few GnRH cells in male mice exhibited galanin-IR. These results are consistent with earlier reports in rats. Unlike in rats, the proportion of GnRH cells coexpressing galanin in mice was high following ovariectomy (OVX) and the treatment of OVX mice with estrogen decreased the number of GnRH cells with galanin-IR. The GnRH system can be considered more active during proestrous and following OVX since the output of luteinizing hormone is elevated during these phases in females. Since the induction of galanin-IR in GnRH cells is more pronounced in OVX and proestrous mice, the expression of galanin-IR in GnRH cells in mice appears to be an activation-dependent phenomenon rather than a direct effect of estrogen. However, in OVX mice treated with steroids to induce an LH surge the number of GnRH cells with galanin-IR was not proportionately increased. The possible reasons for this discrepancy are also discussed.     

Quantitative analysis of synaptic input to gonadotropin-releasing hormone neurons in normal mice and hpg mice with preoptic area grafts

Mutant hypogonadal (hpg) mice with a truncated gene for the precursor to gonadotropin-releasing hormone (GnRH) show certain aspects of recovery of reproductive function after receiving grafts of normal preoptic area into the third ventricle. We have previously shown that GnRH neurons from within the grafts can innervate the appropriate neural-hemal target in the host. To determine if in turn these exogenously derived neurons receive a synaptic input comparable to the GnRH neurons in the normal animal we have now carried out a quantitative ultrastructural analysis to compare the synaptic input to GnRH neurons in the normal preoptic area and in the grafts. In almost all cases GnRH cells or dendrites in normal brains and within the grafts received a synaptic input. In normal animals, input to GnRH dendritic profiles was significantly greater (P less than 0.001) than to the somatic plasma membrane and this trend was also observed within the grafts though the difference was not statistically significant. In addition, no statistically significant difference was found between the input to GnRH structures within the grafts and in normal preoptic area. However, a substantial variability in input among grafted animals was evident which was not observed in normal animals. The sources of variability within the grafts are discussed and we suggest that the deficiencies and differences that exist in regulation of gonadotropin secretion among grafted hpg animals may be reflected in aberrant synaptic input.     

OMP-ZsGreen fluorescent protein transgenic mice for visualisation of olfactory sensory neurons in vivo and in vitro

Research into the biology of the mammalian olfactory system would be greatly enhanced by transgenic reporter mice with cell-specific fluorescence. To this end we previously generated a mouse whose olfactory ensheathing cells (OECs) express DsRed driven by the S100? promoter. We present here a transgenic reporter mouse whose olfactory sensory neurons express ZsGreen, driven by the olfactory marker protein (OMP) promoter. ZsGreen was very strongly expressed throughout the cytoplasm of olfactory sensory neurons labelling them in living cells and after fixation. Labelled sensory neurons were seen in all olfactory regions in the nose and fluorescent axons coursed through the lamina propria and into the main and accessory bulbs. We developed methods for culturing embryonic and postnatal olfactory sensory neurons using these mice to visualise living cells in vitro. ZsGreen was expressed along the length of axons providing exceptional detail of the growth cones. The ZsGreen fluorescence was very stable, without fading during frequent imaging. The combination of OMP-ZsGreen and S100?-DsRed transgenic mice is ideal for developmental studies and neuron-glia assays and they can be bred with mutant mice to dissect the roles of various molecules in neurogenesis, differentiation, axon growth and targeting and other aspects of olfactory sensory neuron and glia biology.     

GABA- and glycine-induced Cl channels in cultured mouse spinal neurons require the same energy to close

Fluctuation analysis has been used to study the influence of temperature on the conductance and mean open time of Cl- permeable ion channels induced by gamma-aminobutyric acid (GABA) and glycine in the membrane of tissue cultured mouse spinal neurons. The conductance of GABA- and glycine-activated channels increased slightly at higher temperatures (Q10 approximately 1.3). The mean open times of these channels decreased at higher temperatures (Q10 approximately 3). Arrhenius plots for the open times of GABA- and glycine-induced channels were parallel and showed no obvious transition behavior over the temperature range 16-40 degrees C.     

Generation of embryonic stem cells and transgenic mice expressing green fluorescence protein in midbrain dopaminergic neurons

We have generated embryonic stem (ES) cells and transgenic mice with green fluorescent protein (GFP) inserted into the Pitx3 locus via homologous recombination. In the central nervous system, Pitx3-directed GFP was visualized in dopaminergic (DA) neurons in the substantia nigra and ventral tegmental area. Live primary DA neurons can be isolated by fluorescence-activated cell sorting from these transgenic mouse embryos. In culture, Pitx3-GFP is coexpressed in a proportion of ES-derived DA neurons. Furthermore, ES cell-derived Pitx3-GFP expressing DA neurons responded to neurotrophic factors and were sensitive to DA-specific neurotoxin N-4-methyl-1, 2, 3, 6-tetrahydropyridine. We anticipate that the Pitx3-GFP ES cells could be used as a powerful model system for functional identification of molecules governing mDA neuron differentiation and for preclinical research including pharmaceutical drug screening and transplantation. The Pitx3 knock-in mice, on the other hand, could be used for purifying primary neurons for molecular studies associated with the midbrain-specific DA phenotype at a level not previously feasible. These mice would also provide a useful tool to study DA fate determination from embryo- or adult-derived neural stem cells.     

Embryonic development of gonadotropin-releasing hormone neurons in the sockeye salmon

Immunocytochemistry and in situ hybridization were used to test the hypothesis that gonadotropin-releasing hormone (GnRH) neurons are formed in the olfactory placode during embryonic development in a salmonid, Oncorhynchus nerka. The development of GnRH neurons and the pituitary cell types was examined from 19 through 910 days after fertilization. Immunoreactive GnRH was first detected at 19 days in the cells of the olfactory placode. GnRH immunoreactivity was not detected in any other structure of the central nervous system at this age. By day 24, GnRH-immunoreactive neurons were seen in the apical, intermediate, and basal layers of the olfactory placode. From days 30 through 51, GnRH neurons were seen emerging from the epithelium, along the olfactory nerve, and at the rostral olfactory bulb. By day 41, GnRH immunoreactivity was lost in the nasal epithelium. In the 72–day-old fish, most of the GnRH neuronal population was found in ganglia of the nervus terminalis, at the cribriform bone (gCB), and at the rostral olfactory bulb (gROB). On day 293, a decrease in GnRH-immunoreactive neurons in the gCB and gROB was concomitant with an initial appearance of GnRH-immunoreactive neurons and fibers along the caudoventral olfactory bulb. By day 462, the distribution of GnRH neurons and fibers was almost similar to adults. In maturing adults (910 days), GnRH-immunoreactive neurons were rarely seen in the nasal regions, but were primarily found in the basal forebrain. GnRH fibers were widespread in the brain, proximal pars distalis, and in the pars intermedia of the pituitary. Our study supports the notion that neurons expressing salmon-GnRH mRNA and peptide originate in the medial olfactory placode and migrate into the basal forebrain during development. The midbrain neurons did not express salmon-GnRH mRNA or peptide in the larval and juvenile fish. © Wiley-Liss, Inc.     

Purification of Purkinje cells by fluorescence-activated cell sorting from transgenic mice that express green fluorescent protein

The cerebellar Purkinje cell has been the focus of numerous studies involving the analysis of development and information processing in the nervous system. Purkinje cells represent less than 0.1% of the total cell content of the cerebellum. To facilitate studies of molecules that are expressed in such a small proportion of neurons, we have established procedures for the purification of these cells. Transgenic mice were developed in which the expression of green fluorescent protein (GFP) was controlled by the L7 promoter. In adult cerebellum, GFP fluorescence was only detected in Purkinje cells, where it filled dendrites, soma and axons. GFP fluorescence was detected in Purkinje cells as early as embryonic day 17 and increased during development in vivo and in dissociated cerebellar culture. Mirroring endogenous L7 expression, high levels of GFP were observed in retinal rod bipolar cells. Lower levels of GFP were seen in olfactory periglomerular cells, neurons in the interpeduncular nucleus, and superior colliculus neurons. Cerebella from transgenic mice were dissociated by mild enzymatic treatment and Purkinje cells were isolated by fluorescence-activated cell sorting (FACS). By selecting optimal parameters, a fraction of viable Purkinje cells that was 94% pure was obtained. These results indicate that FACS is a powerful tool for isolating Purkinje cells from postnatal L7-GFP transgenic mice. GFP-positive neurons will also be useful in the real-time observation of dendritic morphogenesis and axonal outgrowth during development, or after neuronal activity in vitro.     

Reduced startle reactivity and plasticity in transgenic mice overexpressing corticotropin-releasing hormone.

BACKGROUND: Corticotropin-releasing hormone (CRH) hyperactivity in transgenic mice overexpressing CRH in the brain (CRH-OE(2122)) appears to be associated with chronic stress-like alterations, including increased CRH content in the hypothalamus, changes in hypothalamus-pituitary-adrenal axis regulation, and increased heart rate and body temperature. In the present study, we investigated if sensory information processing of startling auditory stimuli was affected in CRH-OE(2122) mice. METHODS: CRH-OE(2122) mice (on C57BL/6J background) were subjected to a number of procedures probing sensory information processing mechanisms, including the acoustic startle response, habituation, and prepulse inhibition of startle. RESULTS: CRH-OE(2122) mice displayed reduced acoustic startle reactivity and increased motor activity during startle testing compared to wild-type mice. Furthermore, transgenic mice did not show habituation of the startle response after repeated exposure to the auditory stimulus, or habituation across procedures. CRH-OE(2122) mice exhibited robust impairments of prepulse inhibition in two different paradigms. CONCLUSIONS: The results in CRH-OE(2122) mice indicate that chronic CRH hyperactivity is associated with reductions in startle reactivity, habituation, and prepulse inhibition. The latter two abnormalities are also observed in schizophrenia patients. We conclude that chronic CRH excess may reduce behavioral reactivity to environmental stimuli and impair information processing mechanisms.     

Coitus-induced activation of c-fos and gonadotropin-releasing hormone in hypothalamic neurons in female rabbits

Copulation induces hypothalamic release of neuropeptides and catecholamines, especially gonadotropin-releasing hormone (GnRH) and norepinephrine, in female rabbits. The forebrain distribution of GnRH cells and the cellular events responsible for the coitally induced GnRH surge have not been identified. We characterized the expression of c-fos mRNA before (0 min) and up to 60 min after coitus in forebrain tissues of mated and nonmated females and compared these findings with those in which single- and double-labeled GnRH/Fos protein cells were identified by immunocytochemistry (ICC). Enhanced expression of fos-mRNA occurred 30 min after coitus, especially in the anteroventral periventricular nucleus (AVPV), the encapsulated portion of the bed nucleus of the stria terminalis (BNSTe) and the ventrolateral hypothalamus (VLH); this increased fos-mRNA activity remained elevated at 60 min in the AVPV and VLH, and was reflected by Fos protein expression 90 min postcoitus. Both ICC Fos-labeled and ICC GnRH-labeled cells were widely distributed throughout the forebrain with postcoital increased double-labeling in the preoptic-septal areas, the anterior-medial hypothalamus and the VLH. The increased number of dual-labeled and unchanged number of single-labeled GnRH cells after coitus suggest some GnRH neurons were non-detected before coitus. Many dual-labeled neurons were adjacent to Fos-labeled cells, suggesting enhanced interneuronal input to GnRH cells after coitus. Collectively, the results suggest that coitus activates hypothalamic GnRH neurons via several loci that include the AVPV, BNSTe and VLH. The distinct anatomical location of the AVPV, BNSTe and VLH further suggests that coital signals may reach the hypothalamus via separate neural pathways that are likely developed within the brainstem.     

面神经损伤大鼠脑内移植绿色荧光蛋白转基因胎鼠神经干细胞的

背景：目前面神经损伤后的修复主要集中在外周神经干,但面神经损伤后会导致部分中枢运动神经元凋亡。现阶段关于干细胞植入面神经损伤大鼠脑后对面神经核团内凋亡神经元的影响相关报道甚少。 目的：观察大鼠面神经损伤后,脑内移植绿色荧光蛋白转基因胎鼠的神经干细胞的成活和迁移情况。 设计、时间及地点：随机对照动物实验,于2008-07/12在昆明医学院神经科学研究所完成。 材料：孕14~16 d的绿色荧光蛋白转基因蛋白小鼠1只,用于制备转基因神经干细胞。清洁级SD雄性大鼠24只,随机分为3组：面神经损伤转基因细胞组12只、面神经损伤细胞培养液组6只、面神经正常转基因细胞组6只。 方法：面神经损伤转基因细胞组、面神经损伤细胞培养液组大鼠建立面神经切断模型。造模后1周,行转基因神经干细胞立体定向移植,注射点为前囟后方11.30 mm、背侧9.00 mm、正中线位置。面神经损伤转基因细胞组、面神经正常转基因细胞组各注入10 μL转基因神经干细胞悬液(含5×106 个细胞),面神经损伤细胞培养液组注入10 μL神经干细胞培养液,4周后制作脑组织冰冻切片。 主要观察指标：荧光显微镜观察移植部位绿色荧光蛋白阳性神经干细胞的存活情况,及其向损伤侧面神经核团周围迁移的情况。 结果：①移植处：面神经损伤转基因细胞组、面神经正常转基因细胞组均可见数量不等的绿色荧光蛋白阳性细胞,其中部分绿色荧光蛋白阳性细胞位于血管内;面神经损伤细胞培养液组未见绿色荧光蛋白阳性细胞。②面神经核团周围：仅面神经损伤转基因细胞组可见数量不等的绿色荧光蛋白阳性细胞迁移于损伤侧面神经核团周围,而健侧面神经核周围未见绿色荧光蛋白阳性细胞;余2组双侧面神经核周围均未见绿色荧光蛋白阳性细胞。③移植处与面神经核团周围之间：未见绿色荧光蛋白阳性细胞相连。 结论：神经干细胞移植入面神经损伤大鼠脑内后,可以向损伤侧面神经核周围迁移。