Effect of bacterial endotoxin on migration of gonadotropin-releasing, hormone producing neurons in rat embryogenesis

The effect of bacterial lipopolysaccharide endotoxin (LPS), immune system activator, on differentiation and migration of gonadotropin-releasing, hormone producing neurons in rat embryogenesis has been studied. Intraperitoneal introduction of LPS (18 jg/kg) to pregnant rats on the 12th day of pregnancy led to 50% decrease in total number of GRH-neurons in the forebrain of 17-day-old embryos and 17% decrease in 19-day-old embryos. At the same time, the number of GRH-neurons in the nasal area of the head of 17- and 19-day-old embryos increased by 40 and 50%, respectively, whereas it increased by 20% in olfactory bulbs of 17-day-old embryos and did not changed in olfactory bulbs of 19-day-old embryos. Neither the total number of neurons nor their distribution patterns were affected by the introduction of LPS into pregnant rats on the 15th day of pregnancy. Singular localization of GRH-neurons in embryo forebrain was observed after LPS administration, whereas the neurons were located by groups of 3-4 cells in rostral areas. Therefore, at the early stages of pregnancy, LPS was shown to suppress initial stages of differentiation and migration of GRH producing neurons. The effects observed in our study may be mediated by LPS-induced, proinflammatory cytokines.     

Migration and differentiation of neurons producing gonadotropin-releasing hormone under conditions of serotonin excess in the brain of mouse embryos

The work has been carried out on mice of the Tg8 line with knockout of gene of monoamineoxidase A with an increase of serotonin and noradrenaline content in the brain, and on mice of the C3H line with unchanged genome and normal concentration of monoamines. An immunocytochemical study has been performed of development of neurons producing gonadotropin-releasing hormone (GnRH) under conditions of excess of serotonin and noradrenaline in the mice in embryogenesis. The GnRH-neurons were revealed at the 18th day of embryonic development in telencephalon along trajectory of their migration from olfactory bulbs to the retrochiasmatic area. In telencephalon of mouse embryos of the Tg8 line, a redistribution of the GnRH-neurons along their migration trajectory was observed as compared with embryos of the C3H line mice. The percent of the GnRH-neurons in the Tg8 mouse embryos in caudal parts of the migration trajectory was lower than in rostral parts, the opposite distribution of the neurons being observed in the C3H line mouse embryos; at the excess of serotonin and noradrenaline in the Tg8 line mouse embryos, the total amount of GnRH-neurons in the brain was lower than in the C3H mice. In males of the Tg8 line mice under conditions of excess of serotonin and noradrenaline the optical density of neurons, which correlated with the GnRH concentration in the cell, was higher than in control mice. Thus, in the Tg8 mice under conditions of the serotonin and noradrenaline excess, migration of the GnRH-neurons to their final anlage in hypothalamus is accelerated as well as the total number of the GnRH-neurons decreases, which indicates a decrease of proliferation of cells-precursors and the earlier differentiation of neurons.     

Effect of serotonin on differentiation of neurons producing vasoactive intestinal polypeptide in the rat suprachiasmatic nucleus

This study was aimed to evaluate the morphogenetic influence of serotonin on the differentiating vasoactive intestinal polypeptide (VIP) neurons of the suprachiasmatic nucleus (SCN) in rats. The comparative morpho-functional analysis of VIP neurons was made in adult rats which developed under normal metabolism of serotonin or in its deficiency. The serotonin deficiency was induced in foetuses by injections of p-chlorophenilalanine to pregnant mothers. Control rats received the saline. According to our data, there was no difference in the VIP mRNA concentration and most probably in VIP synthesis in the SCN in adult rats following prenatal serotonin depletion compared to the control. However, the serotonin deficiency resulted in increase of the VIP concentration in cell bodies and of the VIP neurones number. The size of the VIP-neurones did not change in pharmacologically treated rats compared to the controls showing no functional hypertrophy of the neurones as a result of the activation of specific syntheses. From the above data, it follows that serotonin provides an imprinting influence differentiating the VIP neurones, contributing most probably to development of the VIP release mechanism.     

Cholinergic afferents to gonadotropin-releasing hormone neurons of the rat

Gonadotropin-releasing hormone-synthesizing neurons represent the final common pathway in the hypothalamic regulation of reproduction and their secretory activity is influenced by a variety of neurotransmitters and neuromodulators acting centrally in synaptic afferents to gonadotropin-releasing hormone neurons. The present study examined the anatomical relationship of cholinergic neuronal pathways and gonadotropin-releasing hormone neurons of the preoptic area. The immunocytochemical detection of choline acetyltransferase or vesicular acetylcholine transporter revealed a fine network of cholinergic fibers in this region. At the light microscopic level, the cholinergic axons formed appositions to the gonadotropin-releasing hormone immunoreactive cell bodies and dendrites. Results of electron microscopic studies confirmed the absence of glial interpositions in many of these neuronal contacts. Classical cholinergic synapses, which belonged to the asymmetric category, were only observed rarely on gonadotropin-releasing hormone neurons. The lack of synaptic density in most contacts corroborates previous observations on the cholinergic system elsewhere in the brain. Further, it suggests a dominantly non-synaptic route also in this cholinergic neuronal communication. This study provides direct neuromorphological evidence for the involvement of the cholinergic system in the afferent neuronal regulation of gonadotropin-releasing hormone neurons. The sources of cholinergic afferents and the receptorial mechanisms underlying this interaction will require further clarification.     

Fos-induction in gonadotropin-releasing hormone neurons receiving vasoactive intestinal polypeptide innervation is reduced in middle-aged female rats

A hallmark of reproductive aging in rats is a delay in the initiation and peak, and a decrease in the amplitude, of both proestrous and steroid-induced surges of LH and a decrease in the number of GnRH neurons that express Fos during the surge. The altered timing of the LH surge and the decline in Fos expression in GnRH neurons may be due to changes in the rhythmic expression of vasoactive intestinal polypeptide (VIP), a neuropeptide that carries time-of-day information from the circadian pacemaker, located in the suprachiasmatic nuclei (SCN), to GnRH neurons. The goals of our study were to determine if aging alters 1) the innervation of GnRH neurons by VIP and 2) the ability of VIP to activate GnRH neurons by examining the effects of aging on the number of GnRH neurons apposed by VIP fibers and the number of GnRH neurons that receive VIP input that express Fos. Immunocytochemistry for GnRH and VIP; or GnRH, VIP, and Fos was performed on tissue sections collected from young (2-4 mo), regularly cycling females and middle-aged (10-12 mo) females in constant estrus. The number of GnRH neurons, GnRH neurons apposed by VIP fibers, and GnRH neurons that express Fos and apposed by VIP fibers were counted in both age groups. Our results clearly demonstrate that aging does not alter the number of GnRH neurons that receive VIP innervation. However, the number of GnRH neurons that receive VIP innervation and coexpress Fos decreases significantly. We conclude that the age-related delay in the timing of the LH surge is not due to a change in VIP innervation of GnRH neurons, but instead may result from a decreased sensitivity of GnRH neurons to VIP input.     

Effect of the intraventricular administration of gonadotropin-releasing hormone analogue on immune response and unspecific resistance in rats

Intraventricular administration of the synthetic analogue of gonadotropin-releasing hormone (surfagon) results in increased humoral immune response and neutrophile activation including phagocytic and special functional indices in rats. Since testosterone blood level is not changed it may be concluded that the surfagon effect is mediated by nervous mechanisms. The conclusion is also confirmed by the obtained data of the brain stress-limiting system activation manifested by the decrease of oxidative stress.     

Reelin provides an inhibitory signal in the migration of gonadotropin-releasing hormone neurons

Gonadotropin-releasing hormone (GnRH) neurons, a small number of cells scattered in the hypothalamic region of the basal forebrain, play an important role in reproductive function. These cells originate in the olfactory placode and migrate into the basal forebrain in late embryonic life. Here, we show that reelin, which is expressed along the route of the migrating cells, has an inhibitory role in guiding GnRH neurons to the basal forebrain. Only a small (approximately 5%) subpopulation of these neurons expresses one of the reelin receptors (ApoER2/Lrp8), and all GnRH neurons appear to lack the intracellular adaptor protein Dab1, suggesting that the function of reelin is not mediated by the conventional signal transduction pathway. The importance of reelin in the establishment of GnRH neurons in the hypothalamus was confirmed by our finding that the brains of developing and adult reeler mice of both sexes contained a markedly reduced number of these neuroendocrine neurons. Furthermore, the testes of adult males showed dilation of seminiferous tubules and reduction in their density when compared with controls. Mutants lacking the reelin receptors ApoER2 and Vldlr, and scrambler mice lacking Dab1, showed a normal complement of GnRH neurons in the hypothalamus, confirming that the effect of reelin in their migration is independent of Dab1.     

Effect of postnatal treatment with a gonadotropin-releasing hormone antagonist on sexual maturation of male rats

The role of postnatal pituitary-testicular activity in sexual maturation at puberty was studied in male rats. Rats were injected twice daily with a potent gonadotropin-releasing hormone antagonist (N-Ac-4-Cl-D-Phe1, 4-Cl-D-Phe2, D-Trp3, D-Phe6, D-Ala10-NH2-GnRH) (GnRH-Ant.), 2 mg/kg, on Days 1-15 of life, and killed on Day 48, 56 or 90 of life. The treatment delayed the onset of puberty (monitored by balano-preputial separation) by 8 days (from the age of 48 to 56 days). The weights of testes, seminal vesicles and ventral prostates were reduced by 50-60% on days 48 and 56 of life, but only the testis weights remained suppressed by Day 90. Levels of serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH), but not those of prolactin (Prl), were elevated 2-to-4-fold in the treated animals at the three ages studied. Serum and testicular testosterone (T) and the receptors for LH and Prl were suppressed in the peripubertal animals (48 and 56 days), but serum T was elevated and the receptor levels were normal in the 90-day group. The testicular FSH receptors were 50% suppressed at all ages studied. Only minor changes were observed in testicular histology when studied at 48 and 56 days. The 85-day-old animals treated with GnRH-Ant. were infertile when mated with females.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct and indirect regulation of gonadotropin-releasing hormone neurons by estradiol

Estrogen signaling to GnRH neurons is critical for coordinating the preovulatory surge release of LH with follicular maturation. Until recently it was thought that estrogen signaled GnRH neurons only indirectly through numerous afferent systems. This minireview presents new evidence indicating that GnRH neurons are directly regulated by estradiol (E2), primarily through estrogen receptor (ER)-beta, and indirectly through E2-sensitive neurons in the anteroventral periventricular (AVPV) region. The data described suggest that E2 generally represses GnRH gene expression but that this repression is transiently overcome by indirect E2-dependent signals relayed by AVPV neurons. We also present evidence that the AVPV neurons responsible for relaying E2 signals to GnRH neurons are multifunctional gamma aminobutyric acid-ergic/glutamatergic/neuropeptidergic neurons.     

Activation of A-type gamma-aminobutyric acid receptors excites gonadotropin-releasing hormone neurons

Gamma-aminobutyric acid (GABA), acting through GABA(A) receptors (GABA(A)R), is hypothesized to suppress reproduction by inhibiting GnRH secretion, but GABA actions directly on GnRH neurons are not well established. In green fluorescent protein-identified adult mouse GnRH neurons in brain slices, gramicidin-perforated-patch-clamp experiments revealed the reversal potential (E(GABA)) for current through GABA(A)Rs was depolarized relative to the resting potential. Furthermore, rapid GABA application elicited action potentials in GnRH neurons but not controls. The consequence of GABA(A)R activation depends on intracellular chloride levels, which are maintained by homeostatic mechanisms. Membrane proteins that typically extrude chloride (KCC-2 cotransporter, CLC-2 channel) were absent from the GT1-7 immortalized GnRH cell line and GnRH neurons in situ or were not localized to the proper cell compartment for function. In contrast, GT1-7 cells and some GnRH neurons expressed the chloride-accumulating cotransporter, NKCC-1. Patch-clamp experiments showed that blockade of NKCC hyperpolarized E(GABA) by lowering intracellular chloride. Regardless of reproductive state, rapid GABA application excited GnRH neurons. In contrast, bath application of the GABA(A)R agonist muscimol transiently increased then suppressed firing; suppression persisted 4-15 min. Rapid activation of GABA(A)R thus excites GnRH neurons whereas prolonged activation reduces excitability, suggesting the physiological consequence of synaptic activation of GABA(A)R in GnRH neurons is excitation.     

Methods for the isolation of viable gonadotropin-releasing hormone (LRF) neurons

Studies were conducted in order to determine if selected neurons could be isolated from the brain using Sepharose-linked recognition complexes directed against or related to the biosynthetic/neurosecretory product of the desired neuronal population. Immunoreactive LRF neurons were precipitated when dispersed cells of adult male rats were incubated successively in media containing free LRF antiserum followed by the exposure of LRF bound to Sepharose-4B. The radioimmunoassayable LRF content of the isolated cells was 88% of that contained in fresh frozen tissue of a contemporary group of rats and trypan blue exclusion indicated that at least 85% of the neurons were viable. Furthermore, based on immunocytochemistry and cresyl violet staining in combination with immunocytochemistry, the isolated cell fraction appeared to be free from other types of cells and also exhibited assayable LRF release when challenged with potassium. These results suggest that the neuroendocrine properties of hypothalamic neurons may be exploited in order to isolate viable cells for acute in vitro experiments.     

Melanin-concentrating hormone producing neurons: Activities and modulations

Regulation of energy homeostasis in animals involves adaptation of energy intake to its loss, through a perfect regulation of feeding behavior and energy storage/expenditure. Factors from the periphery modulate brain activity in order to adjust food intake as needed. Particularly, “first order” neurons from arcuate nucleus are able to detect modifications in homeostatic parameters and to transmit information to “second order” neurons, partly located in the lateral hypothalamic area. These “second order” neurons have widespread projections throughout the brain and their proper activation leads them to a coordinated response associated to an adapted behavior. Among these neurons, melanin-concentrating hormone (MCH) expressing neurons play an integrative role of the various factors arising from periphery, first order neurons and extra-hypothalamic arousal systems neurons and modulate regulation of feeding, drinking and seeking behaviors. As regulation of MCH release is correlated to regulation of MCH neuronal activity, we focused this review on the electrophysiological properties of MCH neurons from the lateral hypothalamic area. We first reviewed the knowledge on the endogenous electrical properties of MCH neurons identified according to various criteria which are described. Then, we dealt with the modulations of the electrical activity of MCH neurons by different factors such as glucose, glutamate and GABA, peptides and hormones regulating feeding and transmitters of extra-hypothalamic arousal systems. Finally, we described the current knowledge on the modulation of MCH neuronal activity by cytokines and chemokines. Because of such regulation, MCH neurons are some of the best candidate to account for infection-induced anorexia, but also obesity.     

Effect of individual peculiarities on formation of preference of ethanol in female and male Wistar rats

Individual characteristics of animal behavior can serve a prognostic parameter of predisposition to use of alcohol. The goal of the work was to study dynamics of formation of preference of alcohol at early stages in the process of forced alcoholization in male and female rats as well as the accompanying changes in behavior parameters. For 3 months, the rats were submitted to the forced alcoholization with 10 % ethanol. Each week the rats were tested in the "two-glass trial". Individual peculiarities of all animals were evaluated prior to, after 6 weeks, and after the end of the experiment with aid of the "open field test" and "Suok-test". Results showed that the male rats demonstrating by the end of the experiment the significantly higher level of the alcohol preference, at the initial stages of the forced alcoholization demonstrated the significantly lower preference as compared with the remaining ones. These rats also showed the lower levels of the motor and exploratory activities before alcoholization as compared with control. On the contrary, the individuals that by the end of the experiment did not differ from control by the level of the alcohol preference demonstrated prior to alcoholization in the "Suok-test" the higher anxiety level. In female groups, no statistically significant differences were observed both in parameters of the motor and exploratory activities and in the anxiety level in both tests. Thus, in male rats, the prognostic parameter predicting formation of the abuse can serve the degree of alcohol preference at the initial stages.     

Regulation of Ca2+-sensitive adenylyl cyclase in gonadotropin-releasing hormone neurons

In immortalized GnRH neurons, cAMP production is elevated by increased extracellular Ca2+ and the Ca2+ channel agonist, BK-8644, and is diminished by low extracellular Ca2+ and treatment with nifedipine, consistent with the expression of adenylyl cyclase type I (AC I). Potassium-induced depolarization of GT1-7 neurons causes a dose-dependent monotonic increase in [Ca2+]i and elicits a bell-shaped cAMP response. The inhibitory phase of the cAMP response is prevented by pertussis toxin (PTX), consistent with the activation of G(i)-related proteins during depolarization. Agonist activation of the endogenous GnRH receptor in GT1-7 neurons also elicits a bell-shaped change in cAMP production. The inhibitory action of high GnRH concentrations is prevented by PTX, indicating coupling of the GnRH receptors to G(i)-related proteins. The stimulation of cAMP production by activation of endogenous LH receptors is enhanced by low (nanomolar) concentrations of GnRH but is abolished by micromolar concentrations of GnRH, again in a PTX-sensitive manner. These findings indicate that GnRH neuronal cAMP production is maintained by Ca2+ entry through voltage-sensitive calcium channels, leading to activation of Ca2+-stimulated AC I. Furthermore, the Ca2+ influx-dependent activation of AC I acts in conjunction with AC-regulatory G proteins to determine basal and agonist-stimulated levels of cAMP production.     

Influence of serum supplements in culture medium on gonadotropin-releasing hormone effects on colony formation

A number of studies have demonstrated that GnRH has anti-proliferative effects on various carcinomas of breast, ovary, endometrium, prostate, pancreas, and liver origin. In contrast, GnRH increases the proliferative activity of lymphoid tissues and cells, which suggests that GnRH is also an important immunomodulator. In a previous study, we demonstrated that the colony-forming efficiencies of HHUA (derived from human endometrial carcinoma) and Jurkat (derived from human mature leukemia) cells are affected by the GnRH agonist Buserelin, and that the conditioned media of HHUA and Jurkat cells severely affect the Buserelin activity. The latter finding suggests that substances in the culture medium have some relation to the GnRH activity. Therefore, in the present study, to evaluate the effect of serum supplements on the colony-forming efficiency assay, the assay was performed using 3 lots of fetal bovine serum (FBS) and 2 lots of Nu-Serum I, a semi-synthetic serum supplement. The results showed that the colony-forming efficiencies of HHUA and Jurkat cells fluctuated greatly depending on the lot of FBS. In contrast, Buserelin significantly affected the colony-forming efficiency to similar extents in the media containing both the lots of Nu-Serum I. These results strongly suggest that the constituents of the serum supplements also influence the effect of GnRH on cell proliferation. For further studies about the relationship between substances in the culture medium and the GnRH effects on cell proliferation, it will be necessary to use a completely defined medium, and that a semi-synthetic serum supplement such as Nu-Serum I will also be useful.     

Effect of gonadotropin-releasing hormone on fertility in repeat-breeder California dairy cows

To assess the potential benefit to fertility from gonadotropin-releasing hormone (GnRH) administration to third service cows managed in typical California dairy systems, 963 cows were enlisted from 14 dairies served by 6 veterinary practices. The cows were randomly assigned to receive either GnRH (100 mug) or placebo at the time of the third artificial insemination. Fertility data were entered onto a proprietary microcomputer program common to all six practices, and collated independently by a third party. For the duration of the trial (1 yr), GnRH and placebo-treated cows had 43.2 and 39.3% conception rates, respectively (P=0.35). When treatments administered in summer months (July, August, September) were excluded, conception rates were 48.1 and 41.0%, respectively (P<0.1). The conception rates of cows treated with GnRH in August tended to be lower than those of placebo-treated cows (95% logarithmic confidence intervals of odds ratio = -1.139, 0.377). Between-herd variation in benefit from GnRH was evident, with two dairies showing no benefit, one dairy showing a negative effect, and four showing a range of effects from lightly beneficial to significantly beneficial. First-lactation cows did not benefit at any time from GnRH treatment. The data suggest that GnRH administered to third-service dairy cows under California conditions may result in increased conception rates in non-summer months, but that other unidentified variables may have important influence on the outcome of such treatment.     

GABAergic integration of progesterone and androgen feedback to gonadotropin-releasing hormone neurons

Steroid feedback regulates GnRH secretion and previous work has implicated gamma-aminobutyric acid (GABA)ergic neurons as a mediator of these effects. We examined GABAergic postsynaptic currents (PSCs) in green fluorescent protein-identified GnRH neurons from mice exposed to different steroid milieus in vivo. Adult mice were ovariectomized and treated with estradiol (OVX+E, controls) or E plus progesterone (P, OVX+E+P). P decreased PSC frequency, a presynaptic effect, and PSC size, which could be via pre- and/or postsynaptic mechanisms. In contrast, dihydrotestosterone (DHT, OVX+E+DHT) increased both GABAergic PSC frequency and size in GnRH neurons. Tetrodotoxin (TTX), which eliminates action-potential-dependent presynaptic effects, did not alter frequency, suggesting DHT may have increased PSC frequency by increasing connectivity between GABAergic and GnRH neurons. TTX reduced PSC size below control values, indicating DHT may augment presynaptic GABA release but inhibits the postsynaptic GnRH neuron response. In mice treated with both P and DHT (OVX+E+P+DHT), PSC frequency and size were similar to controls, suggesting these steroids counteract one another. These results demonstrate GABAergic neurons participate in integrating and conveying steroid feedback to GnRH neurons, defining a potential central mechanism for steroid regulation of GnRH neurons during the reproductive cycle, and providing one possible mechanism for increased activity of these cells in hyperandrogenic females.