摘要:

The expression and function of large-conductance Ca2+-activated K+ (BK) channels in the GT1-7 line of immortalized gonadotropin-releasing hormone (GnRH) neurons was investigated. Ionic currents were recorded using the patch-clamp technique, cytoplasmic free Ca2+ concentration ([Ca2+]i) was monitored using the fluorescent indicator, fura-2, and GnRH secretion was measured by radioimmunoassay. In cell-attached and inside-out patch-clamp recordings, K+ channels with a single-channel conductance of ∼200 pS were detected. Depolarizing the patch increased the unitary current size and the open probability. In perforated-patch recordings, depolarizing pulses (50 ms) to potentials of –10 to +60 mV from a holding potential of –90 mV elicited outward current with early transient and sustained components. The transient current peaked 2–10 ms after the beginning of each pulse and increased in a voltage-dependent manner. This current was: (1) unaffected by the small-conductance Ca2+-activated K+ channel blocker, apamin (100 nM); (2) reduced by the BK channel blocker, charybdotoxin (5 nM); (3) abolished by the Ca2+ channel blocker, CdCl2 (25 µM), and (4) prolonged by the endoplasmic reticu-lum Ca2+-ATPase inhibitor, thapsigargin (1 µM). Based on the single-channel and whole-cell conductances, the number of channels per patch, the patch area, and the surface area of the cell, each GT1-7 cell contains 30–60 BK channels. The functional role of BK channels in GT1-7 cells was evaluated by measuring the effect of charybdotoxin (100 nM) on basal [Ca2+]i and GnRH secretion, as well as on the [Ca2+]i and GnRH secretory responses to γ-aminobutyric acid (GABA, 100 µM), an excitatory neurotransmitter in this system. Charybdotoxin had no effect on basal [Ca2+]i or GnRH secretion, or on the GABA-evoked [Ca2+]i and GnRH secretory responses. These results indicate that GT1-7 cells express BK channels; however, the physiological role of BK channels in GT1-7 cells re

ISSN:0028-3835    

DOI:10.1159/000126946

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

Neuropeptide Y (NPY) has been shown to stimulate hypothalamic release of luteinizing hormone-releasing hormone (LHRH) both in vitro and in vivo. In female rats, NPY facilitation of LHRH release is greatly augmented in advance of preovulatory LHRH surges, likely via the actions of ovarian steroids. However, the role of NPY in regulating LHRH release in male rats and the effects of testicular hormones on LHRH responses to NPY in males are not well understood. The objective of the present studies was to determine whether NPY stimulates LHRH release in vitro from hypothalamic tissue of male rats, and whether these effects could be modulated by testosterone (T). Mediobasal hypothalamic (MBH) or median eminence (ME) fragments from either sham-operated or castrated male rats (7 days) were placed in superfusion chambers and superfused with M199 for a 30-min baseline, 30-min challenge with NPY (10–7M), and a final 30-min challenge with 56 mM KC1. One-milliliter fractions were collected every 10 min and average LHRH release values over the 30-min periods were compared among groups. NPY (10–7M) produced a significant increase in LHRH release from the MBH and ME from intact animals. In contrast, the same dose of NPY did not stimulate LHRH release from tissues from castrated animals; only with a higher dose of NPY (10–6M) were the effects of NPY on LHRH release significant. Potassium challenge (56 mM KCl) significantly stimulated LHRH release from the ME of both intact and castrate male rats suggesting that all tissues were able to respond to a stimulus, and that castration did not alter the responsiveness of the LHRH neuron to a nonspecific secretagogue. To determine the extent to which T regulates the sensitivity of LHRH responses to NPY, male rats were castrated and implanted with T capsules that maintained either low (1.24 ± 0.06 ng/ml) or high (2.17 ± 0.31 ng/ml) physiological plasma levels of T. Treatment with the higher dose of T restored the ability of NPY to stimulate LHRH release from the ME tissues. These results demonstrate that NPY stimulates LHRH release from the hypothalamus in vitro, and that gonadal steroids, in this case T and/or its metabolites, modulate the responsiveness of the LHRH neuron to NPY. Based on these data from intact and castrate-derived tissues, it appears that steroids are necessary to maintain LHRH responsiveness to NPY receptor stim

ISSN:0028-3835    

DOI:10.1159/000126947

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

Testosterone exerts important feedback effects on the hypothalamus of the ram to influence reproductive functioning. To provide a neuroanatomical basis for understanding this androgen action, the present study has examined androgen receptor (AR) immunoreactivity within the hypothalamus and adjacent brain areas of the intact non-breeding season ram. The largest populations of AR-immunoreactive cells were detected in the medial preoptic area, infundibular and premammillary nuclei in addition to the ventromedial nucleus (VMN) where cells were found distributed throughout its medial and lateral divisions. Smaller numbers of AR-expressing cells were identified in the bed nucleus of the stria terminalis and anterior hypothalamic area (AHA) including the paraventricular, but not the supraoptic, nucleus. Double-labelling immunocytochemistry revealed the presence of AR immunoreactivity in only 2 of 460 gonadotropin-releasing hormone (GnRH) neurons. A very small population of TH-immunoreactive cells located in the lateral aspect of the AHA was found to contain ARs. Dopaminergic cells elsewhere in the hypothalamus, including the infundibular nucleus, did not display AR immunoreactivity. Nearly 50% of AR-expressing cells in the lateral VMN were immunoreactive for somatostatin while less than 5 % of periventricular somatostatin neurons displayed AR immunoreactivity. These results show where ARs are expressed in the ram hypothalamus and indicate the neuroanatomical sites at which androgen may act to influence reproductive function. The absence of ARs in the neuroendocrine GnRH and tuberoinfundibular dopaminergic cells suggests that androgens do not influence the genome of these cells in any direct manner. In contrast, the somatostatin neurons of the VMN appear to be an important target for circulating androgens in the non-breeding season ram.

ISSN:0028-3835    

DOI:10.1159/000126948

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

Progesterone (P) acts in the central nervous system to increase prolactin secretion in estrogen (E)-primed female monkeys. β-Endorphin (BE) and Substance P (SP) are two hypothalamic peptides which increase prolactin secretion when administered to rats and monkeys. Studies were performed to determine if P acts on these two potential prolactin-releasing systems. The presence of a nuclear steroid receptor defines the cell as a target for the cognate hormone. Therefore, the hypothalamic populations of BE and SP neurons were examined for the presence and regulation of nuclear progestin receptors (PR) in spayed, E-treated (28 days) and E + P-treated monkeys (14 days E + 14 days E + P). Hypothalamic blocks were prepared after perfusion fixation with 4% paraformaldehyde. Cryosectioning (10 µm) was followed by double immunocytochemistry (ICC) for PR (black nuclear stain) and either BE or SP (brown cytoplasmic stain). Sections were processed for ICC at 100- or 200-µm intervals through the hypothalamic block. Peptidergic neurons with and without PR were counted in each section. The E + P-treated monkeys exhibited a significant increase in serum prolactin. BE neurons were found only in the arcuate nucleus (ARC) and median eminence (ME). The colocalization of BE and PR equaled 2% in spayed controls, 21 % in the E-treated group and 25 % in the E + P-treated group. SP neurons were located in a dorsomedial hypothalamic (DMH) subpopulation which extended caudally under the mammary nuclei and in a subpopulation located in the ARC and ME. Neither the DMH or submamillary SP neurons contained PR. The percent colocalization of SP and PR in the ARC/ME equaled 5, 26 and 10% in the spayed, E- and E + P-treated groups, respectively. The decrease in PR + SP colocalization with P treatment is probably due to a decrease in SP and not to a decrease in PR immunoreactivity. In summary, E treatment induced PR in BE and SP neurons. Addition of P to the E treatment did not alter the expression of PR in BE neurons, but PR colocalization decreased in SP neurons. Therefore, it is unlikely that SP neurons could transduce the action of P on prolactin secretion in primates, but BE neurons may play an intermediary ro

ISSN:0028-3835    

DOI:10.1159/000126949

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

The volume of the anteroventral periventricular nucleus (AVPv) of the rat hypothalamus is larger in females than in males. A preliminary study from this laboratory found that this sexual dimorphism develops between days 30 and 91. The present study was designed to confirm and extend these findings and to determine the role of endogenous gonadal steroids in the development of the AVPv postnatally. The results indicate that the sexual dimorphism in AVPv volume arises between days 30 and 40 and that the length of the nucleus becomes sexually dimorphic between days 60 and 80. Additionally, both AVPv volume and length increased between days 30 and 80 in females. Castration of male rats on the day of birth sex-reversed AVPv volume in adulthood and AVPv length was sex-reversed by castration of males 5 days after birth; ovariectomy of females at these ages had no effect on either parameter. Moreover, in both males and females, AVPv volume and length were unaffected by gonadectomy at later ages. That the AVPv appears to be influenced by testicular hormones neonatally, but changes structurally around the time of puberty in females, clearly challenges current concepts of sexual differentiation that limit the process to the early postnatal period.

ISSN:0028-3835    

DOI:10.1159/000126950

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

Intraneuronal production of estradiol from testosterone has been shown to play a pivotal role in gender-specific brain development of most vertebrates, and to participate in numerous functions of the adult central nervous system. Previous biochemical and morphological approaches demonstrated that estrogen synthetase (aromatase) is present in specific limbic and hypothalamic structures. On the other hand, less attention has been paid to revealing its subcellular distribution. The possibility of aromatase presence in axonal processes has been indicated by recent biochemical and morphological observations suggesting new insights for the role of aromatase in neural functions. The objective of the present study was to provide morphological evidence for the subcellular location of aromatase in neurons of different vertebrate species including Japanese quail, rat, monkey, and human. Immunocytochemistry using a purified polyclonal antiserum against human placental aromatase localized immunoreactivity to hypothalamic and limbic cell groups in all of these species. Light and electron microscopic examination of vibratome sections revealed the presence of aromatase immunoreactivity throughout the neuronal perikarya, including dendrites and axonal processes. In each species there were numerous boutons which contained labeled small clear synaptic vesicles. Many of these axon terminals formed synapses with immuno-negative and immuno-positive dendrites and perikarya. This study furnishes the first immunolocalization of aromatase in the brains of two primate species, humans and monkeys. The provision of further evidence for estrogen synthesis in axons and axon terminals may help resolve apparent differences between the measurement of aromatase activity and the lack of aromatase-immunopositive cell bodies in previous studies. The present findings may be coupled with recent evidence regarding the molecular biology and the diversity of functional properties of P450 aromatase to indicate previously unexpected effects of brain aromatase at the synaptic level.

ISSN:0028-3835    

DOI:10.1159/000126951

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

A double immunocytochemical procedure, with two different chromogens, was used to compare the respective distribution of estrogen receptor-immunoreactive cells and tyrosine hydroxylase-immunoreactive neurons on the same sections of the preoptic region of adult female rainbow trout (Oncorhynchus mykiss). Estrogen receptor-immunoreactive cells were observed in the anterior preoptic region surrounding the preoptic recess and its large lateral extensions. Tyrosine hydroxylase-immunoreactive cells were consistently detected in the ventral and ventrolateral walls of the preoptic recess, in an area that was named nucleus preopticus pars anteroventralis. Dopamine immunohistochemistry and Dil retrograde transport studies indicated that part of these catecholaminergic neurons are dopaminergic and could project to the pituitary. Double staining studies showed consistently that most estrogen receptor-positive cells located ventral to the large extensions of the preoptic recess are also tyrosine hydroxylase-positive, indicating that this region is a major target for estradiol feedback. The results are discussed in relation to the role of the nucleus preopticus pars anteroventralis in mediating the negative feedback actions of estradiol on the secretion of gonadotrophin (GTH2) secretion. A hypothesis is drawn in order to explain the synchronizing role of estradiol at the time of ovulation in rainbow trout.

ISSN:0028-3835    

DOI:10.1159/000126952

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

The time courses of changes in rat brain neuroactive steroid concentrations and γ-aminobutyric acid type A (GABAA) receptor function elicited by acute stress were investigated in animals exposed to CO2 for 1 min, a treatment known to induce stress in rats and panic attacks in humans. Inhalation of CO2 induced increases in cerebral cortical steroid concentrations, the time dependence of which varied with the steroid examined. Thus, progesterone and deoxycorticosterone showed maximal increases (10- and 4-fold, respectively) 10 min after CO2 inhalation and had returned to basal values by 30 and 60 min, respectively. In contrast, pregnenolone and 3α-hydroxy-5α-pregnan-20-one (allopregnanolone) concentrations showed maximal increases (+174 and +200%, respectively) at 30 min, were still higher than control at 60 min and returned to control values 120 min after stress. Inhalation of CO2 also resulted in increases in plasma steroid concentrations, most of which peaked at 30 min and had returned to control values by 60 min. A parallel analysis of the stress-induced changes in GABAA receptor function, assessed either biochemically by t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to cerebral cortical membranes or behaviorally by the punished responding score in Vogel’s test, showed that the effects of CO2 inhalation on both parameters were maximal (+51 and –40%, respectively) after 10 min; the behavioral reaction returned to normal after 60 min, whereas [35S]TBPS binding had returned to control values 120 min after stress. The results show that: (a) the maximal increase in the brain concentrations of allopregnanolone, a potent and efficacious positive modulator of GABAA receptors, occurred at a time (30 min) when both conflict behavior and [35S]TBPS binding begun to decrease, and (b) both allopregnanolone concentrations and [35S]TBPS binding had returned to control values 120 min after CO2 inhalation. The data are thus consistent with a physiological role of neuroactive steroids in restoring GABAergic tone after

ISSN:0028-3835    

DOI:10.1159/000126953

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

The pulsatile pattern of GH secretion slowly develop in the postnatal period concomitantly with the dual network of GHRH and somatostatin (SRIH) hypothalamic neurons. We investigated whether an early postnatal treatment with a long acting SRIH analogue, octreotide, could affect maturation and subsequent operation of those networks in the adult rat. Octreotide administration (5 µg/rat SC) every other day during the first 10 days of life resulted in growth retardation in the adult. In parallel, the amplitude of plasma GH secretory episodes in free moving unanesthetized animals was markedly reduced. The numbers of arcuate GHRH mRNA-containing and periventricular SRIH-mRNA containing neurons were not affected by the treatment. GHRH mRNA levels per neuron however was decreased by 30%, and median eminence GHRH stores by 50%. SRIH expression in the arcuate nucleus was also diminished, as was the number of 125I-SRIH labeled neurons in that nucleus. The effects of octreotide were compared to the hyposomatotropinemia induced by administration of monosodium glutamate (MSG), every other day during the first 10 days of life. Growth retardation and inhibition of GH secretory episodes in adult rats neonatally treated with MSG were slightly more pronounced than after octreotide. In contrast to octreotide, MSG induced a massive loss of GHRH neurons and a concomitant decrease in 125I-SRIH binding. Somatostatin did not protect GHRH neurons against the neurotoxic action of MSG since octreotide treatment did not further affect any of the parameters impaired by MSG In conclusion, these experiments demonstrate that neonatally injected octreotide cannot counteract the toxic effect of MSG on arcuate neurons. However, a neonatal treatment with the SRIH agonist affects permanently growth rate and GH pulsatility. This effect is mediated in the hypothalamus by permanently impairing the neural networks that control GH secretion

ISSN:0028-3835    

DOI:10.1159/000126954

出版商:S. Karger AG    

年代:1996

数据来源: Karger

摘要:

To investigate the secretory pattern of somatostatin (SS) from the median eminence (ME) in the female rat, as well as estrogenic influence on this secretion, we measured both SS release and hypothalamic content in cycling, 10-day ovariectomized, and ovariectomized rats treated with estradiol for 3 days before. Animals were stereotaxically implanted with a push-pull cannula into the ME, and 10 days later the hypothalamic structure was perfused with artificial cerebrospinal fluid for 120–150 min at a regular flow rate of 17 µl/min. Secretion peaks were observed in the pattern of SS release, whatever the stage of the estrous cycle. The mean amplitude of SS peaks was similar throughout the cycle: 11.7 ± 4.0, 8.6 ± 1.5 and 10.5 ± 1.3 pg at proestrus, estrus and diestrus, respectively, and it was affected neither by ovariectomy (7.4 ± 1.3 pg) nor by estrogen replacement (5.5 ± 1.0 pg). By contrast, mean SS release levels in the proestrus phase were significantly higher than those measured in the other phases: 21.6 ± 2.1 vs. 17.7 ± 1.2 pg/15 min in diestrus (p < 0.05) and vs. 12.0 ± 0.7 pg/15 min in estrus (p < 0.001). Hypothalamic SS content showed variations quite similar to those observed during its release, i.e. with the highest values corresponding to the proestrus phase (1,170.5 ± 224.9 pg/mg of tissue) and to the diestrus (1,156.5 ± 332.1 pg/mg of tissue) and the lowest values in the estrus (511.5 ± 52.9 pg/mg of tissue; p < 0.05 vs. proestrus and diestrus). In addition, the lowest SS content and secretion values were found in ovariectomized animals: 95.5 ± 5.1 pg/mg of tissue (p < 0.001 compared to the values obtained for each stage of the estrous cycle) and 10.0 ± 0.9 pg/15 min (p < 0.001 vs. proestrus and diestrus), respectively. Patterns of SS release and SS hypothalamic content were not modified by estradiol treatment in ovariectomized animals. Our results suggest that (1) whatever the stage of the estrous cycle, SS release from the ME is not uniform and exhibits irregular peaks; (2) mean SS release levels were subjected to gonadal influence; (3) the occurrence of SS peaks seems to be estrogen-independent, and (4) variations in hypothalamic SS content were generally in good agreement with those of neuro

ISSN:0028-3835    

DOI:10.1159/000126955

出版商:S. Karger AG    

年代:1996

数据来源: Karger