Cold-induced glutamate release in vivo from the magnocellular region of the paraventricular nucleus is involved in ovarian sympathetic activation.

We previously reported that centrally-induced sympathetic activation in response to cold stress is associated with a polycystic ovarian condition in rats, and thyrotrophin-releasing hormone (TRH) released locally from the magnocellular region of the paraventricular nucleus (PVN) appears to be involved in this activation. Because TRH neurones express NMDA glutamate receptors, in the present study, we investigated the role of glutamate in the increased release of TRH from magnocellular neurones induced by cold stress and its relationship to ovarian neurotransmission. Animals with a push-pull cannula stereotaxically implanted into the magnocellular portion of the PVN were exposed to cold stress (4 degrees C for 64 h) and subjected to intracerebral perfusion. Perfusate fractions were obtained and analysed by high-performance liquid chromatography to measure glutamate and GABA levels. Glutamate, but not GABA, release increased significantly in animals perfused under cold exposure. In vivo administration of glutamate to the PVN increased TRH release. Injection of MK-801 into the magnocellular portion of the PVN reduced ovarian noradrenaline turnover and led to an increase in catecholamine concentration from the adrenal glands and celiac ganglia. Taken together, the results obtained in the present study strongly suggest that glutamate release from the magnocellular PVN is sensitive to cold stress and that glutamate acts through the NMDA receptor to mediate cold-induced TRH release. This in turn triggers hypothalamic-ovarian pathway activation, which might be responsible for the polycystic condition induced by cold stress and other ovarian pathologies characterised by increased sympathetic discharge.

Cold stress induces metabolic activation of thyrotrophin-releasing hormone-synthesising neurones in the magnocellular division of the hypothalamic paraventricular nucleus and concomitantly changes ovarian sympathetic activity parameters.

Recent studies suggest thyrotrophin-releasing hormone (TRH) serves as a neurotransmitter and thereby provides a functional vegetative connection between the brain and the ovary. In the present study, magnocellular neurones of the paraventricular nucleus (PVN) in animals subjected to cold exposure were studied to determine the hypothalamic origin of the TRH involved in this pathway. In situ hybridisation analysis of hypothalamic tissue showed that cold exposure causes a two-fold increase in the total number of neurones expressing TRH mRNA in the PVN. Immunohistochemical studies showed that TRH peptide is localised to the magnocellular PVN and that the number of TRH immunoreactive cells increases two-fold following 64 h of cold exposure. Double-immunostaining for MAP-2 and TRH revealed that TRH peptide is localised in the perikarya of the magnocellular neurones. TRH release was measured in vivo from the magnocellular portion of the PVN using push-pull perfusion. Although controls exhibited a very low level of TRH release, animals subjected to cold showed a pulsatile-like TRH release profile with two different patterns of release: (i) low basal level with small bursts of TRH release and (ii) a profile with an up to seven-fold increase in TRH release compared to controls. The colocalisation of TRH with the specific somato-dendritic marker MAP-2 in processes of the magnocellular neurones suggested a local release of TRH. Additional studies demonstrated a reduction in ovarian noradrenaline content after 48 h of cold exposure, a feature indicative of nerve activation at the terminal organ. After 64 h of cold exposure, the ovarian noradrenaline returned to control values but the noradrenaline content of the coeliac ganglia was increased, suggesting a compensatory effect originating in the cell bodies of the sympathetic neurones that innervate the ovary. The correlation between the local release of TRH from dendrites within the magnocellular PVN in conditions of cold and the activation of the sympathetic nerves supplying the ovary raises the possibility that TRH contributes to the processing regulating sympathetic outflow and may thereby impact on the functional activity of the ovary.

Age-related changes in brain-derived neurotrophic factor and tyrosine kinase receptor isoforms in the hippocampus and hypothalamus in male rats.

A large amount of aging individuals show diminished cognitive and endocrine capabilities. The main brain areas involved in these changes are the hippocampus and hypothalamus, two regions possessing high plasticity and implicated in cognitive and endocrine functions, respectively. Among neurotrophins (considered as genuine molecular mediators of synaptic plasticity), brain-derived neurotrophic factor (BDNF) exhibits in adult rats, the highest concentrations in the hippocampus and hypothalamus. Most of neuronal effects of BDNF are mediated through high-affinity cell surface BDNF tyrosine kinase receptors (TrkB). Different TrkB isoforms are issued by alternative splicing of mRNA encoding for TrkB (trkB mRNA) generating at least three different TrkB receptors with different signaling capabilities. The goal of this study was to examine simultaneously the expression (mRNAs and proteins) of BDNF and its three specific receptors, in the hippocampus and hypothalamus throughout lifespan in rats. We observed that BDNF essentially increased during the first 2 postnatal weeks in the hippocampus and hypothalamus, with no close correlation to its mRNA levels. In these regions, mRNA encoding for BDNF full-length catalytic receptor (trkB.FL mRNA) showed no important changes throughout life but of the mRNA truncated forms of TrkB receptors (trkB.T1 mRNA and trkB.T2 mRNA) trkB.T1 mRNA strongly increased after birth, then remaining stable during aging. trkB.T2 mRNA gradually decreased from 1 postnatal week becoming undetectable in the hippocampus in old-rats. Proteins issued from these mRNAs showed substantial quantitative modifications with aging. From 2 months old, the BDNF full-length catalytic receptor (TrkB.FL) gradually and significantly decreased in the hippocampus and the hypothalamus. Of the truncated forms of TrkB receptors (TrkB.T1 and TrkB.T2) TrkB.T1, which is essentially localized in glial cells, significantly increased from the first postnatal week in the hippocampus and in the hypothalamus, remaining stable during aging but reduced in old rats. TrkB.T2 which similarly to TrkB.FL has a neuronal localization also gradually decreased in the hippocampus and in the hypothalamus throughout lifespan. These reductions were significant at 21 and 30 days old, respectively. All the changes reported here could contribute to the reduced plasticity of these regions observed in old rats.

Immobilization stress rapidly modulates BDNF mRNA expression in the hypothalamus of adult male rats.

We demonstrated that short times (15 min) of immobilization stress application induced a very rapid increase in brain-derived neurotrophic factor (BDNF) mRNA expression in rat hypothalamus followed by a BDNF protein increase. The early change in total BDNF mRNA level seems to reflect increased expression of the BDNF transcript containing exon III, which was also rapidly (15 min) modified. The paraventricular and supraoptic nuclei, two hypothalamic nuclei closely related to the stress response and known to express BDNF mRNA, were analyzed by in situ hybridization following immobilization stress. In the parvocellular region of the paraventricular nucleus, BDNF mRNA levels increased very quickly as early as 15 min. In contrast, in the two other regions examined, the lateral and ventral magnocellular regions of the paraventricular nucleus, as well as in the supraoptic nucleus, signals above control were increased later, at 60 min. After stress application, plasma adrenocorticotropic hormone and corticosterone levels were strongly and significantly increased at 15 min. These studies demonstrated that immobilization stress challenge very rapidly enhanced BDNF mRNA levels as well as the protein, suggesting that BDNF may play a role in plasticity processes related to the stress response.

Regulation of brain-derived neurotrophic factor transcripts by neuronal activation in rat hypothalamic neurons.

Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family and regulates the survival, differentiation, and maintenance of function in different neuronal populations. BDNF is strongly expressed in hypothalamic neurons, where it exerts long- or short-lasting actions. Because glutamate has been associated with regulations of hypothalamic hormones, we examined the regulation of the four promoters of the BDNF gene by glutamate in fetal hypothalamic neurons. The expression levels of BDNF transcripts were investigated using semiquantitative RT-PCR. BDNF protein was determined by enzyme immunoassay, and BDNF and Trk B (BDNF receptor) gene variations were determined by RNAse protection assay. By RT-PCR, we showed that, under basal conditions, BDNF transcripts from exons I, II, and III but not from IV were expressed in the hypothalamic neurons. Glutamate increased expression of both the protein and the four transcripts via N-methyl-D-aspartate receptors, with maximal stimulations after 3 hr of application for exon I and II mRNAs and after 1 hr for exon III and IV mRNAs. Actinomycin D blocked the increase of all transcripts, whereas cycloheximide treatment inhibited stimulation only of exon I and II mRNAs. Trk B mRNA was rapidly and transiently reduced after glutamate application. Our results demonstrate that glutamate 1) regulates BDNF mRNA expression at an early developmental stage in hypothalamic neurons and 2) exerts a differential regulation of BDNF transcripts.

Immobilization stress rapidly and differentially modulates BDNF and TrkB mRNA expression in the pituitary gland of adult male rats.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin involved in neuronal survival and plasticity that binds to high-affinity receptors named TrkB. In the central nervous system, brain insults, including stress, induce modifications in BDNF messenger RNA (mRNA) expression. The present study attempted to determine in the adult rat pituitary, a peripheral structure relevant for the stress response: (1) whether BDNF and TrkB mRNA expression is influenced by different durations (15, 30, 60, 180 and 300 min) of single immobilization stress; (2) the expression of BDNF transcripts containing the different exons and their possible variations after stress exposure. Plasma corticotropin (ACTH) and corticosterone concentrations were strongly and significantly increased as early as 5 min after the stress stimulus. Using RNAse protection assay and in situ hybridization, a rapid increase in BDNF mRNA occurred at 15 min. This was accompanied by an increase in BDNF protein at 60 min, and by a rapid and significant decrease in TrkB mRNA expression observed at 15 and 30 min after stress application. RT-PCR analysis of BNDF transcripts showed strong basal expression of exons III and IV, whereas transcripts containing exons I and II seemed weakly expressed. After stress application, transcripts containing exons III and IV were rapidly and significantly increased at 30 min, whereas transcripts containing exons I and II remained unchanged. These results show that pituitary BDNF transcripts expression is differentially affected by immobilization stress.

Rapid stimulatory effects of brain-derived neurotrophic factor and neurotrophin-3 on somatostatin release and intracellular calcium rise in primary hypothalamic cell cultures.

Although the long-lasting effects of neurotrophins have been extensively studied, less data are available on their rapid effects, especially on peptide release. In the present report, we investigated rapid effects of neurotrophins on somatostatin release and on intracellular calcium concentration ([Ca(2+)](i)) in primary cultures of hypothalamic neurons. RT-PCR experiments revealed mRNA expression of the three high-affinity neurotrophin receptors tyrosine kinase (Trk) TrkA, TrkB and TrkC, indicating potential responses to their preferential ligands: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3), respectively. We demonstrated that BDNF, and to a lesser extent NT-3, induced significant time- and concentration-dependent somatostatin release, while NGF was devoid of any effect. BDNF or NT-3 induction of somatostatin release was inhibited by the Trk inhibitors K-252a and genistein, whereas K-252b, a less effective inhibitor, had no effect. BDNF- and NT-3-induced somatostatin release depended upon extra- and intracellular Ca(2+) since it was completely abolished in the presence of the Ca(2+) chelators BAPTA (bis-(alpha-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid) or BAPTA-AM (bis-(alpha-aminophenoxy)-ethane-N,N,N',N'-tetraacetoxymethylester), respectively. In addition, BDNF and NT-3 induced a sustained and rapid increase in [Ca(2+)](i) which depended on the extracellular Ca(2+) concentration. MK-801 (dizocilpine) and tetrodotoxin (TTX) entirely blocked neurotrophin-evoked somatostatin release and [Ca(2+)](i) rise in response to BDNF and NT-3 application in most neurons. Neurotrophin-induced [Ca(2+)](i) rise was completely blocked by K-252a. The present results are consistent with: (1) an indirect effect of neurotrophins on somatostatin release via endogenous glutamate release and subsequent NMDA receptor activation, (2) a major indirect effect of neurotrophins on Ca(2+) rise in hypothalamic neurons which very likely occurs through NMDA receptor activation. Taken altogether, these results indicate that BDNF and NT-3 can rapidly affect the activity of hypothalamic neurons.

Effects of alcohol on brain-derived neurotrophic factor mRNA expression in discrete regions of the rat hippocampus and hypothalamus.

Chronic alcohol consumption has adverse effects on the central nervous system, affecting some hippocampal and hypothalamic functions. In this study we tempted to demonstrate that some of these modifications could involve impairment of neurotrophic factors. Three experimental groups of male Sprague Dawley rats were studied: one control group, one chronically treated with alcohol vapor according to a well-established model that induces behavioral dependence, and a third group treated similarly but killed 12 hr after alcohol withdrawal. In all groups, changes in brain-derived neurotrophic factor mRNA expression occurring in the hippocampus and supraoptic nucleus were first analyzed by reverse transcription-polymerase chain reaction and then by in situ hybridization. In parallel, we used ribonuclease protection assay to measure mRNA levels encoding trkB in the two central nervous system regions. We showed that chronic alcohol intoxication decreases brain-derived neurotrophic factor mRNA expression in discrete regions of the rat hippocampus (CA1 region and dentate gyrus) and in the supraoptic nucleus of the hypothalamus. We also showed a global up-regulation of trkB mRNA expression encoding the high-affinity brain-derived neurotrophic factor receptor (TrkB), after applying the same treatment. Following 12 hr of alcohol withdrawal, a significant increase in BDNF mRNA expression was observed in the dentate gyrus and CA3 region of hippocampus and in the hypothalamic supraoptic nucleus. These findings suggest that chronic alcohol intake may modify hippocampal and hypothalamic neuronal functions through modifications in growth factors and its receptors.

Rapid modifications of somatostatin neuron activity in the periventricular nucleus after acute stress.

We have previously reported that stress induces a rapid increase in hypothalamic somatostatin (SS) release. In the present work, we investigated whether SS synthesis is also affected by this treatment. Male rats were subjected to 15-min immobilization (IMO) stress, and measurements of both SS mRNA levels and SS mRNA-containing cells were analyzed in the periventricular nucleus (PeV) by radioactive and nonradioactive in situ hybridization (ISH), respectively. In addition, SS content and total SS mRNA were measured in the whole hypothalamus by radioimmunoassay (RIA) and northern blot analysis, respectively. ISH was conducted by applying either a radioactive-labeled (35S) or a digoxigenin (DIG)-labeled oligonucleotide probe on histological sections containing the periventricular region of the anterior hypothalamic area (AHA). ISH analysis using radioactive label showed a significant increase in SS mRNA levels in stressed rats. In contrast, stress treatment decreased the number of DIG-labeled cells expressing SS mRNA in this region by 35% as compared to the same histological sections from naive control rats. In addition, a significant decrease in the total SS mRNA DIG-labeled area was observed. Finally, SS content and SS mRNA measured in the whole hypothalamus of stressed rats were markedly inhibited as compared to control rats. Our data show that IMO stress induces a significant and rapid increase in SS mRNA level accompanied by a decrease in the number of cells expressing SS mRNA in the PeV-AHA. The present results suggest that a subset of PeV SS neurons, which became silent at the onset of stress, are regulated independently of the remaining whole mass of PeV neurons. This differential control is in line with the cellular heterogeneity described in periventricular SS-producing neurons and with the multiple hypothalamic and pituitary functions assigned to SS.

Negative regulation of brain-derived neurotrophic factor mRNA expression by kainic acid in substantia nigra.

By using in situ hybridization, we have demonstrated that kainic acid (KA) administration decreases brain-derived neurotrophic factor (BDNF) mRNA expression in the substantia nigra pars compacta of adult rat. In addition, RT-PCR analysis indicated that transcripts derived from exons 1a and 1c of the BDNF gene were strongly decreased after KA treatment. Conversely, in dentate gyrus this treatment increased the expression of BDNF mRNAs derived from exons 1b, 1c and 1d. Our data show, contrarily to what is observed in most brain structures, that KA exerts a negative regulatory effect on BDNF mRNA expression in the adult substantia nigra.

Brain-derived neurotrophic factor and neurotrophin-3 enhance somatostatin gene expression through a likely direct effect on hypothalamic somatostatin neurons.

Although neurotrophins (NTs) have been extensively studied as neuronal survival factors in some areas of the central nervous system, little is known about their function or cellular targets in the hypothalamus. To understand their functional significance and sites of action on hypothalamic neurons, we examined the effects of their cognate ligands on neuropeptide content and messenger RNA (mRNA) expression in somatostatin neurons present in fetal rat hypothalamic cultures. Treatments were performed in defined insulin-free medium between days 6 and 8 of culture, since the maximal effects of NTs on somatostatin content and mRNA expression were observed after 48-h incubations. Brain-derived neurotrophic factor and NT-3, but not nerve growth factor, induced a dose-dependent increase in somatostatin content, which was influenced by plating density. The same treatment increased somatostatin mRNA and immunostaining intensity of somatostatin neurons, but had no effect on the number of these labeled neurons. The increased levels of somatostatin (peptide and mRNA) induced by NTs were not blocked by tetrodotoxin or by glutamate receptor antagonists, suggesting that endogenous neurotransmitters (e.g. glutamate) were not involved in these effects. In contrast, the stimulatory effects were completely blocked by K-252a, an inhibitor of tyrosine kinase (Trk) receptors, whereas the less active analog K-252b was ineffective. Double-labeling studies demonstrated that both TrkB or TrkC receptors were located on somatostatin neurons. Our results show that, in rat hypothalamic cultures, brain-derived neurotrophic factor, and NT-3 have a potent stimulatory effect on peptide synthesis in somatostatinergic neurons, likely through direct activation of TrkB and TrkC receptors.

BDNF gene transcripts in mesencephalic neurons and its differential regulation by NMDA.

Brain-derived neurotrophic factor (BDNF) enhances survival and protects dopaminergic neurons from neurotoxicity. We have examined in primary cultures of fetal mesencephalic neurons the expression of BDNF transcripts and its regulation by glutamate receptor agonists. RT-PCR experiments showed in these cultures the expression of mRNA encoding for neurotrophin receptors TrkA, TrkB and TrkC as well as a differential expression of alternative BDNF gene transcripts derived from exons 1a, 1b, 1c and 1d. In adult rat mesencephalon we only detected BDNF mRNAs originated from exons 1a and 1c. Quantitative RT-PCR analysis of mesencephalic neurons revealed that NMDA, but not kainate treatment, significantly increased BDNF mRNAs derived from exons 1b and 1d. Our data indicate the existence in mesencephalic neurons of different and alternative promoters within the BDNF gene that may be differentially regulated by glutamate receptor stimulation.

Effect of acute, but not chronic ethanol treatment on somatostatin secretion in rat hypothalamic neurons.

To examine the possible involvement of somatostatin in growth hormone modifications induced by ethanol, we examined: (1) the effects of chronic ethanol exposure of cultured hypothalamic neurons on somatostatin content and mRNA levels; (2) the acute effect of ethanol on somatostatin release stimulated by N-methyl-D-aspartate (NMDA). The results showed that 8 days of ethanol exposure (10-100 mM) did not decrease somatostatin content or somatostatin mRNA levels. Ethanol treatment alone had no significant effect on cell morphology or on protein content. In contrast, acute application of ethanol in 8 day-old cultures significantly reduced (50 mM) or completely blocked (100 mM) somatostatin release elicited by 50 microM NMDA without modifying basal release. We conclude that chronic ethanol treatment to concentrations up to 100 mM has no effect on somatostatin biosynthesis in fetal rat hypothalamic neurons, while weaker concentrations decrease NMDA-induced somatostatin release.

Expression of mRNAs encoding BDNF and its receptor in adult rat hypothalamus.

We used a digoxigenin-UTP-labeled cRNA probe with in situ hybridization and Northern blot analysis to investigate the localization of brain derived neurotrophic factor (BDNF) mRNA and expression of its different transcripts in adult rat hypothalamus. As the BDNF gene is under the control of alternative multiple promoters, which provide tissue-specific gene expression, we studied whether these transcripts were expressed in adult hypothalamus. Our data revealed two novel sources of hypothalamic BDNF mRNA: the supraoptic and periventricular nuclei. In addition, we observed the expression of transcripts from exons, I, II and III as well as the presence of 1.6 and 4.2 kb BDNF mRNAs. Finally, our findings confirmed expression of mRNA encoding neurotrophins receptors in the hypothalamus.

Estradiol enhances prostaglandin E2 receptor gene expression in luteinizing hormone-releasing hormone (LHRH) neurons and facilitates the LHRH response to PGE2 by activating a glia-to-neuron signaling pathway.

Prostaglandin E2 (PGE2) mediates the stimulatory effect of norepinephrine (NE) on the secretion of luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling reproductive function. In rodents, this facilitatory effect requires previous exposure to estradiol, suggesting that the steroid affects downstream components in the cascade that leads to PGE2-induced LHRH release. Because astroglia are the predominant cell type contacting LHRH-secreting nerve terminals, we investigated the involvement of hypothalamic astrocytes in the estradiol facilitation of PGE2-induced LHRH release. A subpopulation of LHRH neurons was found to express the mRNA encoding the PGE2 receptor subtype EP1-R, which is coupled to calcium mobilization. The LHRH-producing cell line GT1-1 also contains EP1-R mRNA and, to a lesser extent, the three alternatively spliced forms of EP3-R mRNA (alpha, beta, and gamma) that encode receptors linked to inhibition and stimulation of cAMP formation. Hypothalamic astrocytes treated with estradiol produced a conditioned medium that when applied to GT1-1 cells resulted in a selective upregulation of EP1-R and EP3gamma-R mRNAs. The conditioned medium also enhanced the LHRH response to EP1-R and EP3-R agonists and the cAMP response to EP3-R activation. Thus, one mechanism by which estradiol facilitates the effect of neurotransmitters acting via PGE2 to stimulate LHRH release is by enhancing the glial production of substances that upregulate PGE2 receptors on LHRH neurons. The existence of such a mechanism underscores the emerging importance of glial-neuronal communication in the control of brain neurosecretory activity.

The transforming growth factor alpha gene family is involved in the neuroendocrine control of mammalian puberty.

The concept is proposed that the central control of mammalian female puberty requires the interactive participation of neuronal networks and glial cells of the astrocytic lineage. According to this concept neurons and astrocytes control the pubertal process by regulating the secretory activity of those neurons that secrete luteinizing hormone-releasing hormone (LHRH). LHRH, in turn, governs sexual development by stimulating the secretion of pituitary gonadotropins. Astrocytes affect LHRH neuronal function via a cell-cell signaling mechanism involving several growth factors and their corresponding receptors. Our laboratory has identified two members of the epidermal growth factor/transforming growth factor (EGF/TGF alpha) family as components of the glial-neuronal interactive process that regulates LHRH secretion. Transforming growth factor alpha (TGF alpha) and its distant congener neu-differentiation factor, NDF, are produced in hypothalamic astrocytes and stimulate LHRH release via a glial intermediacy. The actions of TGF alpha and NDF on hypothalamic astrocytes involve the interactive activation of their cognate receptors and the synergistic effect of both ligands in stimulating the glial release of prostaglandin E2 (PGE2). In turn, PGE2 acts directly on LHRH neurons to stimulate LHRH release. A variety of experimental approaches has led to the conclusion that both TGF alpha and NDF are physiological components of the central mechanism controlling the initiation of female puberty.

Targeting transforming growth factor alpha expression to discrete loci of the neuroendocrine brain induces female sexual precocity.

Precocious puberty of cerebral origin is a poorly understood disorder of human sexual development, brought about by the premature activation of those neurons that produce luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling sexual maturation. An increased production of transforming growth factor alpha (TGF alpha) in the hypothalamus has been implicated in the mechanism underlying both normal and precocious puberty. We have now used two gene delivery systems to target TGF alpha overexpression near LHRH neurons in immature female rats. Fibroblasts infected with a retroviral construct in which expression of the human TGF alpha gene is constitutively driven by the phosphoglycerate kinase promoter, or transfected with a plasmid in which TGF alpha expression is controlled by an inducible metallothionein promoter, were transplanted into several regions of the hypothalamus. When the cells were in contact with LHRH nerve terminals or in the vicinity of LHRH perikarya, sexual maturation was accelerated. These results suggest that precocious puberty of cerebral origin may result from a focal disorder of TGF alpha production within the confines of the LHRH neuron microenvironment.

Hypothalamic astrocytes respond to transforming growth factor-alpha with the secretion of neuroactive substances that stimulate the release of luteinizing hormone-releasing hormone.

Previous studies demonstrated the involvement of transforming growth factor-alpha (TGF alpha), a member of the epidermal growth factor (EGF) family, in the developmental regulation of hypothalamic LHRH release. Although both TGF alpha and EGF stimulate LHRH release, they do not appear to act directly on LHRH neurons, as no EGF/TGF alpha receptors are detected on these cells in vivo. Instead, the stimulatory effect of TGF alpha on LHRH release seems to require a glial intermediacy. The present study identifies one of the glial molecules involved in this process. In vitro exposure of purified hypothalamic astrocytes to TGF alpha or EGF in a defined medium led to activation of the cyclooxygenase-mediated pathway of arachidonic acid metabolism, as indicated by an increase in PGE2 release, but failed to affect lipooxygenase-mediated metabolism, as assessed by the lack of increase in leukotriene C4 production; addition of TGF alpha- (T-CM) or EGF-conditioned medium to cultures of LHRH-producing GT1-1 cells stimulated LHRH release. In contrast, direct exposure of GT1-1 cells to the growth factors was ineffective. Incubation of the cells in medium conditioned by untreated astrocytes (CM) was also ineffective. Blockade of either EGF receptor signal transduction or cyclooxygenase activity in the astrocytic cultures prevented both TGF alpha-induced PGE2 formation in astrocytes and the stimulatory effect of T-CM on LHRH release. Immunoneutralization of PGE2 actions or selective removal of the PG from T-CM also prevented T-CM-induced LHRH release. Addition of exogenous PGE2 restored the effect. Thus, PGE2 is one of the glial molecules involved in mediating the stimulatory effect of TGF alpha on LHRH release. The effectiveness of PGE2 in eliciting LHRH release was, however, greatly reduced when PG was delivered to GT1-1 cells in astrocyte-defined medium instead of CM. Thus, astrocytes appear to produce a yet to be identified substance(s) that facilitates the stimulatory effect of PGE2 on LHRH output. We postulate that the ability of TGF alpha to enhance LHRH release depends on the potentiating interaction of PGE2 with these additional glial-derived molecules.

RNase Protection Assay

The RNase protection assay is a highly sensitive technique developed to detect and measure the abundance of specific mRNAs in samples of total cellular RNA. The assay utilizes in vitro transcribed 32P-labeled antisense RNA probes that are hybridized in solution to their complementary cellular mRNAs. This is followed by digestion of nonhybridizing (single-stranded) RNA species with RNases, removal of the RNases by treatment with proteinase K, phenol extraction of the cRNA:mRNA complexes, and electrophoretic isolation of the hybridizing cRNA fragments. Since one can synthesize "sense" mRNAs having the same sequence as the target cellular mRNA, appropriate standard curves can be generated and used to quantitate the changes in tissue mRNA levels. Because the assay requires perfect sequence complementarity for full protection, it not only serves as a quantitative tool but also provides conclusive evidence for the existence of a specific mRNA in a given tissue. The procedure described here is a modification of that originally described by M. Gilman [1993, in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., Eds.), Vol. 1, pp. 4.7.1-4.7.6, Greene and Wiley-Interscience, New York].

Neuroendocrine and autonomous mechanisms underlying thermoregulation in cold environment.

This review focuses on the central regulation of thermoregulatory responses with special attention to the participation of thyrotropin-releasing hormone (TRH) in both autonomous and endocrine responses to a cold environment. Besides a direct projection of TRH neurons from paraventricular nuclei (PVN) to the median eminence, and the subsequent activation of the thyroid axis, there are direct projections from the PVN to the autonomic preganglionic neurons controlling autonomous responses. There projections convey information to peripheral targets involved in thermogenesis through the dorsal vagal complex and the spinal cord, for parasympathetic and sympathetic neurotransmissions respectively. Furthermore, cold exposure increases TRH mRNA levels in the PVN but also in dorsal motor and caudal raphe nuclei, thus providing strong evidence for a functional link between autonomous and neuroendocrine systems involved in thermoregulation. The review also focuses on neuroendocrine regulation of cold-induced TRH/TSH release associated with modifications in somatostatin release, with special reference to the participation of several central neurotransmitters (catecholamines, serotonin or GABA) or the influence of sex steroids.