A receptor in pituitary and hypothalamus that functions in growth hormone release.

Small synthetic molecules termed growth hormone secretagogues (GHSs) act on the pituitary gland and the hypothalamus to stimulate and amplify pulsatile growth hormone (GH) release. A heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPC-R) of the pituitary and arcuate ventro-medial and infundibular hypothalamus of swine and humans was cloned and was shown to be the target of the GHSs. On the basis of its pharmacological and molecular characterization, this GPC-R defines a neuroendocrine pathway for the control of pulsatile GH release and supports the notion that the GHSs mimic an undiscovered hormone.

Mobilisation of intracellular Ca2+ by mGluR5 metabotropic glutamate receptor activation in neonatal rat cultured dorsal root ganglia neurones.

The ability of metabotropic glutamate receptor activation to mobilise intracellular calcium was investigated in cultured dorsal root ganglion (DRG) neurones from neonatal rats using the calcium sensitive fluorescent dye Fura-2. L-glutamate (10 microM) caused sustained and oscillatory increases in intracellular calcium concentration ([Ca2+]i) in a subpopulation of cultured DRG neurones. The oscillatory responses were not blocked by combined application of the ionotropic glutamate receptor antagonists MK 801 (2 microM) and CNQX (20 microM). Oscillations in [Ca2+]i were also observed following application of the nonselective metabotropic glutamate receptor (mGluR) agonist, trans-(1S,3R)-1-aminocyclopentane-1S, 3R-dicarboxylic acid (1S,3R)-ACPD, 20 microM) and the mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, 500 microM). These responses were blocked by the selective Group I mGluR antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) (100 microM) and Ca2+ release channel inhibitors ryanodine (100 microM) and dantrolene (10 microM). The predominantly Group II agonist (2S,2'R,3'R)-2-(2'3'-dicarboxy-cyclopropyl)glycine (DCG-IV, 100 microM) failed to produce Ca2+ transients alone but suppressed responses to CHPG. Reverse transcriptase PCR techniques, using primers specific to Group I mGluRs, revealed the presence of mGluR5 but not mGluR1 mRNA in these cells. Therefore, glutamate can cause a slowly activating and reversible mobilisation of [Ca2+]i in sensory neurones by activation of ionotropic receptors, and can induce oscillatory calcium transients by selectively activating metabotropic glutamate receptors that are likely to be of the mGluR5 subtype.

Expression of B1 and B2 bradykinin receptor mRNA and their functional roles in sympathetic ganglia and sensory dorsal root ganglia neurones from wild-type and B2 receptor knockout mice.

Bradykinin has been implicated in nociception and inflammation. To examine the relative significance of B1 and B2 bradykinin receptor subtypes in sympathetic and sensory ganglia, the electrophysiological effects of bradykinin analogues and the expression of receptor subtype mRNA were examined in wild-type and "B2 knockout" mice from which the B2 receptor gene had been deleted. In wild-type mice the B2 receptor agonist bradykinin depolarized superior cervical ganglia (SCG) and activated inward currents in dorsal root ganglia (DRG) neurones. Responses to the B1 receptor agonist, [des-Arg10]-kallidin, were seen only in SCG that had been pre-treated with interleukins and the peptidase inhibitor captopril, but not in DRG neurones. The up-regulation of responses to [des-Arg10]-kallidin and substance P were blocked by indomethacin and, thus, were dependent upon cyclo-oxygenase activity. The effects of bradykinin were abolished in SCG and DRG's from B2 knockout mice and this was correlated with the absence of B2 receptor mRNA in ganglia from these animals. However, despite the presence of B1 receptor mRNA in interleukin treated SCG from B2 knockout mice, no depolarizing effects of the B1 receptor agonist [des-Arg10]-kallidin were observed. The successful elimination of bradykinin responses and B2 mRNA in sympathetic and sensory ganglia from B2 knockout mice, confirms that B2 receptors are the predominant functional bradykinin receptor subtype in these tissues and that B1 receptor mRNA is expressed in both sympathetic and sensory ganglia from these animals.

Experimental hemiparkinsonism in the rat following chronic unilateral infusion of MPP+ into the nigrostriatal dopamine pathway--I. Behavioural, neurochemical and histological characterization of the lesion.

1-Methyl-4-phenylpyridinium ion (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, has been chronically infused (10 micrograms/24 h for 7 days) via osmotic minipumps into the left median forebrain bundle of the rat in order to determine whether it can induce permanent damage to the nigrostriatal dopamine system. Its effects were assessed over a period of 6 months post lesion. Four to 5 days following minipump implantation, all MPP+-treated animals displayed spontaneous ipsilateral postural bias indicating a marked imbalance in striatal dopamine and degeneration of the ipsilateral nigrostriatal dopamine pathway. After 3-5 weeks, MPP+-infused animals showed dose-related ipsilateral and contralateral circling in response to methamphetamine (1-5 mg/kg i.p.) and apomorphine (0.05-0.25 mg/kg s.c.) respectively. In vivo, using bilateral monitoring of striatal dopamine in MPP+-infused animals at 2 and 4 months by push-pull perfusion, both basal and methamphetamine- (2.5 mg/kg i.p.) stimulated release of dopamine was undetectable in the ipsilateral striatum, indicating a complete loss of dopamine terminals. In contrast, in the contralateral striatum of these animals and in striata of saline-infused animals, there were 4-5-fold increases in dopamine release in response to methamphetamine. Six months after lesion, animals infused with MPP+ continue to exhibit robust rotational behaviour in response to methamphetamine and apomorphine. In the ipsilateral striatum of the MPP+-infused animals the tissue concentrations of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, were all undetectable; however, the levels of noradrenaline, serotonin and its metabolite, 5-hydroxyindoleacetic acid, were not significantly different from control values. In contrast to the striatum, MPP+ had no significant effect on the levels of dopamine and its metabolites in the ipsilateral nucleus accumbens; in addition, the levels of noradrenaline and serotonin and its metabolite were comparable to control levels. Histological examination revealed a marked loss of cells and severe gliosis in the substantia nigra pars compacta of MPP+-infused animals. The present results provide evidence that direct infusion of MPP+ into the medial forebrain bundle of the rat can lead to a complete loss of dopamine neurons in the pars compacta of the substantia nigra with ensuing behavioural, neurochemical and biochemical changes characteristic of the lesion.(ABSTRACT TRUNCATED AT 400 WORDS)

Temporal changes in the messenger RNA levels of cellular immediate early genes and neurotransmitter/receptor genes in the rat neostriatum and substantia nigra after acute treatment with eticlopride, a dopamine D2 receptor antagonist.

The cellular immediate early genes are involved in the transcriptional events associated with the dopaminergic regulation of neurotransmitter expression within neurons of the neostriatum. To characterize these events in detail, quantitative in situ hybridization histochemistry was used to assess the temporal effects of acute dopamine receptor blockade with eticlopride, a dopamine D2 receptor antagonist, on the messenger RNA expression of the immediate early genes and neurotransmitters/receptors in the caudate-putamen and ventral tegmental area/substantia nigra pars compacta of the rat. Groups of rats were injected with a single dose of either isotonic saline or eticlopride (0.5 mg/kg i.p.) and killed at various time intervals ranging from 5 min to 24 h and frozen brain sections processed by in situ hybridization histochemistry. Using computerized image analysis, the changes in messenger RNA expression for c-fos, c-jun, jun B, jun D, nerve growth factor I-A and nerve growth factor I-B and for neurotensin, glutamate decarboxylase, proenkephalin, the dopamine D1 receptor and the short and long isoforms of the D2 receptor were examined in the caudate-putamen. In the ventral tegmental area and substantia nigra pars compacta, the messenger RNA expression of the above early response genes and that for neurotensin, tyrosine hydroxylase, cholecystokinin and the D2 receptor isoforms were also examined. In the neostriatum, eticlopride caused a rapid increase in c-fos messenger RNA with significantly increased levels at 10 min (P < 0.01). The levels peaked at 30 min and thereafter declined to control levels. A similar profile was observed for jun B messenger RNA, although levels were still significantly (P < 0.01) elevated at 1 h and declined to basal levels thereafter. No significant changes were observed for c-jun, jun D, nerve growth factor I-A and nerve growth factor I-B messenger RNAs. In the dorsolateral neostriatum, there was an increase in proneurotensin messenger RNA 10 min after eticlopride, this increase becoming significant (P < 0.01) at 60 min. Levels were maximal at 2-6 h and decreased after 12 h to basal levels. There were small increases in proenkephalin messenger RNA, but these were not significant (P < 0.05) until 6 h after the injection. Eticlopride did not have any significant effects on the messenger RNA levels for glutamate decarboxylase, the D1 receptor and the short and long isoforms of the D2 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of messenger RNAs for glutamic acid decarboxylase, preprotachykinin, cholecystokinin, somatostatin, proenkephalin and neuropeptide Y in the adult rat superior colliculus.

The mammalian superior colliculus is an important subcortical integrator of sensorimotor behaviours. It is multi-layered, each layer containing specific neuronal types and possessing distinct input/output relationships. Here we use in situ hybridisation methods to map the distribution of seven neurotransmitters/neuromodulator systems in adult rat superior colliculus. Coronal sections were probed for preprotachykinin, cholecystokinin, somatostatin, proenkephalin, neuropeptide Y and the enzymes glutamic acid decarboxylase and choline acetyltransferase, markers for GABA and acetylcholine respectively. Cells expressing glutamic acid decarboxylase messenger RNA were the most abundant, the highest density being found in the superficial layers. Many cells containing proprotachykinin messenger RNA were found in stratum zonale and the upper two-thirds of stratum griseum superficiale; cells were also located in deeper tectal laminae, particularly caudomedially. Most cholecystokinin messenger RNA expressing cells were located in the superficial layers with a prominent band in the middle third of stratum griseum superficiale. Cells expressing moderate to high levels of somatostatin messenger RNA formed a dense band in the lower third of stratum griseum superficiale/upper stratum opticum; two less distinct tiers of labelling were seen in deeper layers. These in situ hybridisation data reveal three distinct sub-laminae in rat stratum griseum superficiale. Cells expressing moderate to low levels of proenkephalin messenger RNA were located in lower stratum griseum superficiale/upper stratum opticum and intermediate laminae. A cluster of enkephalinergic cells was located medially in the deep tectal laminae. Expression of neuropeptide Y messenger RNA was relatively low and mostly confined to cells in stratum griseum superficiale and stratum opticum. No choline acetyltransferase messenger RNA was detected. This in situ analysis of seven different neurotransmitters/neuromodulator systems sheds new light on the neurochemical organisation of the rat superior colliculus. The data are related to what is known anatomically and physiologically about intrinsic and extrinsic tectal circuitry, and the potential involvement of different neuropeptides in these circuits is discussed. The work forms the basis for future developmental studies examining the effects of transplantation and visual deprivation/deafferentation on tectal neurochemistry and function.

Cholecystokinin-dependent regulation of host dopamine inputs to striatal grafts.

Intrastriatal infusions of cholecystokinin-8-sulphate in the rat exerts a dose-dependent inhibition of dopamine-release from nigrostriatal terminals in the neostriatum, as measured by push-pull perfusion. This effect is abolished by excitotoxic lesions of the neostriatum, which, along with behavioural, electrophysiological and receptor binding studies, suggests that cholecystokinin exerts its action indirectly on dopamine release via receptors located on intrinsic striatal neurons. Grafts of embryonic striatum implanted in the lesioned striatum become innervated by host-derived dopamine axons and restore the response of those host neurons to cholecystokinin infusion. This suggests that the innervation of the grafts by dopaminergic axons of the host brain does not simply provide a tonic input to the grafts, but rather represents a phasic input that is under dynamic local regulation by graft-host feedback influences from the transplanted neurons themselves.

Increased messenger RNA expression of the 695 and 751 amino acid isoforms of the beta-amyloid protein precursor in the thalamus of 17-year-old cynomolgus (Macaca fascicularis) monkeys.

The levels of expression of messenger RNAs of the 695 and 751 amino acid isoforms of the beta-amyloid protein precursor in the brains of three-year-old and 17-year-old cynomolgus monkeys (Macaca fascicularis) were visualized and quantified by in situ hybridization histochemistry using 35S-labelled oligonucleotide probes. The analysis was carried out on coronal brain sections taken through the hippocampus and thalamus at the level of the geniculate nuclei. High densities of beta-amyloid protein precursor695 and beta-amyloid protein precursor751 messenger RNAs were found in the medial aspects of the mediodorsal, centromedian and parafascicular nuclei of the 17-year-old monkeys. The messenger RNA levels of the 695 and 751 isoforms were about two- and seven-fold, respectively, those found in the same nuclei of the three-year-old animals. The levels of these messenger RNA transcripts in the 17-year-old monkeys were not significantly different from those in the three-year-old animals in other brain areas e.g. the temporal cortex, entorhinal cortex and hippocampus. No Alzheimer's disease-like neuropathology in terms of diffuse or senile beta-amyloid plaques, dystrophic neurites or neurofibrillary tangles were detectable by specific innumohistochemical procedures in the above thalamic nuclei of the 17-year-old animals. In addition no reactive gliosis was seen in the thalamus of these monkeys.(ABSTRACT TRUNCATED AT 250 WORDS)

The messenger RNAs for the N-methyl-D-aspartate receptor subunits show region-specific expression of different subunit composition in the human brain.

The expression of the messenger RNAs encoding N-methyl-D-aspartate receptor subunits in neurologically normal post-mortem human brain was studied by in situ hybridization. In the caudate, putamen and nucleus accumbens strong hybridization signals were observed for N-methyl-D-aspartate R1-1 messenger RNA but much weaker signals for N-methyl-D-aspartate R1-3 and N-methyl-D-aspartate R1-4, N-Methyl-D-aspartate R1-2 was not detectable. N-methyl-D-aspartate R2B was the only N-methyl-D-aspartate R2 subunit detected in these nuclei. In the hippocampus the messenger RNAs for both N-methyl-D-aspartate R1-1 and N-methyl-D-aspartate R1-4 were strongly expressed in the dentate gyrus, CA3-CA1 pyramidal cells, subiculum, entorhinal cortex and perirhinal cortex. Much lower expression was seen for N-methyl-D-aspartate R1-2 and N-methyl-D-aspartate R1-3. The messenger RNAs for both N-methyl-D-aspartate R2A and N-methyl-D-aspartate R2B, but not N-methyl-D-aspartate R2C, subunits were expressed in the hippocampus. In the temporal cortex all N-methyl-D-aspartate RI isoforms were expressed (N-methyl-D-aspartate R1-1 and N-methyl-D-aspartate R1-4 being the most abundant) and N-methyl-D-aspartate R2A and N-methyl-D-aspartate R2B but not N-methyl-D-aspartate R2C were also moderately expressed. In the brain stem N-methyl-D-aspartate R1-4 was strongly expressed in various nuclei including the locus coeruleus, nucleus centralis superior and deep pontine nuclei. Only weak expression was seen for N-methyl-D-aspartate RI-1 and N-methyl-D-aspartate R1-3 but not N-methyl-D-aspartate RI-2; of the N-methyl-D-aspartate R2 subunits only N-methyl-D-aspartate R2C was found to be expressed in these nuclei. In the cerebellum all the N-methyl-D-aspartate I isoforms were expressed (mostly N-methyl-D-aspartate R1-4) in the Purkinje layer which also expressed N-methyl-D-aspartate R2A and N-methyl-D-aspartate R2C. In the molecular layer cells were found expressing N-methyl-D-aspartate R1-4 and N-methyl-D-aspartate R2B and cells in the granule layer were found to express N-methyl-D-aspartate R1-1, N-methyl-D-aspartate R1-3 and N-methyl-D-aspartate R1-4 and N-methyl-D-aspartate R2C only. Preliminary studies indicated that the messenger RNA for the N-methyl-D-aspartate R2D subunit was not expressed in the above areas of brain. These results give the first demonstration of the distribution of N-methyl-D-aspartate receptor subunit messenger RNAs in the human brain. The region-specific expression of subunit combinations suggests a heterogeneity of N-methyl-D-aspartate receptors with diverse physiological/pathophysiological roles and provides a rationale for the development of discriminatory N-methyl-D-aspartate receptor antagonists to target selective neuronal populations.

Quantitative comparison of pretreatment regimens used to sensitize in situ hybridization using oligonucleotide probes on paraffin-embedded brain tissue.

Paraffin embedding of tissue is generally perceived to dramatically reduce RNA detectability. As a consequence, in situ hybridization on paraffin-embedded tissue is largely confined to detection of high-copy RNA species (e.g., viral RNA) and/or to detection using typically more sensitive cDNA probes or riboprobes. In this study, several procedures for in situ hybridization on paraffin-embedded rat tissue using oligonucleotide probes complementary to cellular transcripts were developed and quantitatively compared. Certain pretreatments showed marked increases in sensitivity compared to untreated sections. Furthermore, through quantitative assessment using image analysis, sensitivity of optimal pretreatments was equal to that of routinely used fresh-frozen, postfixed tissue sections. The development of such techniques permitting in situ hybridization to be carried out on paraffin-embedded tissue allows a comparison of protein and mRNA distribution to be made in adjacent sections and provides the potential for double labeling by in situ hybridization and immunohistochemistry which may not be possible on post-fixed frozen sections.

Decreased tyrosine hydroxylase mRNA but not cholecystokinin mRNA in the pars compacta of the substantia nigra and ventral tegmental area of aged rats.

Quantitative in situ hybridization histochemistry was used to determine the age-related changes in tyrosine hydroxylase (TH) mRNA and cholecystokinin (CCK) mRNA in the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc) of the rat. Coronal sections (10 microns) were cut in a cryostat through the VTA and SNc of brains from 3 months and 33 month old Sprague-Dawley rats and immediately adjacent sections hybridized with 35S-labelled 45-mer oligonucleotide probes specific for either the rat TH or CCK genes. The mRNA levels of each gene were estimated by computerised densitometric analysis of the signal on X-ray film autoradiograms and estimation of the number of mRNA expressing cells as well as the density of expression per cell (grain density) was made from high resolution emulsion autoradiograms. Analysis of the TH mRNA on X-ray film autoradiograms indicated that the levels averaged 25% lower in the SNc (P < 0.01) and 18% lower in the VTA (P < 0.05) of the old rats. However, analysis of the emulsion autoradiograms showed that this reduction in TH mRNA in the VTA and SNc in the old rats was not due to a loss of TH mRNA expressing cells but due to a reduction in the hybridization signal per expressing cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular localization of corticotropin releasing factor mRNA in the ovine brain.

In this study in situ hybridization histochemistry was used to determine the regional distribution and cellular localization of corticotropin releasing factor (CRF) mRNA in the sheep brain. The highest densities of labelled cell bodies were found in the paraventricular nucleus (PVN) of the hypothalamus and in the inferior olivary nuclei in the brain stem. Labelled cells were also found in every major cortical field as well as in the vicinity of the locus coeruleus and parabrachial nucleus and nucleus of the solitary tract. No CRF mRNA-expressing cells were found in the supraoptic nucleus or other diencephalic nuclei or in telencephalic and mesencephalic nuclei. The dense population of CRF mRNA-expressing cells in the PVN support the major role of CRF in the modulation of adrenocorticotropin (ACTH) and cortisol secretion. Moreover, the widespread distribution of CRF mRNA transcripts would suggest that there are distinct populations of CRF neurons with extrahypophysiotropic roles involved in the coordination and integration of endocrine, autonomic and behavioural responses in response to stress as well as in the control of complex cognitive and motor tasks.

Distribution and cellular localization of preproenkephalin mRNA in the ovine brain and pituitary.

In this study in situ hybridization histochemistry was used to determine the regional and cellular localization of preproenkephalin (PPE) mRNA in the sheep brain and pituitary. Coronal brain sections were hybridized with an 35S-labelled synthetic 45-mer deoxyribonucleotide probe complementary to a portion of the bovine PPE gene. The specificity of the probe was confirmed by Northern blot analysis. The highest density of labelled cell bodies was found in the nucleus accumbens, caudate-putamen, olfactory tubercle, the central nucleus of the amygdala, the paraventricular nucleus of the hypothalamus, the suprachiasmatic nucleus and in the gigantocellular division of the medullary reticular formation. Labelled cells were also found in the olfactory bulb, prefrontal cortex, piriform cortex and cerebral cortex and in the vicinity of the locus coeruleus, parabrachial nucleus and the nucleus of the solitary tract. In the pituitary a dense PPE mRNA signal was observed in the intermediate lobe; cells in the anterior or neural lobe did not express PPE mRNA. The widespread distribution of cells containing PPE mRNA transcripts within the ovine brain agrees with a similar distribution in the rat. The data suggest that PPE neurons may be involved in diverse physiological functions including the processing of sensory and nociceptive information and in the regulation of endocrine and motor responses.

Distribution of novel CGRP1 receptor and adrenomedullin receptor mRNAs in the rat central nervous system.

Recent cloning studies have isolated receptors which confer specific responsiveness to calcitonin gene related peptide (CGRP) and the related peptide adrenomedullin. Using in situ hybridisation, we demonstrate the heterogenous distribution of the mRNAs of two proposed CGRP1 receptors (RDC-1 and calcitonin receptor-like receptor, CRLR) in the rat brain. Adrenomedullin receptor mRNA was weakly expressed, principally in the cerebellum. These findings may assist in the determination of the function of these largely uncharacterised receptors.

Distribution of 5-ht(5A), 5-ht(5B), 5-ht(6) and 5-HT(7) receptor mRNAs in the rat brain.

The precise involvement of 5-ht(5A), 5-ht(5B), 5-ht(6) and 5-HT(7) receptors in the pleiotropic actions of 5-HT remain incompletely known. To gain insights into their physiological function(s), localization of mRNAs encoding these subtypes was carried out using in situ hybridization on rat brain sections. Localization was heterogeneous. For example, 5-ht(5A) mRNA was widely expressed while 5-ht(5B) mRNA was predominantly expressed in habenula, hippocampus and inferior olive. 5-ht(6) mRNA was abundant in olfactory tubercles and caudate putamen, and highest levels of 5-HT(7) mRNA were observed in multiple thalamic nuclei. These data suggest that these receptors may have distinct functional roles within the serotonergic system.

Expression of c-fos, jun D and pp60c-src+ mRNAs in the developing and grafted rat striatum.

Expression of the mRNAs of the proto-oncogenes pp60c-src+, c-fos and jun D were studied using in-situ hybridisation histochemistry in the developing striatum and in striatal grafts. The temporal patterns of mRNA expression were monitored in the striatum of the normal developing rat from the 12th day of gestation (E12) to 10 days postnatally, and were compared to the changes in gene expression observed in E13-E14 primordial striatal tissue grafts 7, 15 and 30 days after implantation in the ibotenic acid-lesioned striatum of adult rats. During development, all three proto-oncogenes were most highly expressed just before birth, at E19. Striatal expression of all three proto-oncogenes was markedly reduced after birth and remained at a low level through to adulthood. A different mode of expression was observed in the transplanted striatum which was unique to each particular gene. jun D and pp60c-src+ were expressed for a longer time period in the grafted primordial cells than in normal development, whereas no c-fos expression could be detected in the grafts. These results suggest that transplantation of embryonic neural cells into the host brain may affect the normal developmental regulation of such cells and their expression of some proto-oncogenes.

Regional and cellular localization of calcitonin gene-related peptide-receptor component protein mRNA in the guinea-pig central nervous system.

Recent cloning studies have isolated proteins which confer responsiveness to calcitonin gene-related peptide (CGRP). In this study, we have determined the central nervous system (CNS) distribution of the mRNA of one such protein, termed CGRP-receptor component protein (RCP), by in situ hybridization. CGRP-RCP mRNA was widely expressed in the guinea-pig CNS, being particularly abundant in cerebellum and hippocampus. These data should assist in the determination of the potential physiological function(s) of this protein in the CNS.

Acute action of oestrogen on medial preoptic gamma-aminobutyric Acid neurons: correlation with oestrogen negative feedback on luteinizing hormone secretion.

Abstract The acute effects of oestrogen on the medial preoptic area (MPOA) gamma-aminobutyric acid (GABA) system were examined by delivering an intravenous bolus of 17beta-oestradiol (5 mug/100 g body wt) to conscious ovariectomized rats implanted with microdialysis probes. Fifteen-min blood samples were taken to determine the time-course of negative feedback effects of oestrogen on luteinizing hormone (LH) secretion. Two h after administration of 17beta-oestradiol, GABA release from the MPOA was significantly elevated compared with vehicle-treated controls (P<0.05). The rise in GABA levels continued until the end of the experiment, 4 h after 17beta- oestradiol, at which time it was over 50% higher than controls (P<0.01). The pulsatile pattern of LH secretion was significantly depressed 2 and 3 h after administration of 17beta-oestradiol compared with controls (P<0.05). To determine the effects of the 17beta-oestradiol treatment on pituitary responsiveness to LH-releasing hormone (LHRH), a further group of rats were given exogenous LHRH (50ng/100g body wt, intravenously) before and 3 h after vehicle or 17beta-oestradiol treatment and blood samples taken to determine the effect on LH secretion. The maximal LH response to LHRH in 17beta-oestradiol-treated rats was approximately 50% that of control-treated values. This study demonstrates the acute and potent action of 17beta-oestradiol on GABA release in the MPOA and lends support to a genomic site of action for oestrogen in modulating neural elements regulating GABA release from the MPOA. These results, showing a parallel decrease in LH secretion with increased GABA levels in the MPOA, suggest a role for GABA elements within the MPOA as a site of oestrogen negative feedback on LH secretion.

EDG receptors as a therapeutic target in the nervous system.

EDG receptors are a family of closely related G-protein-coupled receptors, so-called since the first family member to be cloned is encoded by an endothelial differentiation gene. Of the six family members identified, five use lysophospholipids as their endogenous ligands. The sixth receptor, EDG-6, remains an orphan. These receptors activate multiple secondary-messenger pathways involving coupling to Gi, Gq/11, and G12/13 trimeric guanine nucleotide-binding proteins and are thought to play an important role in cell growth, development and maintenance, and cytoskeletal-dependent changes. EDG receptors are expressed in most mammalian cells and tissues, each subtype having a distinct distribution pattern, raising the possibility of tissue-specific biological roles that could be explored in drug-discovery programs. In this study the distribution of EDG-receptor mRNA within the nervous system has been investigated. As seen in peripheral tissues, these receptors appear to be discretely localized within specific brain regions and cell types. For example, EDG-1, -3, -4 receptors are confined to neuronal cells, EDG-2 receptors to white matter tracts, while EDG-5 receptors appear to be expressed in various cell types, including neuronal cells, white matter tracts, and ependymal cells. EDG-6-receptor mRNA was not detected in the nervous system. Speculation as to the role of these receptors in physiological/pathophysiological processes, particularly those involving cell development, proliferation, maintenance, migration, differentiation, plasticity, and apoptosis can be made from such distribution studies. EDG receptors located in brain neuronal cells might, for example, influence apoptosis and be involved in cell rescue following ischemic damage or during the early stages of progressive neurodegenerative diseases. Those restricted to oligodendrocytes might play a crucial role in myelination and offer a potential target in the treatment of demyelinating diseases, such as multiple sclerosis. In order to explore the role of these receptors, it is necessary to identify selective compounds. To this end we have developed an agonist-induced [35S]GTP gamma S binding assay using an HEK cell line expressing a pertussis-toxin-insensitive human-EDG-2-receptor-rat-Gi alpha 1-fusion protein. Such as assay system overcomes the problems associated with the almost ubiquitous responsiveness of mammalian cells to lysophospholipid. This assay lends itself to high throughput application, opening up the possibility of identifying compounds to further probe the therapeutic potential of EDG receptor manipulation.

Molecular and functional diversity of the expanding GABA-A receptor gene family.

Fast inhibitory neurotransmission in the mammalian CNS is mediated primarily by the neurotransmitter gamma-aminobutyric acid (GABA), which, upon binding to its receptor, leads to opening of the intrinsic ion channel, allowing chloride to enter the cell. Over the past 10 years it has become clear that a family of GABA-A receptor subtypes exists, generated through the coassembly of polypeptides selected from alpha 1-alpha 6, beta 1-beta 3, gamma 1-gamma 3, delta, epsilon, and pie to form what is most likely a pentomeric macromolecule. The gene transcripts, and indeed the polypeptides, show distinct patterns of temporal and spatial expression, such that the GABA-A receptor subtypes have a defined localization that presumably reflects their physiological role. A picture is beginning to emerge of the properties conferred to receptor subtypes by the different subunits; these include different functional properties, differential modulation by protein kinases, and the targeting to different membrane compartments. These properties presumably underlie the different physiological roles of the various receptor subtypes. Recently we have identified a further member of the GABA-A receptor gene family, which we have termed theta, which appears to be most closely related to the beta subunits. The structure, function, and distribution of theta-containing receptors, and receptors containing the recently reported epsilon subunit, are described.