GABA inhibits migration of luteinizing hormone-releasing hormone neurons in embryonic olfactory explants

During development, a subpopulation of olfactory neurons transiently expresses GABA. The spatiotemporal pattern of GABAergic expression coincides with migration of luteinizing hormone-releasing hormone (LHRH) neurons from the olfactory pit to the CNS. In this investigation, we evaluated the role of GABAergic input on LHRH neuronal migration using olfactory explants, previously shown to exhibit outgrowth of olfactory axons, migration of LHRH neurons in association with a subset of these axons, and the presence of the olfactory-derived GABAergic neuronal population. GABAA receptor antagonists bicuculline (10(-5) M) or picrotoxin (10(-4) M) had no effect on the length of peripherin-immunoreactive olfactory fibers or LHRH cell number. However, LHRH cell migration, as determined by the distance immunopositive cells migrated from olfactory pits, was significantly increased by these perturbations. Addition of tetrodotoxin (10(-6) M), to inhibit Na+-transduced electrical activity, also significantly enhanced LHRH migration. The most robust effect observed was dramatic inhibition of LHRH cell migration in explants cultured in the presence of the GABAA receptor agonist muscimol (10(-4) M). This study demonstrates that GABAergic activity in nasal regions can have profound effects on migration of LHRH neurons and suggests that GABA participates in appropriate timing of LHRH neuronal migration into the developing brain.     

GABAergic neurons in the embryonic olfactory pit/vomeronasal organ: maintenance of functional GABAergic synapses in olfactory explants

In previous work, we showed a robust gamma-aminobutyric acid (GABAergic) synaptic input onto embryonic luteinizing hormone-releasing hormone (LHRH) neurons maintained in olfactory explants. In this study, we identify GABAergic neurons in olfactory pit (OP) of embryonic mice in vivo and study, using patch-pipet whole-cell current and voltage clamp techniques, synaptic interactions of these neurons in explant cultures. In vivo, glutamate decarboxylase (GAD, the enzyme which synthesizes GABA) mRNA was first detected in nasal regions on Embryonic Day (E) 11.5. From E12.5 to E13.5, robust GAD expression was localized to cells primarily in the ventral aspect of the OP. GAD mRNA was not detected over dorsally located cells in olfactory sensory or respiratory epithelium. In addition, GAD mRNA was not observed in cells along olfactory axons. GAD mRNA was dramatically reduced in the OP/vomeronasal organ by E16.5. Using antibodies against both GABA and GAD, immunopositive axonal-like tracts were detected in the nasal septum on E12.5. GABAergic staining decreased by E13.5. To examine synaptic interactions of these GABAergic cells, embryonic olfactory explants were generated and maintained in serum-free media. As explants spread, neuron-like cells migrated into the periphery, sometimes forming ganglion-like clusters. Cells were recorded, marked intracellularly with Lucifer Yellow and post-fixation, immunocytochemically examined. Forty-six cells, typically multipolar, were GABAergic, had resting potentials around -50 mV, and exhibited spontaneous action potentials which were generated by spontaneous depolarizing GABAergic (GABAA) synaptic activity. OP neurons depolarized in response to GABA by increasing Cl- conductance. The biophysical properties of OP-derived GABAergic neurons were distinct from those reported for olfactory receptor neurons but similar to embryonic LHRH neurons. However, unlike LHRH neurons, GABAergic neurons did not migrate large distances in olfactory explants or appear to leave the olfactory pit in vivo.     

Granulocyte-macrophage colony stimulating factor suppresses LHRH release by inhibition of nitric oxide synthase and stimulation of gamma-aminobutyric acid release

Several cytokines produced by immune cells act within the hypothalamus and/or on the pituitary to produce the pattern of pituitary hormone secretion that characterizes infection. Granulocyte-macrophage colony stimulating factor (GMCSF) was first described as a hematopoietic cytokine; however, its synthesis is also stimulated during infection, and it has been found in glia in the brain. Previous research indicates that interleukin-1 inhibits release of luteinizing hormone-releasing hormone (LHRH) both in vivo and in vitro. In the present study, we determined that GMCSF inhibited the release of LHRH in vitro and evaluated the mechanisms involved. After a 1-hour preincubation in Krebs-Ringer bicarbonate glucose buffer (KRB), medial basal hypothalamic explants were incubated in KRB together with recombinant murine GMCSF for 0.5 h in a Dubnoff metabolic shaker (50 cycles/min) in an atmosphere of 95% O2/5% CO2. LHRH release into the media was determined by radioimmunoassay. At concentrations of 10(-12) and 10(-11) M, GMCSF significantly inhibited LHRH release. There was a U-shaped dose-response curve and LHRH release was not inhibited at lower or higher cytokine concentrations. The inhibition was specific since it was completely blocked by GMCSF antiserum. Since sodium nitroprusside (NP; 300 microM), a releaser of nitric oxide (NO), stimulates LHRH, presumably by acting within the LHRH neurons, we examined the effect of GMCSF (10(-11) M) on NP-induced LHRH release. It completely suppressed NP-induced release of LHRH. Bicuculline (10(-5) M), a gamma-aminobutyric acid (GABA) receptor antagonist, partially reversed the inhibitory effects of GMCSF on LHRH release. This dose completely reversed the suppression of LHRH release induced by GABA. The present results indicate that the inhibitory effects of GMCSF on LHRH release are partially caused by blockade of NO-induced LHRH release by its activation of GMCSF receptors on GABAergic neurons. The stimulated release of GABA acts on the GABA-a receptors on the LHRH terminals to inhibit their response to NO. At the end of the experiment, NO synthase (NOS) activity was measured in the tissue homogenate by the citrulline method. NOS activity was highly significantly reduced by GMCSF (10(-11) M) indicating that part of its suppressive action on LHRH release is mediated by reduction in NOS activity in the medial basal hypothalamus.     

Gene expression in a subpopulation of luteinizing hormone-releasing hormone (LHRH) neurons prior to the preovulatory gonadotropin surge

Gene expression in luteinizing hormone-releasing hormone (LHRH) neurons was analyzed during the periovulatory period to (1) characterize temporal patterns of LHRH gene expression and their relationship(s) to gonadotropin surges, and (2) determine if any such changes are uniform or dissimilar at different rostrocaudal levels of the basal forebrain. The number of neurons expressing mRNA for the decapeptide, and the relative degree of expression per cell were analyzed using in situ hybridization and quantitative image analysis. Rats were killed at 1800 hr on metestrus (Met), 0800 hr, 1200 hr, 1800 hr, and 2200 hr on proestrus (Pro), or 0200 hr, 0800 hr, and 1800 hr on estrus (E; n = 5-6 rats/group). All sections were processed for LHRH mRNA in a single in situ hybridization assay. Sections were atlas matched and divided into four rostrocaudal groups for analysis: vertical limb of the diagonal band of Broca (DBB), rostral preoptic area/organum vasculosum of the lamina terminalis (rPOA/OVLT), medial preoptic area (mPOA), and suprachiasmatic/anterior hypothalamic area (SCN/AHA). Plasma LH and FSH levels from all animals were analyzed by RIA. The labeling intensity per cell was similar among all time points at all four rostrocaudal levels. The number of cells expressing LHRH mRNA, however, varied as a function of time of death during the estrous cycle, and this temporal pattern varied among the four anatomical regions. At the level of the mPOA, the number of cells was highest at 1200 hr on Pro, and then declined and remained low throughout the morning of E. At the level of the rPOA/OVLT, the greatest number of LHRH neurons was noted later in Pro, at 1800 hr, dropping rapidly to lowest numbers at 2200 hr. No significant changes in LHRH cell number occurred at the DBB or SCN/AHA levels. At all anatomical levels, the secondary surge of FSH was unaccompanied by any change in the number of neurons expressing LHRH mRNA. These data demonstrate that (1) the number of detectable LHRH mRNA-expressing cells fluctuates during the periovulatory period and (2) peak numbers of LHRH-expressing cells are attained in the mPOA before the onset of the LH surge and before peak LHRH cell numbers are seen at more rostral levels. A model is proposed in which gene expression in this subpopulation of LHRH neurons may be activated by preovulatory estrogen secretion and acutely reduced following the proestrous surge of progesterone.     

Nitric oxide is involved in the genesis of pulsatile LHRH secretion from immortalized LHRH neurons

Neuronal networks controlling endocrine events present synchronous activity which is required for maintaining physiological functions, including reproduction. Although pulsatile hormone secretion is of paramount importance, the mechanism(s) by which secretory episodes are generated remain largely unknown. Nitric oxide (NO) has become the prototype of a new family of signaling molecules in the body. Nitric oxide diffuses from the source cell and controls activity of neighboring neurons as well as itself as a retrograde messenger. Cells of the luteinizing hormone-releasing hormone (LHRH) neuronal network, the key component in the control of reproduction, are scattered and loosely arranged in the anterior hypothalamus. A diffusible neurotransmitter could provide a means for establishing synchronous activation of the LHRH neuronal network leading to physiologically-relevant pulsatile LHRH secretion. In this study, we demonstrate that immortalized LHRH-producing neurons (GT1-7 cells) express NO synthase (NOS) mRNA and protein. Furthermore, GT1-7 cells are NADPH-diaphorase-positive (a marker of NOS activity) and the histochemical reaction can be abolished by treatment with a competitive NOS blocker. The presence of citrulline in these cells provides further evidence for the biological activity of NOS. These observations indicate that an active NO synthesizing machinery is present in immortalized LHRH neurons. In addition, we show that LHRH secretion is enhanced by NO in a cGMP-dependent manner. Since pulsatile LHRH secretion from immortalized LHRH neurons in vitro is abolished by NOS blockers and NO scavengers, it appears that NO is a unique neurotransmitter that is necessary to set LHRH neurons in phase to establish synchronized pulsatile LHRH secretion.     

Luteinizing hormone-releasing hormone gene expression differs in young and middle-aged females on the day of a steroid-induced LH surge

LHRH mRNA levels were examined in young and middle-aged female rats at 4 times (10:00 h, 14:00 h, 18:00 h and 20:00 h) on the day of a steroid-induced LH surge by in situ hybridization with a digoxigenin-labeled riboprobe. Young, but not middle-aged females, exhibited dynamic temporal changes in the number of LHRH mRNA positive neurons detected in the organum vasculosum of the lamina terminalis-preoptic area (OVLT-POA) continuum. Specifically, fewer LHRH mRNA positive neurons were detected at 18:00 h compared with the number detected at 14:00 h and 20:00 h (P < 0.01) in the OVLT-POA of young females. All LHRH mRNA positive neurons present in 4 anatomically matched sections through the rostral POA of young and middle-aged animals were digitized for detailed computer-assisted analysis of the hybridization reaction product. The mean hybridization area (P < 0.00025) and integrated optical density per cell (P < 0.006) were reduced in middle-aged compared to young females consistent with a relative age-related decline in LHRH mRNA levels. Moreover, an age-related reduction in cellular and/or regional hybridization area was noted at each of the time points examined (P < 0.05-P < 0.001). These data confirm earlier reports of dynamic changes in LHRH mRNA levels on the day of an LH surge. Furthermore, they support a role for age-related alterations in LHRH gene expression in the disruption of regular estrous cyclicity in middle-aged females.     

Central administration of antisense oligodeoxynucleotides to neuropeptide Y (NPY) mRNA reveals the critical role of newly synthesized NPY in regulation of LHRH release

Compelling evidence shows that the episodic and cyclic secretion of hypothalamic luteinizing hormone releasing hormone (LHRH), the primary stimulator of pituitary LH release, is subject to regulation by neuropeptide Y (NPY). We have reported earlier that sequential treatment of ovariectomized (ovx) rats with estrogen and progesterone to stimulate a preovulatory-type LH surge elevated the levels of both NPY and preproNPY mRNA levels in the hypothalamus concomitant with dynamic changes in LHRH activity. The present study was designed to determine whether these elevations in NPY content and gene expression represent new synthesis of NPY that is crucial to elicit LHRH discharge. Ovx, steroid-primed rats received intracerebroventricular injections of an unmodified 20-mer oligodeoxynucleotide (oligo) complementary to the NPY mRNA sequence. Control rats were injected similarly with either saline or the sense or missense oligos. Results showed that control rats displayed a characteristic surge-type elevation in plasma LH levels that was not affected by the administration of missense or sense oligos. However, in rats injected with the antisense oligo, the steroid-induced LH surge was completely blocked. In an additional experiment, NPY peptide levels were measured in microdissected hypothalamic sites following the injection of antisense or missense oligos. NPY antisense oligo administration blocked the significant increases in NPY levels in the median eminence-arcuate area, the medial preoptic area and lateral preoptic area seen in control rats. These results suggest that sequential ovarian steroid treatment augments NPY synthesis in the hypothalamus and this newly synthesized NPY is critical for induction of the LHRH and LH surge.     

Spatiotemporal appearance of developing LHRH neurons in the rat brain

To obtain insight into the development of the heterogeneous intracerebral populations of luteinizing hormone-releasing hormone (LHRH) neurons, their spatiotemporal appearance was examined at different stages in normal rat embryos, in nasal epithelial explants in vitro, and in intrauterine nasal-operated embryos. Following the appearance of nerve cell adhesion molecule in the nasal placode at embryonic day (E) 12.5, LHRH neurons, generated in the nasal placode at E13.5, penetrated the forebrain vesicle (FV) by E14.5-15.5. After E16.5, as the FV elongated to form the olfactory bulb, the migrating neurons traversed posteriorly through the interhemispheric space to penetrate the septopreoptic (S-P) area. By E18.5, LHRH neurons were detected in the preoptic-diagonal band (P-D) area as well as in the S-P region, along with some scattered extrahypothalamic LHRH neurons. To determine the source of these neurons, we separately cultured dissected parts of E12.5 nasal pit epithelium. Neuronal generation was predominantly from the medial wall epithelium (NAP), but some LHRH neurons originated in the roof epithelium. Cocultures of the NAP (E12.5) with the FV, median eminence-arcuate complex, Rathke's pouch, mesencephalon, or medulla oblongata from E14.5 embryos revealed the ability of LHRH cells to penetrate all of these tissues. Uni- or bilateral nasal destruction was conducted at E16.5 or E15.5, respectively, and examined at E18.5 and E21.5. In the operated embryos, most LHRH neurons were present in the P-D system and some in the S-P area. This finding suggests that the neurons generated before E15.5 are primarily predisposed to form the P-D system, whereas those derived afterward form the S-P system.     

Quantification of the GABA shunt and the importance of the GABA shunt versus the 2-oxoglutarate dehydrogenase pathway in GABAergic neurons

We investigated the activity of the cerebral GABA shunt relative to the overall cerebral tricarboxylic acid (TCA) cycle and the importance of the GABA shunt versus 2-oxoglutarate dehydrogenase for the conversion of 2-oxoglutarate into succinate in GABAergic neurons. Awake mice were dosed with [1-(13)C]glucose, and brain extracts were analyzed by 13C NMR spectroscopy. The percent enrichments of GABA C-2 and glutamate C-4 were the same: 5.0 +/- 1.6 and 5.1 +/- 0.2%, respectively (mean +/- SD). This, together with previous data, indicates that the flux through the GABA shunt relative to the overall cerebral TCA cycle flux equals the GABA/glutamate pool size ratio, which in the mouse is 17%. It has previously been shown that under the experimental conditions used in this study, the 13C labeling of aspartate from [1-(13)C]-glucose specifically reflects the metabolic activity of GABAergic neurons. In the present study, the reduction in the formation of [13C]aspartate during inhibition of the GABA shunt by gamma-vinyl-GABA indicated that not more than half the flux from 2-oxoglutarate to succinate in GABAergic neurons goes via the GABA shunt. Therefore, because fluxes through the GABA shunt and 2-oxoglutarate dehydrogenase in GABAergic neurons are approximately the same, the TCA cycle activity of GABAergic neurons could account for one-third of the overall cerebral TCA cycle activity in the mouse. Treatment with gamma-vinyl-GABA, which increased GABA levels dramatically, caused changes in the 13C labeling of glutamate and glutamine, which indicated a reduction in the transfer of glutamate from neurons to glia, implying reduced glutamatergic neurotransmission. In the most severely affected animals these alterations were associated with convulsions.     

Morphine amplifies norepinephrine (NE)-induced LH release but blocks NE-stimulated increases in LHRH mRNA levels: comparison of responses obtained in ovariectomized, estrogen-treated normal and androgen-sterilized rats

In these studies we examined the temporal effects of intracerebroventricular (i.c.v.) infusions of norepinephrine (NE) on plasma LH and on LHRH mRNA levels in the organum vasculosum of the lamina terminalis (OVLT) and in neurons located in the rostral (r), middle (m) and caudal (c) preoptic areas (POA) of ovariectomized, estrogen-treated rats. Thereafter, we compared these responses to those which occur in androgen-sterilized rats (ASR). NE infusions not only increased plasma LH concentrations but within 1 h after NE, LHRH mRNA levels also were increased significantly in the OVLT and rPOA but not in the mPOA or cPOA. By 4 h, these message levels still were elevated in the OVLT and rPOA and they now also were significantly higher than control values in the mPOA and cPOA. While NE also increased LH secretion in ASR, the plasma LH concentrations obtained were markedly blunted compared to control values. Moreover, NE infusions did not alter single cell levels of LHRH mRNA in any region of the rostral hypothalamus. Previously, we have reported that morphine (s.c.) markedly amplifies NE-induced LH release and questioned whether these responses are accompanied by concomitant augmented increases in LHRH mRNA levels. Morphine alone did not affect basal LHRH mRNA or plasma LH levels. However, when rats were pretreated with morphine (-15 min) and NE was infused i.c.v. at 0 time, significant amplification of LH release occurred but, unexpectedly, morphine completely blocked NE-induced increases in LHRH mRNA levels in all of the neurons we examined. Morphine also amplified LH release in ASR but these responses were significantly less than those obtained in control rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimisation of a heterogeneous non-competitive flow immunoassay comparing fluorescein, peroxidase and alkaline phosphatase as labels

GABA, somatostatin and enkephalin are neurotransmitters of enteric interneurons and comprise part of the intrinsic neural circuits regulating peristalsis. Within the relaxation phase of reflex peristalsis, nitric oxide (NO) is released by inhibitory motor neurons and perhaps enteric interneurons as well. Previously, we identified by GABA transaminase (GABA-T) immunohistochemistry, a subpopulation of GABAergic interneurons in the human colon which also contain NO synthase activity and hence produce NO. In this study, we have examined further the capacity for cotransmission within the GABAergic innervation in human colon. The expression of two important neuropeptides within GABAergic neurons was determined by combined double-labelled immunocytochemistry using antibodies for GABA-T, enkephalin and somatostatin, together with the demonstration of NO synthase-related NADPH diaphorase staining in cryosectioned colon. Both neuropeptides were found in GABAergic neurons of the colon. The evidence presented herein confirms the colocalization of NO synthase activity and GABA-T immunoreactivity in subpopulations of enteric neurons and further allows the neurochemical classification of GABAergic neurons of the human colon into three subsets: (i) neurons colocalizing somatostatin-like immunoreactivity representing about 40% of the GABAergic neurons, (ii) neurons colocalizing enkephalin-like immunoreactivity, about 9% of the GABAergic neurons and (iii) neurons colocalizing NO synthase activity, about 23% of the GABAergic neurons. This division of GABAergic interneurons into distinct subpopulations of neuropeptide or NO synthase containing cells is consistent with and provides an anatomical correlate for the pharmacology of these transmitters and the pattern of transmitter release during reflex peristalsis.     

Luteinizing hormone-releasing hormone quantified in tissues and slice explant cultures of postnatal rat hypothalami

LH-releasing hormone (LHRH) peptide from postnatal rat preoptic area (POA)/hypothalamic tissues in vivo and slice explant cultures maintained in vitro was quantitated using an enzyme-linked immunosorbant assay. Moreover, messenger RNA (mRNA) copy number was calculated in LHRH neurons maintained in culture using in situ hybridization histochemistry with autoradiographic film analysis. POA/hypothalami from postnatal day 5-6 pups averaged 1250 pg of LHRH, with approximately 28% of peptide residing within rostral tissues where most LHRH perikarya reside. Explant cultures maintained 18 days in vitro contained 30.4-92.0 pg/slice with a whole animal total of 244.8 pg. Considering cell numbers in vivo and in vitro, LHRH neurons in whole animal produce 1.0 pg of LHRH/cell, whereas those in culture average 2.0 pg/cell. Furthermore, LHRH mRNA copies/cell in organotypic culture was estimated conservatively at 1410 copies/cell, a relatively high number. This work shows that, compared with whole animal, cultures have substantial LHRH stores, indicating maturation of synthetic activity and/or formation of new terminals in vitro. High LHRH mRNA copy number also suggests a high rate of peptide biosynthesis. Our analysis, demonstrating the dynamic potential of LHRH neurons, suggests that subtle changes in LHRH mRNA expression in all cells or a subpopulation can dramatically alter the LHRH system biosynthetic capacity.     

Modulating mechanisms of neuroendocrine cell activity: the LHRH pulse generator

1. Luteinizing hormone-releasing hormone (LHRH), synthesized in specialized neurons in the hypothalamus, is the prime regulator of reproduction. In its absence, reproductive development is arrested and disorders of LHRH secretion result in several reproductive dysfunctions. 2. The LHRH neuronal network plays a paramount role in the regulatory loop controlling gonadal homeostasis. LHRH input to the pituitary gland maintains gonadotropin secretion, which, in turn, is responsible for gonadal trophism. Steroidal and peptidergic hormones from the gonad close the regulatory system by establishing negative (male and females) and positive (females) feedback loops. 3. Interestingly, LHRH input to the pituitary is intermittent rather than continuous. In fact, continuous exposure to LHRH results in paradoxical hypogonadism. Several studies in animals have provided direct evidence for episodic secretion of LHRH into the hypophyseal portal system. However, the nature of the system(s) responsible for the generation of the LHRH pulsatile profile is not currently known. The recent observation that immortalized LHRH neurons secrete LHRH in a pulsatile manner suggests that the pulse generating mechanism resides within the LHRH neuronal network. 4. In this overview, we compile several lines of evidence supporting this notion and put this characteristic of LHRH neurons in perspective with gonadal influences both internal and external to the LHRH neuronal network. Some recent data regarding the site of action of gonadal steroids on the LHRH neuronal system, the functional significance of galanin colocalization with LHRH, and the role of nitric oxide in the pulse generating mechanism are also discussed.     

Immunocytochemical distribution of LHRH neurons and processes in the rat: hypothalamic and extrahypothalamic locations

The distribution of luteinizing hormone-releasing hormone (LHRH)-immunoreactive perikarya and processes was examined, in the untreated rat, with the unlabeled antibody enzyme method of immunocytochemistry on thick 50 micron vibratome sections. LHRH neurons were primarily observed in the preoptico-anterior hypothalamic and septal areas. Projections from these cell bodies to the median eminence form three distinct pathways, one laterally along the course of the optic tracts, one medially through the periventricular stratum of the third ventricle, and one through the tractus infundibularis. In addition, some of these cell bodies project to the organum vasculosum of the lamina terminalis (OVLT) and the subfornical organ (SFO). LHRH immunoreactive neurons were also noted in the anterior olfactory regions; they project along the medial olfactory tract to the olfactory bulb.     

Vulnerability and plasticity of the GABA system in the pilocarpine model of spontaneous recurrent seizures

Several similarities exist between the alterations observed in the chronic pilocarpine model of recurrent seizures in the rat and those found in human temporal lobe epilepsy. The present studies are focused on changes in the GABA system in this model. Following the initial pilocarpine-induced seizures, a substantial loss of glutamic acid decarboxylase (GAD) mRNA-containing neurons has been found in the hilus of the dentate gyrus (Obenaus et al., J. Neurosci., 13 (1993) 4470-4485), and, recently, a loss of GAD mRNA-labeled neurons has also been found in stratum oriens of CA1. Yet numerous other GABA neurons remain within the hippocampal formation, and there appear to be multiple compensatory changes in these neurons. Labeling for GAD65 mRNA and associated protein is substantially increased in the remaining GABA neurons at 2-4 months after the initial seizure episode. Such increased labeling suggests that the remaining GABA neurons are part of a functional circuit and may be responding to the need for increased activity. Alterations also occur in at least one subunit of the GABA-A receptor. Labeling for the alpha(5) subunit mRNA is substantially decreased in CA1 and CA2 of pilocarpine-treated rats during the chronic, seizure-prone period. These findings emphasize the complexity of changes in the GABA system and indicate a need for evaluating the functional consequences of each of the changes. The initial loss of specific groups of GABA neurons could be a critical first step in the gradual development of epileptiform activity. While many of the subsequent changes in the GABA system may be considered to be compensatory, significant deficits of GABAergic function could remain.