Distribution of vesicular glutamate transporter mRNA in rat hypothalamus

Two isoforms of the vesicular glutamate transporter, VGLUT1 and VGLUT2, were recently cloned and biophysically characterized. Both VGLUT1 and VGLUT2 specifically transport glutamate into synaptic vesicles, making them definitive markers for neurons using glutamate as a neurotransmitter. The present study takes advantage of the specificity of the vesicular transporters to afford the first detailed map of putative glutamatergic neurons in the rat hypothalamus. In situ hybridization analysis was used to map hypothalamic distributions of VGLUT1 and VGLUT2 mRNAs. VGLUT2 is clearly the predominant vesicular transporter mRNA found in the hypothalamus; rich expression can be documented in regions regulating energy balance (ventromedial hypothalamus), neuroendocrine function (preoptic nuclei), autonomic tone (posterior hypothalamus), and behavioral/homeostatic integration (lateral hypothalamus, mammillary nuclei). Expression of VGLUT1 is decidedly more circumspect and is confined to relatively weak labeling in lateral hypothalamic regions, neuroendocrine nuclei, and the suprachiasmatic nucleus. Importantly, dual-label analysis revealed no incidence of colocalization of VGLUT1 or VGLUT2 mRNAs in glutamic acid decarboxylase (GAD) 65-positive neurons, indicating that GABA neurons do not express either transporter. Our data support a major role for hypothalamic glutamatergic neurons in regulation of all aspects of hypothalamic function.     

Distribution of GAD-immunoreactive neurons in the diencephalon of the african lungfish Protopterus annectens: colocalization of GAD and NPY in the preoptic area

The distribution of GABAergic neurons was investigated in the diencephalon of the African lungfish, Protopterus annectens, by using specific antibodies directed against glutamic acid decarboxylase (GAD). A dense population of immunoreactive perikarya was observed in the periventricular preoptic nucleus, whereas the caudal hypothalamus and the dorsal thalamus contained only scattered positive cell bodies. Clusters of GAD-positive cells were found in the intermediate lobe of the pituitary. The diencephalon was richly innervated by GAD-immunoreactive fibers that were particularly abundant in the hypothalamus. In the periventricular nucleus, GAD-positive fibers exhibited a radial orientation, and a few neurons extended processes toward the third ventricle. More caudally, a dense bundle of GAD-immunoreactive fibers coursing along the ventral wall of the hypothalamus terminated into the median eminence and the neural lobe of the pituitary. Double-labeling immunocytochemistry revealed that GAD and neuropeptide tyrosine (NPY)-like immunoreactivity was colocalized in a subpopulation of perikarya in the periventricular preoptic nucleus. The proportion of neurons that coexpressed GAD and NPY was higher in the caudal region of the preoptic nucleus. The distribution of GAD-immunoreactive elements in the diencephalon and pituitary of the African lungfish indicates that GABA may act as a hypophysiotropic neurohormone in Dipnoans. The coexistence of GAD and NPY in a subset of neurons of the periventricular preoptic nucleus suggests that GABA and NPY may interact at the synaptic level.     

The spatial relationship of gamma-aminobutyric acid (GABA) neurons and gonadotropin-releasing hormone (GnRH) neurons in larval and adult sea lamprey,Petromyzon marinus

In this study we examined the spatial relationship of GABA-containing and GnRH-containing neurons by immunocytochemistry and in situ hybridization in larval and adult brains of sea lamprey, Petromyzon marinus. In immunocytochemical studies, GABA-containing neurons were detected early in lamprey development, by day 20 post-fertilization. At this time point, one population of GABA-containing neurons was visualized in the hypothalamus and preoptic area, and another population was located in the olfactory bulb of the telencephalon. By day 30 after fertilization, after the GABA neurons were detected, GnRH-containing neurons were visualized in the preoptic area/rostral hypothalamus region, adjacent to the GABA-containing neurons in the wall of the third ventricle. Similarly, in adult lamprey brains distinct populations of both GABA- and GnRH-containing neurons were located in the hypothalamus adjacent to the third ventricle. To further establish a proximate relationship between GABA and GnRH, the mRNA for glutamate decarboxylase (GAD), the enzyme catalyzing GABA synthesis from glutamate, and GnRH were examined by in situ hybridization in the brains of larval lamprey. These studies also showed that GnRH and GAD are produced in cell populations in and around the third ventricle of the hypothalamus. This close spatial relationship of GABA neurons and GnRH neurons provides a basis for a regulatory role of GABA on GnRH neurons in the sea lamprey.     

Comparative localization of mRNAs encoding two forms of glutamic acid decarboxylase with nonradioactive in situ hybridization methods

Nonradioactive in situ hybridization methods with digoxigenin-labeled cRNA probes were used to localize two glutamic acid decarboxylase (GAD) mRNAs in rat brain. These mRNAs encode two forms of GAD that both synthesize GABA but differ in a number of characteristics including their molecular size (65 and 67 kDa). For each GAD mRNA, discrete neuronal labeling with high cellular resolution and low background staining was obtained in most populations of known GABA neurons. In addition, the current method revealed differences in the intensity of labeling among neurons for each GAD mRNA, suggesting that the relative concentrations of each GAD mRNA may be higher in some groups of GABA neurons than in others. Most major classes of GABA neurons were labeled for each GAD mRNA. In some groups of GABA neurons, the labeling for the two mRNAs was virtually identical, as in the reticular nucleus of the thalamus. In other groups of neurons, although there was substantial labeling for each GAD mRNA, labeling for one of the mRNAs was noticeably stronger than for the other. In most brain regions, such as the cerebellar cortex, labeling for GAD67 mRNA was stronger than for GAD65 mRNA, but there were a few brain regions in which labeling for GAD65 mRNA was more pronounced, and these included some regions of the hypothalamus. Finally, some groups of GABA neurons were predominantly labeled for one of the GAD mRNAs and showed little or no detectable labeling for the other GAD mRNA, as, for example, in neurons of the tuberomammillary nucleus of the hypothalamus where labeling for GAD67 mRNA was very strong but no labeling for GAD65 mRNA was evident. The findings suggest that most classes of GABA neurons in the central nervous system (CNS) contain mRNAs for at least two forms of GAD, and thus, have dual enzyme systems for the synthesis of GABA. Higher levels of one or the other GAD mRNA in certain groups of GABA neurons may be related to differences in the functional properties of these neurons and their means of regulating GABA synthesis. © 1993 Wiley-Liss, Inc.     

Up-regulation of GAD65 and GAD67 in remaining hippocampal GABA neurons in a model of temporal lobe epilepsy.

In the pilocarpine model of chronic limbic seizures, subpopulations of glutamic acid decarboxylase (GAD)-containing neurons within the hilus of the dentate gyrus and stratum oriens of the CA1 hippocampal region are vulnerable to seizure-induced damage. However, many gamma-aminobutyric acid (GABA) neurons remain in these and other regions of the hippocampal formation. To determine whether long-term changes occur in the main metabolic pathway responsible for GABA synthesis in remaining GABA neurons, the levels of mRNA and protein labeling for the two forms of GAD (GAD65 and GAD67) were studied in pilocarpine-treated animals that had developed spontaneous seizures. Qualitative and semiquantitative analyses of nonradioactive in situ hybridization experiments demonstrated marked increases in the relative amounts of GAD65 and GAD67 mRNAs in remaining hippocampal GABA neurons. In addition, immunohistochemical studies demonstrated parallel increases in the intensity of terminal labeling for both GAD65 and GAD67 isoforms throughout the hippocampal formation. These increases were most striking for GAD65, the isoform of GAD that is particularly abundant in axon terminals. These findings demonstrate that, in a neuronal network that is capable of generating seizures, both GAD65 and GAD67 are up-regulated at the gene and protein levels in the remaining GABA neurons of the hippocampal formation. This study provides further evidence for the complexity of changes in the GABA system in this model of temporal lobe epilepsy.     

Sequence and expression of glutamic acid decarboxylase isoforms in the developing zebrafish

We describe the isolation two glutamic acid decarboxylase (GAD) cDNAs from zebrafish with over 84% identity to human GAD65 and GAD67. In situ hybridization studies revealed that both GAD65 and GAD67 were expressed in the early zebrafish embryo during the period of axonogenesis, suggesting a role for GABA prior to synapse formation. Both GAD genes were detected in the telencephalon, in the nucleus of the medial longitudinal fasciculus in the midbrain, and at the border regions of the rhombomeres in the rostral hindbrain. In the caudal hindbrain, only GAD67 was detected (in neurons with large-caliber axons). In the spinal cord, both GAD genes were detected in dorsal longitudinal neurons, commissural secondary ascending neurons, ventral longitudinal neurons, and Kolmer-Agduhr neurons. Immunohistochemistry for γ-aminobutyric acid (GABA) revealed that GABA is produced at all sites of GAD expression, including the novel cells in the caudal hindbrain. These results are discussed in the context of the hindbrain circuitry that supports the escape response. We conclude that fish, like mammals, have two GAD genes. The zebrafish GAD65 and GAD67 are present in identified neurons in the forebrain, midbrain, hindbrain, and spinal cord, and they catalyze the production of GABA in the developing embryo. J. Comp. Neurol. 396:253–266, 1998. © 1998 Wiley-Liss, Inc.     

The distribution of GABA-containing perikarya,fibers, and terminals in the forebrain and midbrain of pigeons,with particular reference to the basal ganglia and its projection targets

Immunohistochemical techniques were used to study the distributions of glutamic acid decarboxylase (GAD) and γ-aminobutyric acid (GABA) in pigeon forebrain and midbrain to determine the organization of GABAergic systems in these brain areas in birds. In the basal ganglia, numerous medium-sized neurons throughout the striatum were labeled for GABA, while pallidal neurons, as well as a small population of large, aspiny striatal neurons, labeled for GAD and GABA. GAD+ and GABA+ fibers and terminals were abundant throughout the basal ganglia, and GABAergic fibers were found in all extratelencephalic targets of the basal ganglia. Most of these targets also contained numerous GABAergic neurons. In pallial regions, approximately 10-12% of the neurons were GABAergic. The outer rind of the pallium was more intensely labeled for GABAergic fibers than the core. The olfactory tubercle region, the ventral pallidum, and the hypothalamus were extremely densely labeled for GABAergic fibers, while GABAergic neurons were unevenly distributed in the hypothalamus. GABAergic neurons and fibers were abundant in the dorsalmost part of thalamus and the dorsal geniculate region, while GABAergic neurons and fibers were sparse (or lightly labeled) in the thalamic nuclei rotundus, triangularis, and ovoidalis. Further, GABAergic neurons were abundant in the superficial tectal layers, the magnocellular isthmic nucleus, the inferior colliculus, the intercollicular region, the central gray, and the reticular formation. GABAergic fibers were particularly abundant in the superficial tectal layers, the parvocellular isthmic nucleus, the inferior colliculus, the intercol-licular region, the central gray, and the interpeduncular nucleus. These results suggest that GABA plays a role as a neurotransmitter in nearly all fore- and midbrain regions of birds, and in many instances the observed distributions of GABAergic neurons and fibers closely resemble the patterns seen in mammals, as well as in other vertebrates. © 1994 Wiley-Liss, Inc.     

Regulation of glutamic acid decarboxylase 65 and 67 gene expression by ovarian steroids: identification of two functionally distinct populations of GABA neurones in the preoptic area

GABA neurones in the preoptic area (POA) are critical for oestradiol (E2)-dependent surge release of luteinizing hormone (LH); however, it is not clear which population(s) of POA GABA neurones is involved. The goals of the present studies were: (i) to determine whether E2 regulates GABA neurones similarly in two subdivisions of the POA that play a role in LH surge release, the rostral POA region that contains the organum vasculosum of the lamina terminalis (rPOA/OVLT), and the region containing the anteroventral periventricular nucleus (AVPV) and medial preoptic nucleus (MPN) and (ii) to determine whether GABA neurones in either or both regions exhibit temporal changes consistent with a role in the regulation of LH surge release. To accomplish these goals, we measured glutamic acid decarboxylase (GAD) 65 and 67 mRNA levels at several time points in ovariectomized (OVX), E2-treated OVX rats exhibiting LH surge release, and in E2-treated OVX rats in which LH surge release was blocked by prior administration of progesterone (P4). Our findings demonstrate that, despite their close proximity, GABA neurones in the AVPV/MPN region are regulated differently from those in the rPOA/OVLT. Only neurones in the AVPV/MPN region show temporal changes in GAD 67 mRNA expression that appear to be linked to positive-feedback effects of E2 on luteinizing hormone-releasing hormone (LHRH) and LH release. Our findings also indicate that a morning rise and an afternoon fall in GAD 67 mRNA levels marks two E2-dependent signals required for LHRH and LH surge release. Finally, our results suggest that there are distinct E2-induced signals to the rPOA/OVLT and AVPV/MPN regions and that these signals differentially regulate GAD 65 and 67 gene expression.     

Immunohistochemical localization of glucocorticoid receptors in the forebrain of the rainbow trout (Oncorhynchus mykiss)

The distribution of glucocorticoid receptor-expressing cells was studied in the forebrain of the rainbow trout by means of antibodies produced against a fusion protein made of the NH₂-terminal fragment of the rainbow trout glucocorticoid receptor fused in frame with glutathione-S-transferase. The results indicate that glucocorticoid receptor-expressing cells are located in many brain regions from the telencephalon to the spinal cord, with the highest density in the neuroendocrine component of the brain, the preoptic region and the mediobasal hypothalamus, and in the periventricular zone of the optic tectum. In virtually all cases, the labeling was located in the nucleus of the cells, although on very rare occasions, a slight labeling of the cytoplasm was detected. Concerning the preoptic region, the most striking feature was the high density of glucocorticoid receptors in the magnocellular preoptic nucleus, known to contain corticotrophin-releasing factor (CRF)-, vasotocin-, and isotocin-expressing cells. Colocalization experiments showed that 100% of the CRF-immunoreactive neurons in the preoptic nucleus express glucocorticoid receptors. In the mediobasal hypothalamus, the highest expression was found in the nucleus lateralis tuberis and parts of the nucleus recessus lateralis. Concerning the pituitary, the glucocorticoid receptor was consistently found in the rostral pars distalis, with the exception of the prolactin cells, and in the proximal pars distalis, which in trout contains thyrotrophs, gonadotrophs, and somatotrophs. In the hindbrain, expression of glucocorticoid receptors were localized mainly in the periventricular regions. J. Comp. Neurol. 401:395–410, 1998. © 1998 Wiley-Liss, Inc.     

In situ characterization of gonadotropin- releasing hormone-I, -III, and glutamic acid decarboxylase expression in the brain of the sea lamprey, Petromyzon marinus

The distribution of lamprey gonadotropin-releasing hormone (GnRH)-I and -III has been extensively characterized by immunocytochemistry in the forebrain of the sea lamprey, Petromyzon marinus. However, the cellular location of lamprey GnRH-III mRNA expression by in situ hybridization in the lamprey brain has not been determined. We show for the first time the location of expression of lamprey GnRH-III, as well as provide a more comprehensive in situ study of lamprey GnRH-I and glutamic acid decarboxylase (GAD; GABA-synthesizing enzyme) mRNA expression in the brain of the lamprey in different reproductive life stages. Colorimetric and dual-label fluorescent amplification methods of in situ hybridization were used on brain tissue sections of adult, juvenile, and larval sea lamprey. In each life stage of the lamprey, expression of lamprey GnRH-I was shown in the preoptic area (POA) and the hypothalamus forming the characteristic arc-like cell population extending from the preoptic nucleus (NPO) to the neurohypophysis. Lamprey GnRH-III expression was also seen in the POA of each life stage in close proximity to lamprey GnRH-I mRNA containing neurons. GAD expression was shown in distinct cell clusters in and around the POA, in the olfactory bulb, in the dorsal thalamus beneath the habenular region, and also in the ventral-medial hypothalamus stretching from the periventricular region to the anterior portion of the rhombencephalon. Using dual-label in situ hybridization, we have shown that lamprey GnRH-I and -III mRNA are colocalized in the same cells in the POA in adult lampreys. Dual-label in situ hybridization also showed close proximity of GAD mRNA containing neurons and GnRH containing neurons in the POA. These data suggest that gamma-aminobutyric acid (GABA) may directly affect GnRH release in the brain of the sea lamprey.     

Hypothalamic control of sleep

A sleep-promoting function for the rostral hypothalamus was initially inferred from the presence of chronic insomnia following damage to this brain region. Subsequently, it was determined that a unique feature of the preoptic hypothalamus and adjacent basal forebrain is the presence of neurons that are activated during sleep compared to waking. Preoptic area "sleep-active" neurons have been identified by single and multiple-unit recordings and by the presence of the protein product of the c-Fos gene in the neurons of sleeping animals. Sleep-active neurons are located in several subregions of the preoptic area, occurring with high density in the ventrolateral preoptic area (vlPOA) and the median preoptic nucleus (MnPN). Neurons in the vlPOA contain the inhibitory neuromodulator, galanin, and the inhibitory neurotransmitter, GABA. A majority of MnPN neurons activated during sleep contain GABA. Anatomical tracer studies reveal projections from the vlPOA and MnPN to multiple arousal-regulatory systems in the posterior and lateral hypothalamus and the rostral brainstem. Cumulative evidence indicates that preoptic area neurons function to promote sleep onset and sleep maintenance by inhibitory modulation of multiple arousal systems. Recent studies suggest a role for preoptic area neurons in the homeostatic aspects of the regulation of both rapid eye movement (REM) and non-REM (NREM) sleep and as a potential target for endogenous somnongens, such as cytokines and adenosine.     

Oestrogen, progesterone and serotonin converge on GABAergic neurones in the monkey hypothalamus

There are dense populations of inhibitory GABA neurones in regions of the primate hypothalamus that have been implicated in the neuroendocrine control of prolactin and luteinizing hormone (LH) secretion. A subpopulation of GABA neurones that express nuclear oestrogen and progestin receptors reside in the arcuate and infundibular nuclei. We questioned whether oestrogen or progesterone regulate the expression of GAD67, the rate limiting enzyme in GABA synthesis, in these regions. Female monkeys were spayed and treated with placebo, oestrogen, progesterone or oestrogen plus progesterone for 28 days and GAD67 mRNA was examined with single in situ hybridization. In the arcuate nucleus, there was no change in GAD67 mRNA expression with hormone treatment. However, in the infundibular region, oestrogen alone and oestrogen plus progesterone significantly suppressed GAD67 mRNA expression compared to spayed controls. In addition, expression of serotonin (5-HT)2C receptor mRNA overlaps markedly with the expression of GAD67 mRNA in the same region. We tested the hypothesis that GABA neurones express 5-HT2C receptors using double in situ hybridization. The highest concentrations of double-labelled cells were detected in the medial preoptic region, the arcuate nucleus and the infundibular region. The suprachiasmatic and ventromedial nuclei contained predominantly 5-HT2C mRNA expressing cells. The nucleus of the diagonal band of Broca and the globus pallidus contained predominantly GAD67 mRNA expressing cells. The bed nucleus of the stria terminalis, the paraventricular and dorsomedial nuclei contained different ratios of single-labelled cells. Together these data suggest (i) that oestrogen decreases expression of GAD67 mRNA in the infundibular region which could lead to decreased GABA synthesis, but addition of progesterone had no further effect and (ii) that GABA neurones in the same region also express mRNA for the stimulatory 5-HT2C receptor which could promote GABA release during serotonin input.     

Regulation of forebrain GABAergic stress circuits following lesion of the ventral subiculum

Ventral subiculum (vSUB) lesions enhance corticosterone responses to psychogenic stressors via trans-synaptic influences on paraventricular nucleus (PVN) neurons. Synaptic relays likely occur in GABA-rich regions interconnecting the vSUB and PVN. The current study examines whether vSUB lesions compromise stress-induced c-fos induction and GABA biosynthetic capacity in putative limbic-hypothalamic stress relays. Male Sprague-Dawley rats received bilateral ibotenate or sham lesions of the vSUB. Animals were divided into two groups, with one group receiving exposure to novelty stress and the other left unstressed. Exposure to novelty stress increased c-fos mRNA expression in the PVN to a greater degree in vSUB lesion relative to shams, consistent with an inhibitory role for the vSUB in the HPA stress response. However, c-fos induction was not affected in other forebrain GABAergic stress pathways, such as the lateral septum, medial preoptic area or dorsomedial hypothalamus. vSUB lesions increased GAD65 or GAD67 mRNA levels in several efferent targets, including anterior and posterior subnuclei of the bed nucleus of the stria terminalis and lateral septum. Lesions did not effect stress-induced increases in GAD65 expression in principal output nuclei of the amygdala. The current data suggest that loss of vSUB innervations produces a compensatory increase in GAD expression in subcortical targets; however, this up-regulation is insufficient to block lesion-induced stress hyperresponsiveness, perhaps driven by amygdalar disinhibition of the PVN.     

Localization of mRNAs encoding two forms of glutamic acid decarboxylase in the rat hippocampal formation

The mRNAs for two forms of glutamic acid decarboxylase (GAD65 and GAD67) were localized in the rat hippocampal formation by nonradioactive in situ hybridization methods with digoxigeninlabeled cRNA probes. Some neurons in all layers of the hippocampus and dentate gyrus were readily labeled for each GAD mRNA, and the patterns of labeling for GAD65 and GAD67 mRNAs were very similar. All major groups of previously described GAD-and GABA-containing neurons appeared to be labeled for each GAD mRNA. Such findings suggest that most GABA neurons in the hippocampal formation contain both GAD mRNAs. When the labeling of neurons in the hippocampal formation and cerebral cortex was compared in the same sections, the intensity of neuronal labeling for GAD67 mRNA was generally similar in the two regions. However, the intensity of labeling for GAD65 mRNA was generally stronger for many neurons in the hippocampal formation than for most neurons in the cerebral cortex. Neurons in the hilus of the dentate gyrus were particularly well labeled for GAD65. The nonradioactive labeling for the GAD mRNAs was confined to the cytoplasm of neuronal cell bodies, and this allowed a clear visualization of the relative number and location of labeled neurons. Several distinct patterns of GAD mRNA-containing neurons were observed among different regions of the hippocampal formation. In the hilus of the dentate gyrus, GAD mRNA-containing neurons were numerous in the regions deep to the granule cell layer as well as in more central parts of the hilus. Within CA3, the densities (quantities) of labeled neurons varied among the regions. In the inner or hilar segment of CA3, the density of labeled neurons was often lower than that in the outer part of CA3 where numerous labeled neurons were distributed throughout all layers. In CA1, GAD mRNA-labeled neurons were distributed in a relatively laminar pattern with the highest density in stratum pyramidale and moderate densities in stratum oriens and at the interface between strata radiatum and lacunosum-moleculare. Lower densities were found within the latter two layers. The prominent localization of the two GAD mRNAs in the hippocampal formation suggests that dual system for GABA synthesis is necessary for normal GABAergic function in this brain region. Most putative GABA neurons contain relatively high levels of GAD67 mRNA as might be expected if this GAD form is responsible for the synthesis of GABA for metabolic and baseline synaptic function. The relatively high levels of GAD65 mRNA in many hippocampal neurons, particularly those of the dentate hilus, may indicate that these neurons have a well-developed reserve system for GAD and GABA synthesis. © 1994 Wiley-Liss, Inc.     

Forebrain GABAergic projections from the dorsal raphe nucleus identified by using GAD67–GFP knock‐in mice

The dorsal raphe nucleus (DR) contains serotonergic (5‐HT) neurons that project widely throughout the forebrain. These forebrain regions also receive innervation from non–5‐HT neurons in the DR. One of the main groups of non–5‐HT neurons in the DR is γ‐aminobutyric acid (GABA)ergic, but their projections are poorly understood due to the difficulty of labeling these neurons immunohistochemically. To identify GABAergic projection neurons within the DR in the current study, we used a knock‐in mouse line in which expression of green fluorescent protein (GFP) is controlled by the glutamic acid decarboxylase (GAD)67 promotor. Projections of GAD67–GFP neurons to the prefrontal cortex (PFC), nucleus accumbens (NAC), and lateral hypothalamus (LH) were evaluated by using retrograde tract tracing. The location of GAD67–GFP neurons projecting to each of these areas was mapped by rostrocaudal and dorsoventral location within the DR. Overall, 16% of DR neurons projecting to either the PFC or NAC were identified as GAD67–GFP neurons. GAD67–GFP neurons projecting to the PFC were most commonly found ventrally, in the rostral two‐thirds of the DR. NAC‐projecting GAD67–GFP neurons had an overlapping distribution that extended dorsally. GAD67–GFP neurons made a larger contribution to the projection of the DR to the LH, accounting for 36% of retrogradely labeled neurons, and were widespread throughout the DR. The current data indicate that DR GABAergic neurons not only may have the capacity to influence local network activity, but also make a notable contribution to DR output to multiple forebrain targets. J. Comp. Neurol. 520:4157–4167, 2012. © 2012 Wiley Periodicals, Inc.     

Biphasic Response of Spinal GABAergic Neurons after a Lumbar Rhizotomy in the Adult Rat

The expression of γ-aminobutyric acid (GABA) and of the isoforms of the enzyme involved in its synthesis, glutamic acid decarboxylase (GAD), is modified in several rat brain structures in different injury models. The aim of the present work was to determine whether such plasticity of the GABAergic system also occurred in the deafferented adult rat spinal cord, a model where a major reorganization of neural circuits takes place. GABAergic expression following unilateral dorsal rhizotomy was studied by means of non-radioactive in situ hybridization to detect GADs₆₇ mRNA and by immunohistochemistry to detect GAD₆₇ protein and GABA. Three days following rhizotomy the number of GAD₆₇ mRNA-expressing neurons was decreased in the superficial layers of the deafferented horn, while GABA immunostaining of axonal fibres located in this region was highly increased. Seven days after lesion, on the other hand, many GAD₆₇ mRNA-expressing neurons were bilaterally detected in deep dorsal and ventral layers, this expression being correlated with the increased detection of GADs₆₇ immunostained somata and with the reduction of GABA immunostaining of axons. GABA immunostaining was frequently found to be associated with reactive astrocytes that exhibited intense immunostaining for glial fibrillary acidic protein (GFAP) but remained GADs₆₇ negative. These results indicate that degeneration of afferent terminals induces a biphasic response of GABAergic spinal neurons located in the dorsal horn and show that many spinal neurons located in deeper regions re-express GAD₆₇, suggesting a possible participation of the local GABAergic system in the reorganization of disturbed spinal networks.     

Differential regulation of forebrain glutamic acid decarboxylase mRNA expression by aging and stress

In aging brain, degeneration or functional impairment of the hippocampus has been connected with stress dysregulation, serving to disinhibit stress responses and allow for glucocorticoid hypersecretion and its attendant pathophysiology. Hippocampal dysfunction appears to be communicated to paraventricular hypothalamic corticotropin-releasing hormone neurons by way of subcortical GABAergic neurons. As such, hippocampal-hypothalamic relays are likely to play an important role in age-related stress dysfunction. To test this hypothesis, regulation of glutamic acid decarboxylase isoform mRNA was studied in young (3 months), middle aged (15 months) and aged (30 months) Fischer 344/Brown Norway F1 hybrid rats. Basal expression of glutamic acid decarboxylase (GAD) 65 mRNA was increased in the medial preoptic area and posteromedial bed nucleus of the stria terminalis (BST) in aged rats relative to both middle-aged and young groups. Unlike young or middle-aged animals, exposure to chronic intermittent stress decreased GAD65 mRNA levels in the medial preoptic area and posteromedial BST of aged rats. Thus, while aged rats show evidence of elevated basal GABA synthesis, chronic stress causes differential loss of GAD in hippocampal-PVN relays, consistent with reduced PVN inhibition.     

Codistribution of GABA- with acetylcholine-synthesizing neurons in the basal forebrain of the rat

In recent years, GABAergic neurons have been identified in the basal forebrain where cholinergic cortically projecting neurons are located and known to be important in mechanisms of cortical activation. In the present study in the rat, the relationship of the GABA-synthesizing neurons to the acetylcholine-synthesizing neurons was examined by application of a sequential double staining immunohistochemical procedure involving the peroxidase-antiperoxidase technique for glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT). In these double and adjacent single immunostained series of sections, the GAD+ and ChAT+ cells were mapped, counted and measured with the aid of a computerized image analysis system. Through the entire basal forebrain, there was no evidence for colocalization of GAD and ChAT in the same neurons. Instead, a large population of GAD-immunoreactive neurons is codistributed with ChAT-immunoreactive neurons and outnumbers them by a factor of two: approximately 39,000 GAD+ cells to 18,000 ChAT+ cells. Although the GAD+ and ChAT+ neurons lie intermingled within fascicles of the major longitudinal and transverse forebrain fiber systems in subregions of the basal forebrain, the GAD+ cells are more highly concentrated within different sectors of the pathways and regions than the ChAT+ cells. Although GAD+ neurons resemble ChAT+ neurons in certain regions, both being bi- or multipolar and, on average, medium-sized cells, the GAD+ neurons are, in the majority (51%), small-sized cells ( < 15 μm in length) and as a population significantly smaller than the ChAT+ neurons. These results suggest that many GABAergic neurons may represent interneurons in the basal forebrain and potentially exert an inhibitory influence on adjacent cortically projecting cholinergic neurons. Medium- to large GAD+ cells, which resemble similar ChAT+ cells, are also present and represent the majority of the GAD+ cells in the nucleus of the diagonal band of Broca, magnocellular preoptic nucleus, and olfactory tubercle, but represent the minority in the anterior and posterior substantia innominata and globus pallidus. Given their prominent size, such GABAergic cells may also exert an inhibitory influence outside the basal forebrain as projection neurons and potentially in parallel with cholinergic neurons, to certain regions of the cerebral cortex. Accordingly, GABAergic cells may be considered as constituents of the magnocellular basal nucleus and potentially important elements within the ventral extrathalamic relay from the brainstem reticular formation to the cerebral cortex. © 1993 Wiley-Liss, Inc.     

Cells containing immunoreactive estrogen receptor-alpha in the human basal forebrain

The distribution of estrogen receptor protein-alpha (ER-alpha)-containing cells in the human hypothalamus and adjacent regions was studied using a monoclonal antibody (H222) raised against ER-alpha derived from MCF-7 human breast cancer cells. Reaction product was found in restricted populations of neurons and astrocyte-like cells. Neurons immunoreactive for ER-alpha were diffusely distributed within the basal forebrain and preoptic area, infundibular region, central hypothalamus, basal ganglia and amygdala. Immunoreactive astrocyte-like cells were noted within specific brain regions, including the lamina terminalis and subependymal peri-third-ventricular region. These data are consistent with the location of estrogen receptors in the basal forebrain of other species and the known effects of estrogens on the cellular functions of both neurons and supporting elements within the human hypothalamus and basal forebrain.     

Ventrally located commissural neurons express the GABAergic phenotype in developing rat spinal cord.

Early-forming commissural neurons are studied intensively as a model of axonal outgrowth and pathfinding, yet the neurotransmitter phenotype of the majority of these neurons is not known. The present study has determined that a substantial number of commissural neurons express the 65-kDa isoform of glutamic acid decarboxylase (GAD65) as early as embryonic day 12 (E 12). Patterns of GAD65 localization were compared with those of TAG-1, the Transiently expressed Axonal Glycoprotein that is the best known marker of commissural axons. On E13, both GAD65- and TAG-1-labeled commissural axons emanate from similar lateral and ventromedial regions. However, dorsally located TAG-1-positive commissural axons were GAD65-negative. These results suggest that commissural neurons have both gamma-aminobutyric acid (GABA)ergic and non-GABAergic phenotypes. The intensity of GAD65 staining within commissural somata and axons decreased between E14-15 and continued to decline during embryonic development, whereas terminal-like structures in surrounding neuropil increased dramatically. This sudden loss of somatic and axonal GAD65 staining was unexpected and could be interpreted as commissural neurons only transiently expressing the GABAergic phenotype. Further experiments were undertaken to identify commissural neurons with other established GABAergic markers, GAD67 and GABA. When antibody labeling of the two GAD isoforms was compared, GAD67 was detected 1 day later than GAD65, and in a different subcellular distribution. In contrast to GAD65, GAD67 intensely stained somata but labeled few commissural axons. GABA immunoreactivity also was detected in commissural axons 1 day after GAD65, and the labeling pattern between E13 and E16 resembled that of GAD67 rather than GAD65. When GAD and GABA results were compared, it was clear that a number of ventrally located commissural neurons expressed and maintained the GABAergic phenotype during embryonic development. However, the early expression and subcellular redistribution of GAD65 suggests that the GAD isoforms are differentially regulated. The function of the transient GAD65 expression in commissural somata and axons is unknown, but its temporal expression pattern parallels the transient expression of TAG-1, as both are expressed during the early stages of commissural axon outgrowth and pathfinding.