Differential expression of gamma-aminobutyric acid type B receptor-1a and -1b mRNA variants in GABA and non-GABAergic neurons of the rat brain

To understand the heterogeneity of gamma-aminobutyric acid type B receptor (GABABR)-mediated events, we investigated expression of GABABR1a and 1b mRNA variants in GABA and non-GABAergic neurons of the rat central nervous system (CNS), by using nonradioactive in situ hybridization histochemistry and, in combination with GABA immunocytochemistry, double labeling. In situ hybridization with a pan probe, which recognizes a common sequence of both GABABR1a and GABABR1b mRNA variants, demonstrated widespread expression of GABABR1 mRNA at various levels in the CNS. Both GABABR1a and GABABR1b were expressed in the neocortex, hippocampus, dorsal thalamus, habenula, and septum, but only GABABR1a was detected in cerebellar granule cells, in caudate putamen, and most hindbrain structures. A majority of GABA neurons in cerebral cortex showed hybridization signals for both GABABR1a and GABABR1b, whereas those in most subcortical structures expressed either or neither of the two. GABA neurons in thalamic reticular nucleus and caudate putamen hybridized primarily for GABABR1a. Purkinje cells in the cerebellar cortex expressed predominantly GABABR1b. GABA neurons in dorsal lateral geniculate nucleus did not display significant levels of either GABABR1a or GABABR1b mRNAs. These data suggested widespread availability of GABABR-mediated inhibition in the CNS. The differential but overlapping expression of GABABR1 mRNA variants in different neurons and brain structures may contribute to the heterogeneity of GABABR-mediated inhibition. Some GABA neurons possessed, but others might lack the molecular machinery for GABABR-mediated disinhibition, autoinhibition, or both.     

FKBP12 mRNA expression is upregulated by intrinsic CNS neurons regenerating axons into peripheral nerve grafts in the brain

We have examined the expression of the immunophilin FKBP12 in adult rat intrinsic CNS neurons stimulated to regenerate axons by the implantation of segments of autologous tibial nerve into the thalamus or cerebellum. After survival times of 3 days to 6 weeks, the brains were fresh-frozen. In some animals the regenerating neurons were retrogradely labelled with cholera toxin subunit B 1 day before they were killed. Sections through the thalamus or cerebellum were used for in situ hybridization with digoxygenin-labelled riboprobes for FKBP12 or immunohistochemistry to detect cholera toxin subunit B-labelled neurons. FKBP12 was constitutively expressed by many neurons, and was very strongly expressed in the hippocampus and by Purkinje cells. Regenerating neurons were found in the thalamic reticular nucleus and deep cerebellar nuclei of animals that received living grafts. Neurons in these nuclei upregulated FKBP12 mRNA; such neurons were most numerous at 3 days post grafting but were most strongly labelled at 2 weeks post grafting. Regenerating neurons identified by retrograde labelling were found to have upregulated FKBP12 mRNA. No upregulation was seen in neurons in animals that received freeze-killed grafts, which do not support axonal regeneration. We conclude that FKBP12 is a regeneration-associated gene in intrinsic CNS neurons.     

Distribution and neurochemical characterization of neurons expressing GIRK channels in the rat brain

G-protein inwardly rectifying potassium (GIRK) channels mediate the synaptic actions of numerous neurotransmitters in the mammalian brain and play an important role in the regulation of neuronal excitability in most brain regions through activation of various G-protein-coupled receptors such as the serotonin 5-HT(1A) receptor. In this report we describe the localization of GIRK1, GIRK2, and GIRK3 subunits and 5-HT(1A) receptor in the rat brain, as assessed by immunohistochemistry and in situ hybridization. We also analyze the co-expression of GIRK subunits with the 5-HT(1A) receptor and cell markers of glutamatergic, gamma-aminobutyric acid (GABA)ergic, cholinergic, and serotonergic neurons in different brain areas by double-label in situ hybridization. The three GIRK subunits are widely distributed throughout the brain, with an overlapping expression in cerebral cortex, hippocampus, paraventricular nucleus, supraoptic nucleus, thalamic nuclei, pontine nuclei, and granular layer of the cerebellum. Double-labeling experiments show that GIRK subunits are present in most of the 5-HT(1A) receptor-expressing cells in hippocampus, cerebral cortex, septum, and dorsal raphe nucleus. Similarly, GIRK mRNA subunits are found in glutamatergic and GABAergic neurons in hippocampus, cerebral cortex, and thalamus, in cholinergic cells in the nucleus of vertical limb of the diagonal band, and in serotonergic cells in the dorsal raphe nucleus. These results provide a deeper knowledge of the distribution of GIRK channels in different cell subtypes in the rat brain and might help to elucidate their physiological roles and to evaluate their potential involvement in human diseases.     

Localization of GDNF/neurturin receptor (c-ret, GFRalpha-1 and alpha-2) mRNAs in postnatal rat brain: differential regional and temporal expression in hippocampus, cortex and cerebellum

Recent studies have identified a multi-component receptor system for the neurotrophic factor, glial cell line-derived neurotrophic factor (GDNF) and its homolog, neurturin (NTN), comprising the signaling tyrosine kinase, Ret and multiple GPI-linked binding proteins, GDNF family receptor alpha-1 and alpha-2 (GFRalpha-1 and GFRalpha-2). In the present study the localization of c-ret and GFRalpha-1 and GFRalpha-2 mRNAs was assessed in the developing rat brain from postnatal day 4 to 70 by in situ hybridization histochemistry, using specific [35S]-labeled oligonucleotides. GFRalpha-1 and GFRalpha-2 mRNAs were differentially distributed throughout the brain at all ages studied, particularly in cerebral cortex, hippocampus, substantia nigra and regions of the thalamus and hypothalamus - both distributions overlapping but different to that of c-ret mRNA. C-ret mRNA was abundant in areas such as the lateral habenula, reticular thalamic nucleus, substantia nigra pars compacta, cranial motor nuclei, and the Purkinje cell layer of the cerebellum. GFRalpha-1 mRNA was abundant in dorsal endopiriform nucleus, medial habenula, reticular thalamic nucleus, pyramidal and granule cell layers of the hippocampus, substantia nigra pars compacta and in cranial motor nuclei. GFRalpha-2 mRNA was highly expressed in many regions including olfactory bulb, lateral olfactory tract nucleus, neocortical layers IV and VI, septum, zona incerta, and arcuate and interpeduncular nuclei. GFRalpha-2 mRNA was detected in the pyramidal cell layers (CA3) of hippocampus at P4 and P7, but was no longer detectable at P14 and beyond, including P70 (adult). GFRalpha-2 mRNA was also detected in Purkinje cells throughout the cerebellum in young postnatal rats, but was enriched in the posterior lobes at P28 and P70. These localization studies support evidence of GDNF/NTN as target-derived and autocrine/paracrine trophic factors in developing brain pathways and earlier suggestions of unique and complex signaling mechanisms for these factors via a family of receptors. Strong expression of GFRalpha-1 and GFRalpha-2 mRNAs in adult brain suggests possible non-trophic functions of GDNF/NTN, as described for other neurotrophins, such as brain-derived neurotrophic factor.     

Localization of the mouse alpha1A-adrenergic receptor (AR) in the brain: alpha1AAR is expressed in neurons, GABAergic interneurons, and NG2 oligodendrocyte progenitors

alpha(1)-Adrenergic receptors (ARs) are not well defined in the central nervous system. The particular cell types and areas that express these receptors are uncertain because of the lack of high avidity antibodies and selective ligands. We have developed transgenic mice that either systemically overexpress the human alpha(1A)-AR subtype fused with the enhanced green fluorescent protein (EGFP) or express the EGFP protein alone under the control of the mouse alpha(1A)-AR promoter. We confirm our transgenic model against the alpha(1A)-AR knockout mouse, which expresses the LacZ gene in place of the coding region for the alpha(1A)-AR. By using these models, we have now determined cellular localization of the alpha(1A)-AR in the brain, at the protein level. The alpha(1A)-AR or the EGFP protein is expressed prominently in neuronal cells in the cerebral cortex, hippocampus, hypothalamus, midbrain, pontine olivary nuclei, trigeminal nuclei, cerebellum, and spinal cord. The types of neurons were diverse, and the alpha(1A)-AR colocalized with markers for glutamic acid decarboxylase (GAD), gamma-aminobutyric acid (GABA), and N-methyl-D-aspartate (NMDA) receptors. Recordings from alpha(1A)-AR EGFP-expressing cells in the stratum oriens of the hippocampal CA1 region confirmed that these cells were interneurons. We could not detect expression of the alpha(1A)-AR in mature astrocytes, oligodendrocytes, or cerebral blood vessels, but we could detect the alpha(1A)-AR in oligodendrocyte progenitors. We conclude that the alpha(1A)-AR is abundant in the brain, expressed in various types of neurons, and may regulate the function of oligodendrocyte progenitors, interneurons, GABA, and NMDA receptor containing neurons.     

Prenatal alteration and distribution of the GABA(B1) and GABA(B2) receptor subunit mRNAs during rat central nervous system development

The prenatal developmental expression changes and distribution of the gamma-aminobutyric acid (GABA)(B1) and GABA(B2) receptor subunit were investigated using in situ hybridization and RNase protection assay (RPA). We defined a different expression pattern of GABA(B1) subunit mRNA to that of GABA(B2) subunit. GABA(B1) subunit mRNA signals were moderately expressed in the cerebral cortex neuroepithelium of discrete brain regions on gestational day (GD) 11.5 and 12.5 and were highly expressed in the brain and spinal cord on GD 13.5 and 15.5. However, GABA(B2) subunit mRNAs were not detected on GD 11.5 and 12.5 and were first weakly detected on GD 13.5. On GD 15.5, 17.5, and 19.5, these subunit mRNAs were found in the retina, hippocampus, cerebral cortex, spinal cord, and cerebellum area. On GD 19.5 and 21.5, these subunits mRNA signals increased in the cerebral cortex, hippocampus, thalamus, and cerebellum, but decreased in the spinal cord, spinal ganglion, and midbrain, reaching similar levels to that of the adult brain. On GD 21.5, these subunit mRNAs were similarly expressed in almost all brain areas with a higher expression level of GABA(B1) subunit mRNA than GABA(B2) subunit mRNA. Our results found that GABA(B1) and GABA(B2) subunit mRNAs showed different expression patterns, with the GABA(B1) subunit expressed earlier and higher. We suggest that GABA(B1) and GABA(B2) subunits might have a role in the fetal brain during the gestational period for pre- and post-synaptogenesis, proliferation, differentiation, and neuronal maturation, and GABA(B1) subunit may be more important than GABA(B2) subunit during rat prenatal development.     

Distinct localization of GABA(B) receptors relative to synaptic sites in the rat cerebellum and ventrobasal thalamus

Metabotropic gamma-aminobutyric acid receptors (GABA(B)Rs) are involved in modulation of synaptic transmission and activity of cerebellar and thalamic neurons. We used subtype-specific antibodies in pre- and postembedding immunohistochemistry combined with three-dimensional reconstruction of labelled profiles and quantification of immunoparticles to reveal the subcellular distribution of pre- and postsynaptic GABA(B)R1a/b and GABA(B)R2 in the rat cerebellum and ventrobasal thalamus. GABA(B)R1a/b and R2 were extensively colocalized in most brain regions including the cerebellum and thalamus. In the cerebellum, immunoreactivity for both subtypes was prevalent in the molecular layer. The most intense immunoreactivity was found in Purkinje cell spines with a high density of immunoparticles at extrasynaptic sites peaking at around 240 nm from glutamatergic synapses between spines and parallel fibre varicosities. This is in contrast to dendrites at sites around GABAergic synapses where sparse and random distribution was found for both subtypes. In addition, more than one-tenth of the synaptic membrane specialization of spine-parallel fibre synapses were labelled at pre- or postsynaptic sites. Weak immunolabelling for both subtypes was also seen in parallel fibres but only rarely in GABAergic axons. In the ventrobasal thalamus, immunolabelling for both receptor subtypes was intense over the dendritic field of thalamocortical cells. Electron microscopy demonstrated an extrasynaptic localization of GABA(B)R1a/b and R2 exclusively in postsynaptic elements. Quantitative analysis further revealed the density of GABA(B)R1a/b around GABAergic synapses was higher than glutamatergic synapses on thalamocortical cell dendrites. The distinct localization of GABA(B)Rs relative to synaptic sites in the cerebellum and ventrobasal thalamus suggests that GABA(B)Rs differentially regulate activity of different neuronal populations.     

Differential expression of GABA and glutamate-receptor subunits and enzymes involved in GABA metabolism between electrophysiologically identified hippocampal CA1 pyramidal cells and interneurons

PURPOSE: The balance between synaptic excitation and inhibition within the hippocampus is critical for maintaining normal hippocampal function. Even mild reduction in inhibition or enhancement of excitation can produce seizures. Synaptic excitation is produced by pyramidal cells and granule cells, whereas inhibition is produced by a smaller number of interneurons. To understand how two subpopulations of these excitatory and inhibitory neurons are regulated at the molecular level, we analyzed specific mRNA expression profiles for receptors that are significantly involved in synaptic transmission and in the synthesis and storage of the principal inhibitory neurotransmitter, gamma-aminobutyric acid (GABA). Our hypothesis was that differences in gene expression between inhibitory and excitatory neurons in the rat hippocampus might point to specific new targets for seizure pharmacotherapy. METHODS: We combined the techniques of (a) whole-cell patch clamping in rat hippocampal slices, (b) biocytin staining for cell identification, (c) single-cell mRNA amplification, and (d) small-scale cDNA microarray analysis to allow us to obtain expression profiles for candidate genes from identified CA1 pyramidal neurons and interneurons. Electrophysiologic and morphologic data and expression profiles were obtained from 12 stratum pyramidale and seven stratum radiatum cells. RESULTS: Presumed inhibitory neurons expressed significantly more GAD65, GAD67, vGAT, GABA(A)-receptor alpha3, and N-methyl-d-aspartate (NMDA)-receptor IIB mRNA, and presumed excitatory neurons expressed more GABA(A)-receptor alpha1, and NMDA-receptor I mRNA. CONCLUSIONS: Differential expression of candidate neurotransmitter-receptor subunits distinguished CA1 pyramidal neurons from interneurons. These differences may indicate potential new targets for altering the balance of inhibition and excitation in the treatment of epilepsy.     

Nogo-receptor gene activity: cellular localization and developmental regulation of mRNA in mice and humans

Nogo (reticulon-4) is a myelin-associated protein that is expressed in three different splice variants, Nogo-A, Nogo-B, and Nogo-C. Nogo-A inhibits neurite regeneration in the central nervous system. Messenger RNA encoding Nogo is expressed in oligodendrocytes and central and peripheral neurons, but not in astrocytes or Schwann cells. Nogo is a transmembraneous protein; the extracellular domain is termed Nogo-66, and a Nogo-66-receptor (Nogo-R) has been identified. We performed in situ hybridization in human and mouse nervous tissues to map the cellular distribution of Nogo-R gene activity patterns in fetal and adult human spinal cord and sensory ganglia, adult human brain, and the nervous systems of developing and adult mice. In the human fetus Nogo-R was transcribed in the ventral horn of the spinal cord and in dorsal root ganglia. In adult human tissues Nogo-R gene activity was found in neocortex, hippocampus, amygdala, and a subset of large and medium-sized neurons of the dorsal root ganglia. Nogo-R mRNA was not expressed in the adult human spinal cord at detectable levels. In the fetal mouse, Nogo-R was diffusely expressed in brain, brainstem, trigeminal ganglion, spinal cord, and dorsal root ganglia at all stages. In the adult mouse strong Nogo-R mRNA expression was found in neurons in neocortex, hippocampus, amygdala, habenula, thalamic nuclei, brainstem, the granular cell layer of cerebellum, and the mitral cell layer of the olfactory bulb. Neurons in the adult mouse striatum, the medial septal nucleus, and spinal cord did not express Nogo-R mRNA at detectable levels. In summary, Nogo-66-R mRNA expression in humans and mice was observed in neurons of the developing nervous system Expression was downregulated in the adult spinal cord of both species, and specific expression patterns were seen in the adult brain.     

Galanin/GMAP- and NPY-like immunoreactivities in locus coeruleus and noradrenergic nerve terminals in the hippocampal formation and cortex with notes on the galanin-R1 and -R2 receptors

By using immunofluorescence methodology, extensive galanin (GAL) and GAL message-associated peptide (GMAP)-positive terminal networks were observed in the hippocampal formation. The majority of the GAL/GMAP fibers were dopamine β-hydroxylase- (DBH) positive, that is, they were noradrenergic. This finding was established with GAL/GMAP-DBH double-staining and with 6-hydroxy-dopamine treatment, which totally abolished all fibers in which GAL/GMAP and DBH coexisted. Also, reserpine treatment caused a marked depletion of GAL. No evidence for GAL/GMAP coexistence with 5-hydroxytryptamine was obtained. In the ventral hippocampus, GAL/GMAP-, DBH-negative fibers were seen in the stratum oriens, the anterior stratum radiatum, along the granule cell layer and in the strata oriens and alveus. In the locus coeruleus (LC), around 80% of the GMAP-positive neurons contained neuropeptide tyrosine (NPY), and about 40% of the NPY-positive neurons expressed GMAP. GAL-R1 receptor mRNA was expressed in Barrington's nucleus (close to the LC), but was not detected in the hippocampal formation/dorsal cortical areas. GAL-R2 receptor mRNA was found in the granule cell layer in the dentate gyrus. The present results show that most, but not all, immunohistochemically detectable GAL/GMAP in the hippocampal formation/dorsal cortex is present in noradrenergic nerve terminals originating in the LC, which has a robust GAL/GMAP synthesis. The functional role of GAL may be related to noradrenaline, possibly by a presynaptic action. However, the presence of GAL in other systems and of GAL-R2 receptor mRNA in granule cells also indicates other targets. J. Comp. Neurol. 392:227–251, 1998. © 1998 Wiley-Liss, Inc.     

An in situ hybridization study of the distribution of the GABA(B2) protein mRNA in the rat CNS.

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system. GABA exerts its actions through two classes of receptors: GABA(A), multimeric ligand-gated Cl(-) ion channels (a class which has been proposed to include the homomeric variant previously called GABA(C), to be designated GABA(A0r)); and GABA(B), G-protein coupled receptors which regulate Ca(2+) and K(+) channels. Currently, within the GABA(B) receptor family two proteins have been identified through molecular cloning techniques and designated GABA(B1) and GABA(B2). Two N-terminal variants of GABA(B1) were isolated and designated GABA(B1a) and GABA(B1b). The distribution of neurons in the rat CNS expressing the mRNA for the GABA(B1) isoforms have been previously described by in situ hybridization histochemistry. The recent isolation and identification of the GABA(B2) protein by homology cloning has enabled the use of radiolabeled oligonucleotides to detect the distribution of the expression of GABA(B2) mRNA in the rat CNS. The expression of GABA(B2) mRNA was observed to be primarily related to neuronal profiles. The highest levels of GABA(B2) mRNA expression were detected in the piriform cortex, hippocampus, and medial habenula. GABA(B2) mRNA was abundant in all layers of the cerebral cortex, the thalamus and in cerebellar Purkinje cells. Moderate expression was observed in several hypothalamic and brainstem nuclei. In contrast to the distribution of GABA(B1) mRNA, only a weak hybridization signal for GABA(B2) was detected over cells of the basal ganglia, including the caudate-putamen, nucleus accumbens, olfactory tubercle and throughout most of the hypothalamus. Moderate-to-heavy GABA(B2) mRNA expression was also seen over dorsal root and trigeminal ganglion cells. In general, the pattern of GABA(B2) mRNA expression in the rat brain overlaps considerably with the distributions described for both GABA(B1) mRNAs, and is concordant with the distribution described for GABA(B) receptor binding sites. However, differences between GABA(B2) expression levels and GABA(B) binding sites were observed in the basal ganglia.     

Heterogeneous output pathways link the anterior pretectal nucleus with the zona incerta and the thalamus in rat

The anterior pretectal nucleus (APT) and the zona incerta (ZI) are diencephalic nuclei that exert a strong inhibitory influence selectively in higher order thalamic relays. The APT is also known to project to the ZI as well as the thalamus, but anatomical details of the APT-ZI projection have not been described. In the present study, the efferent pathways of the APT were examined in the APT-ZI-thalamus network by using anterograde and retrograde tracing in combination with pre- and postembedding immunocytochemical stainings and in situ hybridization. The vast majority of APT fibers selectively innervated the parvalbumin-positive, ventral part of the ZI, which contains ZI neurons with axons projecting to higher order thalamic nuclei. The APT-ZI pathway consisted of both gamma-aminobutyric acid (GABA)-negative and GABA-positive components; 38.2% of the terminals in the ZI contained GABA, and 8.6% of the projecting somata in the APT were glutamic acid decarboxylase 67 (GAD67) mRNA positive. The combination of parvalbumin immunostaining with retrograde tracing showed that strongly and weakly parvalbumin-positive as well as parvalbumin-negative neurons were all among the population of APT cells projecting to the ZI. Similar heterogeneity was found among the APT cells projecting to the thalamus. Double retrograde tracing from higher order thalamic nuclei and their topographically matched ZI regions revealed hardly any APT neuron with dual projections. Our data suggest that both ZI and the higher order thalamic relays are innervated by distinct, physiologically heterogeneous APT neurons. These various efferent pathways probably interact via the rich recurrent collaterals of the projecting APT cells. Therefore, the powerful, GABAergic APT and ZI outputs to the thalamus are apparently co-modulated in a synergistic manner via dual excitatory and inhibitory APT-ZI connections.     

Localization of the Rev-ErbA orphan receptors in the brain

In the present study, we report the localization of the Rev-ErbAα and β nuclear orphan receptors, two closely related members of the nuclear hormone receptor superfamily, in the brain. Both Rev-ErbA variant mRNAs were highly expressed in the olfactory bulb, the hippocampus, and in the granular cells of the cerebellum, areas enriched also in other nuclear orphan receptors. Furthermore, the α-isoform was found in high amounts in the frontal cortex, the superficial gray layer of the superior colliculus, and the stria terminalis. Lower expression was observed in the nucleus accumbens, the caudate-putamen, and in some thalamic and brainstem nuclei. The β-variant, in contrast, was only moderately expressed in the cortex, mainly in the striate and retrosplenial cortices. In addition, moderate levels of Rev-ErbAβ mRNA were seen in various thalamic, pontine and brainstem nuclei. We conclude that the two Rev-ErbA isoforms share a partly similar pattern of expression in the brain, especially in areas that also contain other nuclear orphan receptors and that otherwise the localization of the two receptor subtypes is differential.     

Heterogeneous axonal arborizations of rat thalamic reticular neurons in the ventrobasal nucleus

The γ-aminobutyric acid (GABA)-containing neurons of the thalamic reticular nucleus (nRt) are a major source of inhibitory innervation in dorsal thalamic nuclei. Individual nRt neurons were intracellularly recorded and labelled in an in vitro rat thalamic slice preparation to investigate their projection into ventrobasal thalamic nuclei (VB). Camera lucida reconstructions of 37 neurons indicated that nRt innervation ranges from a compact, focal projection to a widespread, diffuse projection encompassing large areas of VB. The main axons of 65% of the cells gave rise to intra-nRt collaterals prior to leaving the nucleus and, once within VB, ramified into one of three branching patterns cluster, intermediate, and diffuse. The cluster arborization encompassed a focal region averaging approximately 25,000 μm² and contained a high density of axonal swellings, indicative of a topographic projection. The intermediate structure extended across an area approximately fourfold greater and also contained numerous axonal swellings. The diffuse arborization of nRt neurons covered a large region of VB and contained a relatively low density of axonal swellings. Analysis of somatic size and shape revealed that diffuse arborizations arose from significantly smaller, fusiform-shaped somata. Cytochrome oxidase reactivity or parvalbumin immunoreactivity was used to delineate a discontinuous staining pattern representing thalamic barreloids. The size of a cluster arborization closely approximated that of an individual barreloid. The heterogeneous arborizations from nRt neurons may reflect a dynamic range of inhibitory influences of nRt on dorsal thalamic activity. © 1996 Wiley-Liss, Inc.