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Recent studies suggest that metabotropic glutamate receptors (mGluRs) may play a significant role in regulating basal ganglia functions. In this study, we investigated the localization of mGluR4a protein in the mouse and rat basal ganglia. Polyclonal antibodies that specifically react with the metabotropic glutamate receptor subtype mGluR4a were produced and characterized by Western blot analysis. These antibodies recognized a native protein in wild-type mouse brain with a molecular weight similar to the molecular weight of the band from a mGluR4a-transfected cell line. The immunoreactivity was absent in brains of knockout mice deficient in mGluR4. mGluR4a immunoreactivity was most intense in the molecular layer of the cerebellum. We also found a striking mGluR4a immunoreactivity in globus pallidus, and moderate staining in substantia nigra pars reticulata and entopeduncular nucleus. Moderate to low mGluR4a immunoreactivity was present in striatum and other brain regions, including hippocampus, neocortex, and thalamus. Double labeling with mGluR4a antibodies and antibodies to either a dendritic marker or a marker of presynaptic terminals suggest a localization of mGluR4a on presynaptic terminals. Immunocytochemistry at electron microscopy level confirmed these results, revealing that in the globus pallidus, mGluR4a is mainly localized in presynaptic sites in axonal elements. Finally, quinolinic acid lesion of striatal projection neurons decreased mGluR4a immunoreactivity in globus pallidus, suggesting a localization of mGluR4a on striatopallidal terminals. These data support the hypothesis that mGluR4a serves as a presynaptic heteroreceptor in the globus pallidus, where it may play an important role in regulating g-amino-n-butyric acid (GABA) release from striatopallidal terminals.

Metabotropic glutamate receptors (mGluRs), which couple glutamate to second messengers, have important roles in the regulation of movement by the basal ganglia. We used two polyclonal antisera to mGluR1a and mGluR2/3 and confocal laser microscopy to investigate the localization of these receptors in the basal ganglia of the rat. The mGluRs were visualized in combination with an antibody to tyrosine hydroxylase (TH), an antibody to microtubule-associated protein 2 (MAP2, a dendritic marker), or SV2 (an antibody to a protein associated with presynaptic terminals). In the neostriatum, punctate mGluR1a immunoreactivity (ir) was present in the neuropil. This staining did not colocalize with MAP2-ir or SV2-ir and was not altered by decortication or unilateral 6-hydroxydopamine (6-OHDA) lesions. In the globus pallidus and substantia nigra pars reticulata, however, mGluR1a-ir was tightly clustered along large MAP2-ir dendrites. In contrast to the variations in mGluR1a-ir staining, similar punctate neuropil mGluR2/3-ir staining was observed within all basal ganglia structures. In the neostriatum, these puncta were abundant; unlike mGluR1a, many mGluR2/3-ir puncta colocalized with SV2-ir, and striatal mGluR2/3-ir puncta were markedly reduced in number after decortication. Neither mGluR1a-ir nor mGluR2/3-ir could be detected in TH-ir soma within substantia nigra pars compacta, or in TH-ir striatal terminals. Overall, our observations suggest that mGluR1a and mGluR2/3 receptors have distinct cellular localizations in different components of the basal ganglia circuitry and are likely to subserve distinct functions. Our data support the presence of mGluR2/3 on the terminals of corticostriatal afferents, where they may regulate glutamate release. In contrast, mGluR1a appears to be a postsynaptic receptor of neurons in the neostriatum, globus pallidus, and substantia nigra pars reticulata.

Glutamate is the principal excitatory transmitter of the mammalian brain and plays a particularly important role in the physiology of the basal ganglia structures responsible for movement regulation. Using in situ hybridization with oligonucleotide probes, we examined the expression patterns of the five known kainate type glutamate receptor subunit genes, KA1, KA2 and GluR5-7, in the basal ganglia of adult and developing rat brain. In the adult rat, a highly organized and selective pattern of expression of the kainate subunits was observed in the basal ganglia and associated structures as well as in other regions of the brain. KA2 mRNA was abundant in the striatum, nucleus accumbens, subthalamic nucleus and substantia nigra pars compacta, and was present at lower levels in the globus pallidus and substantia nigra pars reticulata. Neither KA1 nor GluR5 expression was observed in the basal ganglia of adult rats, although these messages were present in other regions. GluR6 was highly expressed in the striatum and subthalamic nucleus and to a lesser extent in the substantia nigra pars reticulata, while no hybridization signal was detectable in the large, presumably dopaminergic neurons of the substantia nigra pars compacta. In contrast, GluR7 was strongly expressed in the substantia nigra pars compacta, was present at lower levels in the striatum, globus pallidus and substantia nigra pars reticulata, and was not detectable in the subthalamic nucleus. During postnatal development, expression of the kainate receptor subunits was characteristically highest on postnatal day 1 and declined to adult levels by day 20; however, in the globus pallidus we did observe the transient expression of KA1 and GluR5 between day 1 and day 10. These results demonstrate that the neuronal structures comprising the basal ganglia express a distinct combination of kainate receptor subunit genes, suggesting that the pharmacological properties of the resultant glutamate receptors are likely to be regionally specific. The organization of expression of these genes is established early in life, which is consistent with the important role they may play in establishing the functions of the motor system.

N-Methyl-D-aspartate (NMDA) receptors are enriched in the neostriatum and are thought to mediate several actions of glutamate including neuronal excitability, long-term synaptic plasticity, and excitotoxic injury. NMDA receptors are assembled from several subunits (NMDAR1, NMDAR2A-D) encoded by five genes; alternative splicing gives rise to eight isoforms of subunit NMDAR1. We studied the expression of NMDA receptor subunits in neurochemically identified striatal neurons of adult rats by in situ hybridization histochemistry using a double-labeling technique. Enkephalin-positive projection neurons, somatostatin-positive interneurons, and cholinergic interneurons each have distinct NMDA receptor subunit phenotypes. Both populations of striatal interneurons examined express lower levels of NMDAR1 and NMDAR2B subunit mRNA than enkephalin-positive neurons. The three striatal cell populations differ also in the presence of markers for alternatively spliced regions of NMDAR1, suggesting that interneurons preferentially express NMDAR1 splice forms lacking one (cholinergic neurons) or both (somatostatin-positive neurons) alternatively spliced carboxy-terminal regions. In addition, somatostatin- and cholinergic-, but not enkephalin-positive neurons express NMDAR2D mRNA. Thus, these striatal cell populations express different NMDAR-subunit mRNA phenotypes and therefore are likely to display NMDA channels with distinct pharmacological and physiological properties. Differences in NMDA receptor expression may contribute to the relative resistance of striatal interneurons to the neurotoxic effect of NMDA receptor agonists.

Excitotoxic cell death is hypothesized to contribute to numerous neuropathologic conditions, including hypoxic/ischemic encephalopathy, hypoglycemia, Parkinson's disease, and Huntington's disease. Neuronal death from excitotoxic lesions has been shown to be an active process, with activation of immediate early gene transcription, resulting in secondary changes in gene expression. Another feature of neurotoxic cell death that has been examined is the presence of DNA fragmentation, which presumably indicates impending nuclear disintegration. A technique has been described for labeling fragmented DNA in situ, allowing precise determination of the anatomic and temporal distribution of neurons after an excitotoxic lesion. To investigate this phenomenon, we performed in situ nick translation on brain tissue from rats that have undergone stereotaxically placed intrastriatal quinolinic acid injections. Furthermore, in these same animals we analyzed the expression of c-fos mRNA to compare the time course and regional distribution of DNA fragmentation with immediate early gene activation after an excitotoxic lesion. Our analysis indicates that c-fos expression increases soon after quinolinic acid injection, is widespread in rat brain, but is effectively absent by 24 h postinjection. DNA fragmentation, however, is limited to striatum and is maximal at 24 h after injection. These results demonstrate the sensitivity of in situ nick translation for the detection of regional neuropathology and illustrate the temporal and spatial relationship of c-fos expression to excitotoxic neuronal death.

Metabotropic glutamate receptors (mGluRs) mediate the effects of glutamate neurotransmission on intracellular second messenger systems. Among the seven distinct mGluR receptor isoforms currently identified, the mGluR5 isoform is expressed particularly prominently in the striatum, where it may contribute to neuronal plasticity, motor behaviors, and excitotoxic injury. mGluR5 mRNA expression in striatal enkephalinergic, somatostatinergic, and cholinergic neurons was examined using double label in situ hybridization techniques. mGluR5 expression is abundant in a large number of medium-sized striatal cells but is absent in a significant minority of neurons. Double label in situ hybridization with 35S-dATP- and digoxygenin-dUTP-tailed oligonucleotide probes demonstrated that mGluR5 message is highly expressed by enkephalinergic striatal neurons but is not detectable in cholinergic or somatostatin interneurons. In addition, some nonenkephalin, presumably substance P, neurons were also strongly labeled for mGluR5. The differential expression of mGluR5 in striatal projection neurons vs. interneurons may contribute to the selective vulnerability of these neurons to disease processes.

The cerebral cortex is the primary source of glutamatergic afferents to the neostriatum. We used in situ hybridization to examine the effect of removal of the glutamatergic input to the striatum by unilateral frontal cortical ablation on the expression of genes encoding subunits from three families of glutamate receptors: N-methyl-D-aspartate receptors (NMDAR1, NMDAR2A, and NMDAR2B); alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors (GluR1-4, flip and flop splice variants); and metabotropic receptors (mGluR1-5). Significant changes were restricted to the dorsolateral quadrant of the ipsilateral striatum, the main projection area of the sensorimotor cortex. The expression of those messages which are normally abundant, NMDAR1, NMDAR2A, GluR1-4 flop and mGluR1, 3 and 5, was decreased in the deafferented dorsolateral striatum by 10-39% at 3 days after cortical ablation and subsequently increased to 120-165% of control at 15 and 60 days. mRNAs encoding the flip isoforms of GluR1-4, mGluR2 and 4, and an alternatively spliced region of NMDAR1 (Insertion I) which are undetectable or present at low levels in the striatum were not induced by cortical ablation. In contrast, both glial fibrillary acid protein and beta-actin mRNA expression were markedly enhanced at 3 and 15 days, returning to near normal at 60 days. Striatal NMDA, AMPA and metabotropic type 1 ligand binding sites were increased as early as 3 days after cortical ablation, reached a peak at 15 days and remained increased for up to 60 days, while metabotropic type 2 binding was slightly but significantly reduced at 3 and 15 days and [3H]kainate binding did not change significantly. These results demonstrate that cortical ablation, and subsequent loss of glutamatergic afferents to the striatum, results in alterations in the expression of genes encoding glutamate receptor subunits in striatal neurons. The regulation of these genes appears to be coordinate, so that the relative abundance of the different messages is preserved.

We examined NMDA-sensitive [3H]glutamate, [3H]AMPA, [3H]kainate and metabotropic-sensitive [3H]glutamate binding sites in neostriatum and substantia nigra pars reticulata (SNr) in rats after unilateral 6-hydroxydopamine lesions of the medial forebrain bundle. One week after the lesion, NMDA, AMPA, kainate and metabotropic receptors were decreased in the ipsilateral neostriatum, whereas at three months NMDA receptors were increased while AMPA, kainate and metabotropic receptors were not changed. In the SNr at one week, only AMPA and metabotropic receptors were significantly decreased whereas three months after the lesion NMDA, AMPA and kainate binding sites were decreased. The early decrease of excitatory amino acid receptors in the striatum is likely to reflect degeneration of dopaminergic fibers, suggesting that specific subpopulations of excitatory amino acid binding sites are located on dopaminergic terminals.

Glutamate is an important neurotransmitter in the circuitry of the basal ganglia. Of the four pharmacological classes of receptors that may mediate the actions of glutamate, the N-methyl-D-aspartate (NMDA) type is of particular interest insofar as it has been implicated in the neural processes underlying long-term synaptic plasticity as well as excitotoxic injury. NMDA ligand binding sites are abundant in the structures of the basal ganglia, and NMDA receptors have been linked to neuronal excitability, neuropeptide gene expression, and regulation of dopamine release in these regions. NMDA receptors are believed to be heterooligomers of subunits from two families: NMDAR1, encoded by a single gene but alternatively spliced to produce eight distinct isoforms (NMDAR1A-H), and NMDAR2, encoded by four separate genes (NMDAR2A-D). We have used in situ hybridization with a total of 13 oligonucleotide probes to examine the expression of these genes in the rat basal ganglia. NMDAR1 subunits are expressed throughout the basal ganglia as well as in the rest of the brain; however, the alternatively spliced amino-terminal region Insertion I is abundantly expressed only in the subthalamic nucleus and is not detectable in the neostriatum, globus pallidus, or substantia nigra pars compacta. In contrast, expression of the carboxy terminus segment Deletion I is prominent in the striatum but is not observed in other elements of the basal ganglia. NMDAR2 subunits also exhibit differential expression: NMDAR2B is abundant in the striatum, but NMDAR2A is present within the striatum only at low levels. NMDAR2C is present in the substantia nigra pars compacta only, while NMDAR2D exhibits an unusual distribution, with high levels of expression in the substantia nigra pars compacta, the subthalamic nucleus, the globus pallidus, and the ventral pallidum. Since each isoform of the NMDAR1 and NMDAR2 subunits can confer distinct properties on the resultant NMDA receptor, these data imply that there is a high degree of regional specialization in the properties of NMDA receptors within the basal ganglia.

Metabotropic glutamate receptors (mGluRs) couple the actions of glutamate to intracellular second messenger systems through G-proteins. The mGluRs play an important role in the regulation of basal ganglia function. Ligand binding studies have revealed that the basal ganglia contain at least two pharmacological types of metabotropic binding sites. Agonists of mGluRs can affect both in vitro electrophysiologic responses of striatal neurons and motor behavior in vivo. Recently, cDNAs encoding five mGluRs have been cloned, each with distinct structural and pharmacological properties. In order to elucidate the function of these receptors in the biology of the extrapyramidal motor system, we have used in situ hybridization to examine the regional and cellular expression patterns of mGluR1-mGluR5 in the adult rat basal ganglia. In the striatum, all of these mGluRs were present in widely varying relative densities and cellular patterns. MGluR5 was particularly prominent, and exhibited a heterogeneous cellular distribution, with labeled and unlabeled populations of neurons. MGluR2 was expressed in a small population of large polygonal striatal neurons. The subthalamic nucleus was the only other basal ganglia structure that expressed mGluR2. Distinct cellular distributions of mGluR expression were also observed within the nucleus accumbens, globus pallidus, ventral pallidum, and substantia nigra pars reticulata. MGluR3 was expressed in glia in all basal ganglia structures, but was observed in neurons only in the striatum, substantia nigra pars reticulata, and very weakly in the subthalamic nucleus. Comparison of the restricted mGluR2 and mGluR3 mRNA distributions with that of metabotropic ligand binding sites supports a possible presynaptic location for these receptors in the basal ganglia. MGluR1 was the only mGluR message prominently expressed in the dopaminergic neurons of the substantia nigra pars compacta, suggesting the involvement of this receptor in the regulation of dopamine release from nigrostriatal terminals.

Metabotropic glutamate receptors (mGluRs) have been implicated in a number of hippocampal functions including learning and memory. Five subtypes have been molecularly and pharmacologically characterized. Using in situ hybridization with oligonucleotide probes selective for these five mGluRs, we have found that each has a unique pattern of expression in the hippocampus and entorhinal cortex. mGluR1 is expressed predominantly in the dentate gyrus and CA3. mGluR2 is enriched in the dentate gyrus and inner layer of the entorhinal cortex. mGluR3 is also expressed in these two structures, but unlike all the other mGluRs, is found in white matter areas as well. mGluR4 is present predominantly in CA2 while mGluR5 is concentrated in most regions of the hippocampus and entorhinal cortex. Comparative analysis of the distributions of these receptors with that of the components of their putative downstream signal transduction mechanisms suggests that mGluR5 may be the main subtype of mGluR which mediates the excitatory actions of glutamate in CA1 and could contribute to the elevation of calcium levels found in CA1 pyramidal neurons in long term potentiation and in ischemic/hypoxic injury. mGluR2 and mGluR3, the main subtypes contributing to the inhibitory actions of glutamate, are absent in CA1. Thus, the mGluR-mediated excitatory actions of glutamate can occur in all regions of the hippocampus whereas the mGluR-mediated inhibitory actions of glutamate may be restricted to the dentate gyrus and CA3.

Spasmodic (spd) is a recessive mouse mutation characterized by a prolonged righting reflex, fine motor tremor, leg clasping, and stiffness. Using an intersubspecific backcross that segregates spd, we placed spd on Chr 11 with the following gene order: Adra-1-3.8 +/- 2.1 cM-Pad-1-6.3 +/- 2.7-(spd, Anx-6, Csfgm, Glr-1, Il-3, Il-4, Il-5, Sparc)-9.1 +/- 2.4-D11 Mit5-2.2 +/- 1.5-Asgr-1. This localization eliminated the alpha 1-adrenergic receptor (Adra-1) and the alpha 1 and gamma 2 subunits of the GABAA receptor as candidate genes. Two other promising candidate genes, annexin VI (Anx-6) and a glutamate receptor (Glr-1), were mapped to within 2.1 cM of the spd locus. Although no recombination was observed between spd and Anx-6 or Glr-1, no evidence was obtained for a lesion in either gene. The presence of normal Anx-6 and Glr-1 mRNA transcripts was confirmed by Northern blot analysis, in situ hybridization, and DNA sequence analysis. The localization of Anx-6 and Glr-1 extends the known synteny homology between human chromosome 5q21-q31 and mouse Chr 11 and reveals the probable chromosomal location of the human counterpart to spd. Synteny homology and phenotypic similarities suggest that spasmodic mice may be a genetic model for the inherited human startle disease, hyperekplexia (STHE).

In prior studies a heteroantiserum to a surface membrane component termed T(H2) was used to define two subsets of human T cells (T(H2) (+) and T(H2) (-)), which were found to express distinct sets of activities in vitro. In the present studies we prepared monoclonal antibodies to surface determinants that are restricted to T cells belonging to each of these two subsets. Two antibodies, termed alphaLeu-2a and alphaLeu-2b, which seem to define the same surface antigen identified by the original T(H2) antiserum, reacted with 57-84% of thymocytes and 22-46% of the erythrocyte-rosette-forming cells (ERF-C) in peripheral blood. Two other monoclonal antibodies, termed alphaLeu-3a and alphaLeu-3b, reacted with the same subpopulation of thymocytes (78-89%) and peripheral blood ERF-C (47-78%) but, unlike alphaLeu-2a and alphaLeu-2b, did not exhibit cross-blocking; i.e., labeling cells with alphaLeu-3a did not inhibit the subsequent binding of alphaLeu-3b. T cells reactive with alphaLeu-2a were shown to be unreactive with alphaLeu-3a, indicating that two separate subpopulations of T cells, Leu-2 (formerly T(H2) (+)) and Leu-3 (T(H2) (-)) T cells, were thereby defined. These two T cell subsets make up the subpopulation of ERF-C (80-95%) previously defined by a monoclonal antibody to a T cell membrane antigen (Leu-1) that has a thymus-dependent distribution on normal lymphocytes but is expressed by some surface-immunoglobulin-positive (sIg(+)) leukemic lymphocytes. None of the Leu antibodies reported here reacted with sIg(+), Leu-1(+) leukemic cells, nor did they react with normal hematopoietic cells or lymphoid cells that had surface markers characteristic of B cells. Studies of the blocking effects of Leu antibodies on killing in cell-mediated lympholysis by effector T cells were carried out in the absence of complement. These experiments established the following points: (i) alphaLeu-2a abolished the killing by cytotoxic T cells of allogeneic phytohemagglutinin-stimulated blasts, (ii) inhibition of killing by alphaLeu-2b was markedly less than inhibition by alphaLeu-2a, and (iii) other antibodies, including alphaLeu-1, alphaLeu-3a, and alphaLeu-3b, had little or no effect on killing in cell-mediated lympholysis. The relevance of these findings to prior studies done in the mouse and in man are discussed.