Glutamate receptor genes in Parkinson's disease.
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Biomedical subjects
A B Young
Publications and source records attributed to A B Young.
Differential expression of mGluR5 metabotropic glutamate receptor mRNA by rat striatal neurons.
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.
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DNA fragmentation and immediate early gene expression in rat striatum following quinolinic acid administration.
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.
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Evidence for a preferential loss of enkephalin immunoreactivity in the external globus pallidus in low grade Huntington's disease using high resolution image analysis.
Previous studies have shown that in advanced cases of Huntington's disease, enkephalin-immunoreactive striatal projections to the external globus pallidus may be more affected than substance P-containing striatal projections to the inner segment of the pallidum [Reiner A. et al. (1988) Proc. natn. Acad. Sci. U.S.A. 85, 5733-5737]. Other immunohistochemical [Ferrante R. J. et al. (1990) Soc. Neurosci. Abstr. 16, 1120] and neurochemical observations [Storey E. and Beal M.F. (1993) Brain 116, 1201-1222] suggest no difference in the loss of these peptide-containing pathways in Huntington's disease. In view of the potential significance of this issue for understanding the neuropathological process in Huntington's disease, we examined the globus pallidus in control and Huntington's disease brains, using a quantitative approach which involved high resolution image analysis of 7 microns frozen sections to determine the overall density of peptide-immunoreactive terminals. Results showed that in the controls there was no significant difference between the density of enkephalin- and substance P-immunoreactive terminals in the external and internal globus pallidus, respectively. In all Huntington's disease brains, including grade 1 cases, enkephalin-immunoreactive terminals in the external globus pallidus were significantly reduced compared to substance P-positive boutons in the internal segment of the adjacent section. In comparison to controls, enkephalin immunoreactivity in all Huntington's disease cases was significantly lower; substance P-immunoreactive terminals in the internal globus pallidus were significantly lower than controls in some of the grade 2 cases and in the grade 3 cases.(ABSTRACT TRUNCATED AT 250 WORDS)
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Detectability of early brain meningitis with magnetic resonance imaging.
RATIONALE AND OBJECTIVES: The ability of high-field (1.5 T) magnetic resonance imaging (MRI) to detect early brain meningitis was evaluated in a canine model. Contrast dose, timing postinjection, and imaging technique (specifically the use of magnetization transfer) were assessed. METHODS: Imaging of five canines was performed at 1.5 T 24 hours after injection of Cowans staphylococcus into the cisterna magna. Two control animals also were imaged using the same protocol, with one animal receiving a cisternal injection of nutrient broth only and the other no injection. Contrast doses of 0.1, 0.3, and 0.8 mmol/kg gadoteridol (Gd HP-DO3A or Pro-Hance) were compared. Scans were performed at 2, 12, and 22 minutes after an initial injection of 0.1 mmol/kg. At each time point, paired T1-weighted scans with and without magnetization transfer (MT) were acquired. Thirty minutes after the initial injection of contrast, a supplemental dose of 0.2 mmol/kg was given (for a cumulative dose of 0.3 mmol/kg). Scans were then repeated at 2, 12, and 22 minutes after this dose was administered. A second supplemental contrast injection of 0.5 mmol/kg (for a cumulative dose of 0.8 mmol/kg) was given at 70 minutes, and immediate postinjection scans with and without MT were acquired. RESULTS: In the animals receiving a cisternal injection of bacteria, the degree of meningeal enhancement was greatest at 0.8 mmol/kg, intermediate at 0.3 mmol/kg, and least at 0.1 mmol/kg. These conclusions were constant whether imaging was performed with or without MT. Scans in control studies did not demonstrate abnormal meningeal enhancement. High-contrast dose, MT, and acquisition of immediate postcontrast scans all resulted in statistically significant improvement. On masked film review, abnormal meningeal enhancement was noted in only 2 of 5 experimental dogs at a dose of 0.1 mmol/kg (regardless of the use of MT) compared with all animals at a dose of 0.3 mmol/kg. In 18 of 37 dogs (paired scans with and without MT), when abnormal enhancement was noted, the use of MT improved the visualization of abnormal meningeal enhancement. CONCLUSIONS: In early brain meningitis, high-contrast dose (0.3 mmol/kg), MT, and scanning immediately after injection improve detection of abnormal meningeal enhancement, thus facilitating the diagnosis of meningitis. Of these factors, contrast dose is the most important.
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The functional anatomy of disorders of the basal ganglia.
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NMDA receptor subunit mRNA expression by projection neurons and interneurons in rat striatum.
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.
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Glutamate receptor expression in rat striatum: effect of deafferentation.
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.
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Glutamate receptors in striatum and substantia nigra: effects of medial forebrain bundle lesions.
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.
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Organization of N-methyl-D-aspartate glutamate receptor gene expression in the basal ganglia of the rat.
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.
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Autoradiographic evidence for increased dopamine uptake sites in striatum of hypoxic mice.
Ligand binding to dopamine uptake sites, D1 and D2 dopamine receptors was measured autoradiographically in brain sections of mice exposed to intermittent hypobaric hypoxia (450 Torr; 4,300 m) for 14 days and compared to sea level controls. Desipramine-insensitive [3H]mazindol, [3H]SCH23390 and [3H]YM-09151-2 were used respectively for the labeling of the three binding sites. After 14 days, the striatum of hypoxic mice showed a significant 21% increase in dopamine uptake sites, one of the loci of action of cocaine. A similar (28%) but non-significant increase was found in the ventral tegmental area. No changes were seen in the activities of D1 or D2 receptors in several areas examined including the substantia nigra and the striatum.
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Localization of non-N-methyl-D-aspartate glutamate receptors in normal and Alzheimer hippocampal formation.
The hippocampi and adjacent temporal cortices of 24 human brains were examined with antibodies to the GluR1, GluR2/3, and GluR4 subunits of the D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-preferring glutamate receptor. GluR1 immunoreactivity was most dense in the dentate gyrus, with lower densities in other hippocampal and cortical regions. GluR2/3 immunoreactivity was the most intense of the three antibodies, with high levels throughout most hippocampal subfields, where it was localized to cell bodies, proximal axons, and dendrites. GluR4 immunoreactivity was very sparse in all regions. In Alzheimer's disease brains, the general pattern of staining was similar to that seen in control brains. GluR1 and GluR4 immunoreactivity was seen in some but not all neuritic plaques. All three antibodies recognized some neurons undergoing neurofibrillary degeneration.
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Differential expression of metabotropic glutamate receptors in the hippocampus and entorhinal cortex of the rat.
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.
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Future directions for research on Alzheimer's disease.
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Metabotropic glutamate receptors are differentially regulated during development.
The postnatal expression of metabotropic glutamate receptors was studied in rat brain by in situ hybridization and autoradiographic binding techniques. The messenger RNAs encoding five metabotropic glutamate receptor subtypes named mGluR1-5 had distinct regional and temporal expression profiles. mGluR1, mGluR2 and mGluR4 messenger RNA expression was low at birth and increased during postnatal development. In contrast, mGluR3 and mGluR5 were highly expressed at birth and decreased during maturation to adult levels of expression. [3H]Glutamate binding competition studies in developing brain disclosed the presence of two types of binding sites with the pharmacological properties of metabotropic glutamate receptors, having high (metabotropic type-1 binding sites; K1 = 8 nM) and low affinity (metabotropic type-2 binding sites; K1 = 50 microM) for quisqualic acid, as in adult rat brain. The densities of metabotropic binding sites changed during development in a complex, regionally specific fashion. Metabotropic type-1 binding sites were present at low levels at birth and gradually increased during the second postnatal week. In the striatum, globus pallidus and cerebellar granule layer, the increase in density of metabotropic type-1 binding sites was transient but persisted in the cerebellar molecular layer. In contrast, metabotropic type-2 binding sites were present at high densities in most regions in the first postnatal week and decreased during the second and third week, particularly in the thalamic reticular nucleus and globus pallidus. Only in the external cortex did both metabotropic type-1 and metabotropic type-2 binding sites increase during development. A striking correspondence between the temporal pattern of expression of specific metabotropic glutamate receptor transcripts and metabotropic binding sites was observed in the reticular nucleus of the thalamus (mGluR3; metabotropic type-2 binding sites) and cerebellum (mGluR1; metabotropic type-1 binding sites) suggesting early translation of these metabotropic glutamate receptor messenger RNAs into receptor proteins. In other regions the relationship between messenger RNA expression and binding sites was less direct: comparison between expression of metabotropic glutamate receptor messenger RNA and binding sites suggests both a pre- and postsynaptic location of some receptor subtypes. These data imply a functional role of mGluR3 and mGluR5 during synaptogenesis and maintenance of adult synapses and of mGluR1, mGluR2 and mGluR4 in mature synaptic transmission.
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3-Nitropropionic acid toxicity in the striatum.
We examined the effects of chronic systemic administration of the mitochondrial toxin 3-nitropropionic acid (3-NP) in doses ranging from 12 to 16 mg/kg/day for 30 days on striatal cytoarchitecture in rats. Administration of 3-NP at a dose of 16 mg/kg/day resulted in large lesions with a central necrotic core that was depleted of both neurons and glia. Glial fibrillary acidic protein (GFAP) gene expression was decreased in the lesion core, whereas the tissue surrounding this area showed a massive increase in signal intensity. Enkephalin and substance P mRNA expression in the striatum showed dose-dependent decreases following administration of 3-NP. A substantial decrease occurred even in animals treated with 3-NP at a dose of 12 mg/kg/day, in which there was little discernible neuronal loss and no increase in GFAP gene expression. In contrast to the decrease in enkephalin and substance P mRNA expression, somatostatin mRNA-expressing neurons were largely preserved. There was no preferential loss of [3H]naloxone patches in the rat striatum following chronic administration of 3-NP. In animals treated with 12-15 mg/kg/day neither the area nor binding density of the patches was changed. To study the effect of 3-NP on N-methyl-D-aspartate (NMDA)-gated Ca2+ channels we used in vivo administration of [3H]MK-801. Three hours after a single injection of 3-NP at a dose of 30 mg/kg there was a three- to fivefold increase in [3H]MK-801 binding in cortex and striatum as compared with saline-treated animals, consistent with an activation of NMDA receptors.
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Metabotropic glutamate receptor mRNA expression in the basal ganglia of the rat.
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.
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Metabotropic glutamate receptor heterogeneity in rat brain.
Metabotropic glutamate receptors (mGluRs) are G protein-linked receptors that operate through the formation of different second messengers. Utilizing quantitative autoradiographic techniques, we have characterized [3H]glutamate binding to mGluRs in discrete regions of adult rat brain. [3H]Glutamate binding, in the presence of high concentrations of alpha-amino-3-hydroxymethyl-4-isoxazolepropionic acid (10 microM), N-methyl-D-aspartate (100 microM), and 2.5 mM calcium chloride (CaCl2), was saturable. Scatchard plots were linear in all regions examined and revealed similar affinity constants of about 500 nM. The largest number of sites was found in the outer cerebral cortical layers (10 pmol/mg of protein). [3H]Glutamate binding was displaced by quisqualate, trans-1-amino-1,3-cyclopentane dicarboxylic acid (t-ACPD) (racemic mixture), and (1S,3R)-ACPD but not by (1R,3S)-ACPD. The guanine nucleotide analogue guanosine-5'-O-(3-thio) triphosphate (100 microM) reduced the binding by affecting the affinity but not the total number of sites, as predicted for G protein-coupled receptor sites. Quisqualate displacement curves were always biphasic and resolved two binding sites, with Ki values in the low nanomolar (15 nM) and micromolar (63 microM) ranges. (1S,3R)-ACPD displaced [3H]glutamate binding both in the absence and in the presence of 2.5 microM quisqualate, suggesting that both high and low affinity quisqualate sites are linked to mGluRs. (1S,3R)-ACPD competition curves were broad (Hill coefficient = 0.73) but monophasic under both conditions, with Ki values in the micromolar range (14-116 microM), suggesting that (1S,3R)-ACPD acts on the two quisqualate sites with similar apparent affinities. The regional distributions of the two sites were different. The highest levels of the high affinity quisqualate binding site were found in the cerebellar molecular layer. The highest levels of the low affinity quisqualate binding sites were found in the outer cerebral cortex. The pharmacological profile and regional distribution suggest that the high and low affinity quisqualate-sensitive components of [3H]glutamate binding sites might correspond to the mGluR1/mGluR5 and mGluR2/mGluR3 subgroups of cloned mGluRs, respectively.
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