Julie A. Kerner

EXPRESSION OF GROUP ONE METABOTROPIC GLUTAMATE RECEPTOR SUBUNIT MRNAS IN NEUROCHEMICALLY IDENTIFIED NEURONS IN THE RAT NEOSTRIATUM, NEOCORTEX, AND HIPPOCAMPUS

Metabotropic glutamate receptors (mGluRs) can be divided into three groups based on sequence homology and pharmacology. We studied expression of group I mGluRs (mGluR1 and mGluR5) in identified neurons of the rat neostriatum, neocortex, and hippocampus using in situ hybridization. Tissue sections were hybridized with radiolabeled RNA probes for mGluR1 or mGluR5 and digoxygenin labeled RNA probes detecting somatostatin (SOM), preproenkephalin (ENK), preprotachykinin (SP), glutamic acid decarboxylase 67 (GAD67), parvalbumin (PARV), or choline acetyltransferase (ChAT) mRNA. In the striatum, mGluR1 hybridization signal was observed in all six neuronal populations. The strongest signal was found in SP-positive neurons, with a lower signal in ENK-positive neurons. All striatal interneurons were labeled less intensely than ENK- and SP-positive projection neurons. For striatal mGluR5 mRNA, both SP- and ENK-positive projection neurons were intensely labeled, but only GAD67-positive interneurons exhibited a significant signal. In the neocortex and hippocampus, mGluR1 and mGluR5 hybridization signals were studied in SOM-, GAD67-, and PARV-positive neurons. Hybridization signal for mGluR1 mRNA was intense in SOM-positive neurons of the cortex, CA1, CA3, and dentate gyrus, and weaker in GAD67-positive neurons of CA3 and dentate gyrus. MGluR5 signals were intensely labeled in SOM-, GAD67- and PARV-positive neuronal populations of the cortex and hippocampus. SOM-positive neurons were more intensely labeled in the hippocampus than cortex.

Expression of NMDAR2D glutamate receptor subunit mRNA in neurochemically identified interneurons in the rat neostriatum, neocortex and hippocampus

NMDA receptors are composed of proteins from two families: NMDAR1, which are required for channel activity, and NMDAR2, which modulate properties of the channels. The mRNA encoding the NMDAR2D subunit has a highly restricted pattern of expression: in the forebrain, it is found in only a small subset of cortical, neostriatal and hippocampal neurons. We have used a quantitative double-label in situ hybridization method to examine the expression of NMDAR2D mRNA in neurochemically defined populations of neurons. In the neostriatum, NMDAR2D was expressed by the interneuron populations marked by preprosomatostatin (SOM), the 67-kDa form of glutamic acid decarboxylase (GAD67), parvalbumin (PARV), and choline acetyltransferase (ChAT) mRNAs but not by the projection neurons expressing beta-preprotachykinin (SP) or preproenkephalin (ENK) mRNAs. In the neocortex, NMDAR2D expression was observed in only a small number of neurons, but these included almost all of the SOM-, GAD67-, and PARV-expressing interneurons. In the hippocampus, NMDAR2D was not present in pyramidal or granule cells, but was abundant in SOM-, GAD67-, and PARV-positive interneurons. NMDAR2D expression appears to be a property shared by interneurons in several regions of the brain. The unique electrophysiological characteristics conveyed by this subunit, which include resistance to blockade by magnesium ion and long channel offset latencies, may be important for the integrative functions of these neurons. NMDAR2D-containing receptor complexes may prove to be important therapeutic targets in human disorders of movement. In addition, the presence of NMDAR2D subunits may contribute to the differential vulnerability of interneurons to excitotoxic injury.

Expression of N-Methyl-D-Aspartate receptor subunit mRNAs in the human brain: Hippocampus and cortex

N‐methyl‐D‐aspartate receptor (NR) activation in the hippocampus and neocortex plays a central role in memory and cognitive function. We analyzed the cellular expression of the five NR subunit (NR1 and NR2A‐D) mRNAs in these regions with in situ hybridization and human ribonucleotide probes. Film autoradiograms demonstrated a distinct pattern of hybridization signal in the hippocampal complex and the neocortex with probes for NR1, NR2A, and NR2B mRNA. NR2C and NR2D probes yielded scattered signals without a distinct organization. At the emulsion level, the NR1 probe produced high‐density hybridization signals across the hippocampal complex. NR2A mRNA was higher in dentate granule cells and pyramidal cells in CA1 and subiculum compared to hilus neurons. NR2B mRNA expression was moderate throughout, with higher expression in dentate granule cells, CA1 and CA3 pyramidal cells than in hilus neurons. In the hippocampal complex, the NR2C probe signal was not different from background in any region, whereas the NR2D probe signal resulted in low to moderate grain densities. We analyzed NR subunit mRNA expression in the prefrontal, parietal, primary visual, and motor cortices. All areas displayed strong NR1 hybridization signals. NR2A and NR2B mRNAs were expressed in cortical areas and layers. NR2C mRNA was expressed at low levels in distinct layers that differed by region and the NR2D signal was equally moderate throughout all regions. Pyramidal cells in both hippocampus and neocortex express NR1, NR2A, NR2B, and, to a lesser extent, NR2D mRNA. Interneurons or granular layer neurons and some glial cells express NR2C mRNA. J. Comp. Neurol. 390:75–90, 1998.

Expression of N-Methyl-D-Aspartate receptor subunit mRNAs in the human brain: Striatum and globus pallidus

N‐methyl‐D‐aspartate receptors (NRs) play an important role in basal ganglia function. By using in situ hybridization with ribonucleotide probes, we investigated the regional and cellular distribution of NR subunit mRNA expression in the human basal ganglia: caudate nucleus, putamen, lateral globus pallidus (LGP), and medial globus pallidus (MGP). Analysis of both film autoradiograms and emulsion‐dipped slides revealed distinct distribution patterns for each subunit. On film autoradiograms, the signal for NR1, NR2B, and NR2C in the striatum (STR) was higher than in globus pallidus (GP). The NR2D probe gave a stronger signal in GP than in STR. For NR2A we found a signal in all regions. Analysis of emulsion‐dipped sections demonstrated that in striatal neurons, the NR2B signal was higher than in GP neurons. In GP neurons, NR2D was more abundant than in striatal neurons. Despite the relatively low signal on film for NR2C in GP, we found a slightly higher signal in GP per neuron than in STR since in the pallidal areas neurons were sparse but intensely labeled. NR1 and NR2A were more evenly distributed over neurons of STR and GP. Between the different parts of STR and GP, we observed only minor differences in the expression of NRs. In MGP a subpopulation of neurons exhibiting low NR2D signals could be separated from the majority of neurons showing an intense NR2D signal. Since the physiological properties of NRs are dependent on subunit composition, these data suggest a high degree of regional specialization of NR properties in the human basal ganglia. J. Comp. Neurol. 390:63–74, 1998.

Cloning and stable expression of the mGluR1b subtype of human metabotropic receptors and pharmacological comparison with the mGluR5a subtype

We isolated and characterized a cDNA encoding the human metabotropic glutamate receptor subtype 1b (hmGluR1b). In situ hybridization studies in human brain regions revealed a higher distribution of mGluR1 mRNA in the dentate gyrus of the hippocampus, the substantia nigra pars compacta and the Purkinje cell layer of the cerebellum compared to other regions studied. We established stable expression of recombinant hmGluR1b in L(tk-) mouse fibroblast and Chinese hamster ovary (CHO-dhfr-) cells. In both expression systems, agonist activation of hmGluR1b stimulated inositol phosphate (InsP) formation and elevation of the cytosolic free calcium ([Ca2+]i), and both responses were blocked by (S)-MCPG. The rank order of potency for agonists was quisqualate > glutamate > (1S,3R)-ACPD in both expression systems. Comparison of the agonist profiles of hmGluR1b and hmGluR5a, both stably expressed in L(tk-) cells, indicated the same rank order of potency (quisqualate > glutamate > or = (RS)-3,5-DHPG > or = (1S,3R)-ACPD), but each of the four agonists were more potent on hmGluR5a than on hmGluR1b. In antagonist studies, (S)-MCPG inhibited the agonist-induced InsP formation and elevation of [Ca2+]i in both hmGluR1b- and hmGluR5a-expressing cells. (S)-4CPG and (S)-4C3HPG both inhibited agonist responses only in hmGluR1b-expressing cells. However, in hmGluR5a-expressing cells the antagonist activity of (S)-4CPG and (S)-4C3HPG was dependent on the agonist used in the study, since they inhibited responses to glutamate but not to quisqualate. Stable cell lines expressing specific subtypes of human mGluRs represent valuable tools for the study of the mechanism of action of mGluRs at the molecular and cellular level and as screening targets for identification of subtype-selective agonists or antagonists.

Simultaneous isotopic and nonisotopic in situ hybridization histochemistry with cRNA probes

In situ hybridization histochemistry is widely used to study gene expression at the mRNA level in tissues and cells. Double label in situ hybridization allows for coexpression studies. We describe a protocol for the simultaneous hybridization of two cRNA probes tagged with and digoxigenin-UTP, respectively, to frozen brain tissue sections. Hybridization signals of digoxigenin-tagged probes appear as purple cytoplasmic staining following detection of digoxigenin residues by an alkaline-phosphatase-(AP)-linked antibody. Signals resulting from hybridization of radiolabeled probes are detected as silver grains overlying cellular profiles in sections coated with autoradiographic emulsion. Grain counting allows for semiquantitatively estimates of the cellular expression levels of transcripts. Suitable cRNA-probes can be derived from linear templates generated by polymerase chain reaction (PCR) using nested primers which contain RNA-polymerase promotor sites. The cRNA-probes are sensitive and allow an application of this protocol to the detection of a wide range of mRNAs of medium or low abundance.

The human N-methyl-D-aspartate receptor 2C subunit: genomic analysis, distribution in human brain, and functional expression.

Abstract: cDNAs encoding four isoforms of the human NMDA receptor (NMDAR) NMDAR2C (hNR2C‐1, ‐2, ‐3, and ‐4) have been isolated and characterized. The overall identity of the deduced amino acid sequences of human and rat NR2C‐1 is 89.0%. The sequences of the rat and human carboxyl termini (Gly925‐Val1,236) are encoded by different exons and are only 71.5% homologous. In situ hybridization in human brain revealed the expression of the NR2C mRNA in the pontine reticular formation and lack of expression in substantia nigra pars compacta in contrast to the distribution pattern observed previously in rodent brain. The pharmacological properties of hNR1A/2C were determined by measuring agonist‐induced inward currents in Xenopus oocytes and compared with those of other human NMDAR subtypes. Glycine, glutamate, and NMDA each discriminated between hNR1A/2C‐1 and at least one of hNR1A/2A, hNR1A/2B, or hNR1A/2D subtypes. Among the antagonists tested, CGS 19755 did not significantly discriminate between any of the four subtypes, whereas 5,7‐dichlorokynurenic acid distinguished between hNR1A/2C and hNR1A/2D. Immunoblot analysis of membranes isolated from HEK293 cells transiently transfected with cDNAs encoding hNR1A and each of the four NR2C isoforms indicated the formation of heteromeric complexes between hNR1A and all four hNR2C isoforms. HEK293 cells expressing hNR1A/2C‐3 or hNR1A/2C‐4 did not display agonist responses. In contrast, we observed an agonist‐induced elevation of intracellular free calcium and whole‐cell currents in cells expressing hNR1A/2C‐1 or hNR1A/2C‐2. There were no detectable differences in the macroscopic biophysical properties of hNR1A/2C‐1 or hNR1A/2C‐2.