Receptor Subunit Messenger RNAs Research Articles

Vascular smooth muscle cells from small human omental arteries express P2X1 and P2X4 receptor subunits.

Stimulation of P2X receptors by ATP in vascular smooth muscle cells (VSMCs) is proposed to mediate vascular tone. However, understanding of P2X receptor-mediated actions in human blood vessels is limited, and therefore, the current work investigates the role of P2X receptors in freshly isolated small human gastro-omental arteries (HGOAs). Expression of P2X1 and P2X4 receptor subunit messenger RNA (mRNA) and protein was identified in individual HGOA VSMCs using RT-PCR and immunofluorescent analysis and using Western blot in multi-cellular preparations. ATP of 10μmol/l and αβ-meATP of 10μmol/l, a selective P2X receptor agonist, evoked robust increases in [Ca(2+)]i in fluo-3-loaded HGOA VSMCs. Pre-incubation with 1μmol/l NF279, a selective P2X receptor antagonist, reduced the amplitude of αβ-meATP-induced increase in [Ca(2+)]i by about 70%. ATP of 10μmol/l and αβ-meATP of 10μmol/l produced similar contractile responses in segments of HGOA, and these contractions were greatly reduced by 2μmol/l NF449, a selective P2X receptor inhibitor. These data suggest that VSMCs from HGOA express P2X1 and P2X4 receptor subunits with homomeric P2X1 receptors likely serving as the predominant target for extracellular ATP.

Differential expression of glycine receptor subunit messenger RNA in the rat following spinal cord injury

Spinal cord injury (SCI) results in alterations in the regulation of many genes that may influence neuronal death and the subsequent loss of motor function and neuropathic pain. The subtype expression mRNA levels of glycine receptors (GlyRs) after SCI are unknown. Using real-time reverse transcription PCR, this study analyzed changes in the mRNA abundance of the four major GlyR subunits (α13 and β) at 6, 24 h and 3, 7 and 10 days after SCI in adult male rats. SCI was induced at the T9 level by transection. Our results show a complicated temporal and spatial pattern of alteration in GlyR mRNA expression levels after SCI. Temporal and spatial variations with different degrees and direction (up or downregulation) of expression alteration were observed. The greatest variation was seen in GlyRα1, whereas GlyRα2 was downregulated in all regions following SCI. This study shows that alteration in GlyR expression starts as early as 6 h after SCI. The most significant points of this research are temporal elevation of GlyRα1 and continuous reduction of GlyRα2. Alterations in GlyR expression within the spinal cord may have a key role in the development of pathological pain. Therefore, control of GlyR expression could represent a novel therapeutic avenue for the development of new painkiller agents in SCI.

Molecular Pathology of Genetic Epilepsies Associated with GABAA Receptor Subunit Mutations

Mutations in ligand-gated ion channel genes associated with idiopathic generalized epilepsies have been reported in excitatory acetylcholine receptor alpha4 and beta2 subunit genes linked to autosomal dominant nocturnal frontal lobe epilepsy and in inhibitory GABA(A) receptor alpha1, beta3, gamma2, and delta subunit genes associated with childhood absence epilepsy, juvenile myoclonic epilepsy, pure febrile seizures, generalized epilepsy with febrile seizures plus, and generalized epilepsy with tonic-clonic seizures. Recent studies suggest that these mutations alter receptor function or biogenesis, including impaired receptor subunit messenger RNA stability, receptor subunit protein folding and stability, receptor assembly, and receptor trafficking.

NMDA receptors are expressed in developing oligodendrocyte processes and mediate injury

Injury to oligodendrocyte processes, the structures responsible for myelination, is implicated in many forms of brain disorder. Here we show NMDA (N-methyl-D-aspartate) receptor subunit expression on oligodendrocyte processes, and the presence of NMDA receptor subunit messenger RNA in isolated white matter. NR1, NR2A, NR2B, NR2C, NR2D and NR3A subunits showed clustered expression in cell processes, but NR3B was absent. During modelled ischaemia, NMDA receptor activation resulted in rapid Ca2+-dependent detachment and disintegration of oligodendroglial processes in the white matter of mice expressing green fluorescent protein (GFP) specifically in oligodendrocytes (CNP-GFP mice). This effect occurred at mouse ages corresponding to both the initiation and the conclusion of myelination. NR1 subunits were found mainly in oligodendrocyte processes, whereas AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptor subunits were mainly found in the somata. Consistent with this observation, injury to the somata was prevented by blocking AMPA/kainate receptors, and preventing injury to oligodendroglial processes required the blocking of NMDA receptors. The presence of NMDA receptors in oligodendrocyte processes explains why previous studies that have focused on the somata have not detected a role for NMDA receptors in oligodendrocyte injury. These NMDA receptors bestow a high sensitivity to acute injury and represent an important new target for drug development in a variety of brain disorders.

Human GABA A receptors on dopaminergic neurons in the pars compacta of the substantia nigra.

The gamma-aminobutyric acid A (GABA(A)) receptor subunit expression of the dopaminergic cells of the substantia nigra (SN) was investigated in the present study. Especially the dopaminergic cells, located in the pars compacta of SN (SNc), are of great neurologic interest, because the functional deficit and depletion of these cells are the correlate of Parkinson's disease. We used a combination of in situ hybridization histochemistry (ISH) and immunohistochemistry (IHC) on sections of human postmortem mesencephalon to investigate the expression of GABA(A) receptor subunit messenger RNAs (mRNAs) and of the receptor protein in dopaminergic SN cells. Immunohistochemical detection of tyrosine hydroxylase (TH), the pivotal enzyme of dopamine synthesis, was used to define the boundaries of SN pars reticulata (SNr) and pars compacta subregions. In SNr, all neurons were labeled by subunit-specific oligonucleotide probes and the amount of GABA(A) receptor mRNA expression was quantified as alpha(1) = beta(2) > gamma(2) > alpha(3). In contrast, in SNc, only around 25% of neurons expressed mRNA transcripts of GABA(A) receptor subunits, quantified as alpha(1) = beta(2) > gamma(2) > alpha(3) > alpha(4) = beta(3). In approximately the same percentage of neurons, which were labeled by alpha(1)-subunit-specific probe, the alpha(1)-subunit also was detected at the protein level by a specific monoclonal antibody. We, therefore, could demonstrate that a subset of dopaminergic neurons in human SNc receive inhibitory synaptic input by means of GABA(A) receptors mainly of the alpha(1)beta(2)gamma(2) subtype. This might represent a negative feedback loop between the striatum and the SNc and be a target of pharmacologic interventions in neurodegenerative diseases such as Parkinson's disease.

P2X3 receptor subunit messenger RNA expression in the female mouse bladder after oophorectomy with or without estrogen replacement

Objective: This investigation was undertaken to determine whether the sensory neuron adenosine triphosphate receptor subunit (P2X3) messenger RNA expression is altered in female mouse bladders after surgical oophorectomy with or without estrogen replacement. Study Design: The mean relative concentrations of the P2X3 receptor in 30 female mouse bladders (10 sham operated, 10 oophorectomized, and 10 oophorectomized with estrogen replacement) were determined with quantitative reverse transcription-polymerase chain reaction analysis. Results: P2X3 expression increased after surgical oophorectomy (0.91 ± 0.16 vs 1.04 ± 0.11, P =.048). However, P2X3 expression after oophorectomy and immediate estrogen replacement did not differ from that of oophorectomy alone (1.11 ± 0.15 vs 1.04 ± 0.11, P =.206). Conclusions: The P2X3 sensory neuron receptor messenger RNA expression is increased after oophorectomy but is not influenced by subsequent estrogen replacement. This has clinical significance because sensory neuron receptors may be associated with certain forms of bladder dysfunction. ( Am J Obstet Gynecol 2002;187:103-6.)

Regulation of uterine gamma-aminobutyric acid(A) receptor subunit expression throughout pregnancy.

Uterine contractions at parturition depend upon a variety of factors, including gamma-aminobutyric acid (GABA)-ergic stimulation. A new subunit of the GABA(A) receptor, pi, has recently been identified as being particularly abundant in the rat uterus. Reduced derivatives of progesterone, such as the 3alpha,5alpha-reduced derivative termed allopregnanolone, modulate GABA(A) receptor activity and neuronal inhibition by modulating the frequency and duration of GABA(A) channel opening. This modulation depends on the specific subunit composition of the GABA(A) receptor. In particular, assembly of recombinant pi and delta GABA(A) receptor subunits into a functional GABA(A) receptor have been reported to reduce sensitivity to allopregnanolone. As allopregnanolone works through the GABA(A) receptor to reduce uterine contraction, we hypothesized that incorporation of the pi-subunit into this receptor in the uterus might change the sensitivity of the GABA(A) receptor to allopregnanolone and modulate parturition. We therefore determined the expression of GABA(A) receptor subunit messenger RNAs (mRNAs) in rat uteri from various gestational ages and determined the physiological properties of the receptors. GABA(A) pi-subunit mRNA abundance was constant throughout gestation, but decreased at the onset of labor. Other GABA(A) subunits fluctuated differently during pregnancy: GABA(A) alpha(1)-subunit mRNA expression increased, whereas alpha(2)- and delta-subunit mRNA expression decreased during pregnancy, and beta(3)-subunit mRNA only appeared on postpartum day 1. We determined how allopregnanolone affected the binding of muscimol, a ligand for the GABA(A) receptor, to rat uterine GABA(A) receptors throughout pregnancy. Allopregnanolone caused the greatest increase in muscimol binding to uterine GABA(A) receptors at 19.5 days gestation and the least increase during labor, a time when pi and alpha(1) receptor subunit mRNA concentrations were low, and delta and alpha(2) receptor subunit mRNA concentrations were high. Thus, the subunit composition of the GABA(A) receptor differs in rat uteri throughout gestation. These changes may also affect the sensitivity of the GABA(A) receptor to allopregnanolone and thus contribute to the regulation of parturition.

Regional distribution of glycine receptor messenger RNA in the central nervous system of zebrafish.

We report the cloning of the zebrafish beta subunit of the glycine receptor and compare the anatomical distribution of three glycine receptor subunit constituents in adult zebrafish brain (alphaZ1, alphaZ2 and betaZ) to the expression pattern of homologous receptor subunits (alpha1, alpha2 and beta) in the mammalian adult CNS. Non-radioactive hybridization was used to map the distribution of the alphaZ1, alphaZ2 and betaZ glycine receptor subunit messenger RNAs in the adult zebrafish brain. The anterior-posterior expression gradient found in adult zebrafish brain was similar to that reported in mammalian CNS. However, the glycine receptor transcripts, notably the alphaZ1 subunit, were more widely distributed in the anterior regions of the zebrafish than in the adult mammalian brain. The isoform-specific distribution pattern was less regionalized in zebrafish than in the rat mammalian CNS. Nevertheless, there was some regionalization of alphaZ1, alphaZ2 and betaZ transcripts in the diencephalic and mesencephalic nuclei where different sensory and motor centers express either alphaZ1/betaZ or alphaZ2 subunits. In contrast to the widespread distribution of the beta subunit in adult mammalian brain, alphaZ2 messenger RNA presented the widest expression territory of all three glycine receptor subunits tested. alphaZ2 messenger RNA was expressed in the absence of alphaZ1 and betaZ messenger RNA in the outer nuclear layer of the retina, the inferior olive and the raphe of the medulla oblongata, as well as in the nucleus of Cajal of the medulla spinalis. In contrast, an identified central neuron of the reticular formation, the Mauthner cell, expresses all three glycine receptor subunits (alphaZ1, alphaZ2 and betaZ). This report extends the already described glycine receptor expression in the vertebrate CNS and confirms the importance of glycine-mediated inhibition in spinal cord and brainstem.

Differential Alterations in Nicotinic Receptor α6 and β3 Subunit messenger RNAs in Monkey Substantia nigra After Nigrostriatal Degeneration

Our previous studies showed that α4, α6, α7, β2, β3 and β4 nicotinic acetylcholine receptor messenger RNAs are present in monkey substantia nigra, with a particularly intense and localized labelling of the α6 and β3 subunit messenger RNAs to this brain region. Because loss of nigrostriatal neurons is a central feature of Parkinson’s disease and evidence suggests that nicotinic agonists potentiate antiparkinsonian effects of l-dopa, experiments were done to determine whether nicotinic receptor subunit messenger RNAs and binding sites were altered in the basal ganglia after nigrostriatal degeneration. Squirrel monkeys ( Saimiri sciureus) were rendered parkinsonian by systemic injection of the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Behavioral studies showed that this treatment decreased baseline motor activity to 36±11% of control. One month after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration, caudate and putamen dopamine levels were reduced to 51±15% and 43±6% of control, respectively, while the number of tyrosine hydroxylase-positive neurons in the substantia nigra was 75±6% of control. Despite the reduction in nigral cell number after nigrostriatal degeneration, there were no changes in α4, α7, β2 and β4 messenger RNA levels in the substantia nigra. In contrast, α6 mRNA levels were significantly increased (143±10%) and the β3 transcript decreased (62±6%) in the substantia nigra after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Declines were also observed in [ 125I]epibatidine binding in both the caudate–putamen and substantia nigra, with no change in α7 receptor binding. These results may suggest a dissociation in the regulation of receptor messenger RNA and binding sites, and/or that there are differential alterations in the different receptor subtypes measured using [ 125I]epibatidine. The changes in the two nicotinic receptor subunit messenger RNAs, α6 and β3, which exhibit a selective localization to the substantia nigra, may indicate that nicotinic receptors containing these subunits are altered after nigrostriatal degeneration.

GABA A receptor subunit messenger RNA expression in the enteric nervous system of the rat: implications for functional diversity of enteric GABA A receptors

GABAergic neurons occur in the myenteric plexus and submucosa and their innervations of the gut, where GABA stimulates motor neurons, and non-neural cells via “central type” GABA A receptors. These receptors occur on half of the neurons in the rat intestine. The GABA A receptor is a ligand-gated chloride channel constructed from different subunit families (α, β, γ, δ, ϵ). In rat these exist as subtypes, α 1–6, β 1–3, γ 1–3 and δ, defining the clinically relevant pharmacological features of GABA A receptors. However, the identity, distribution, and abundance of enteric GABA A receptor subunits are unknown. To identify and map the regional expression of GABA A receptor subunit messenger RNAs in the enteric nervous system, we assayed enteric RNA from the ileum of Sprague–Dawley rats by reverse transcription–polymerase chain reaction for α 1–6, β 1–3, γ 1–3, and δ subunit messenger RNAs. Subunit messenger RNA localization, was probed by in situ hybridization. Reverse transcription–polymerase chain reaction analysis of RNA from myenteric and submucosal nerve layers revealed the expression α 1, α 3, α 5, β 2, β 3, γ 1 and γ 3 subunit messenger RNAs. Little α 4 and α 6 and no α 2, β 1, γ 2 or δ subunit messenger RNA were detected. In situ hybridization revealed that transcripts for α 1, α 3, α 5 and β 2 subunits occur in both myenteric and submucous ganglia. However, β 3 messenger RNA was found only in myenteric plexus. The γ 1 subunit messenger RNA was also restricted to the cells in the myenteric plexus while γ 3 was found in cells of both nerve layers. In this study of the subunit messenger RNA expression profile of GABA A receptors within the enteric nerve layers we show an abundant, diverse and widespread distribution that is unique in comparison to the CNS. The distinctive and heterogeneous distribution of enteric GABA A subunits may be important in the integration of neural control of gut function.

Benzodiazepine-mediated regulation of α1, α2, β1–3 and γ2 GABA A receptor subunit proteins in the rat brain hippocampus and cortex

Prolonged flurazepam exposure regulates the expression of selected (α1, β2, β3) GABA A receptor subunit messenger RNAs in specific regions of the hippocampus and cortex with a time-course consistent with benzodiazepine tolerance both in vivo and in vitro. In this report, the immunostaining density of six specific GABA A receptor subunit (α1, α2, β1–3 and γ2) antibodies was measured in the hippocampus and cortex, among other brain areas, in slide-mounted brain sections from flurazepam-treated and control rats using quantitative computer-assisted image analysis techniques. In parallel with the localized reduction in α1 and β3 subunit messenger RNA expression detected in a previous study, relative α1 and β3 subunit antibody immunostaining density was significantly decreased in flurazepam-treated rat hippocampal CA1, CA3 and dentate dendritic regions, and in specific cortical layers. Quantitative western blot analysis showed that β3 subunit protein levels in crude homogenates of the hippocampal dentate region from flurazepam-treated rats, an area which showed fairly uniform decreases in β3 subunit immunostaining (16–21%), were reduced to a similar degree (18%). The latter findings provide independent support that relative immunostaining density may provide an accurate estimate of protein levels. Consistent with the absence of the regulation of their respective messenger RNAs immediately after ending flurazepam administration, no changes in the density of α2, β1 or β2 subunit antibody immunostaining were found in any brain region. γ2 subunit antibody staining was changed only in the dentate molecular layer. The selective changes in GABA A receptor subunit antibody immunostaining density in the hippocampus suggested that a change in the composition of GABA A receptors involving specific subunits (α1 and β3) may be one mechanism underlying benzodiazepine anticonvulsant tolerance.

Temporal and regional regulation of α1, β2 and β3, but not α2, α4, α5, α6, β1 or γ2 GABA A receptor subunit messenger RNAs following one-week oral flurazepam administration

The effect of prolonged benzodiazepine administration on GABA A receptor subunit (α1–6, β1–3, γ2) messenger RNAs was investigated in the rat hippocampus and cortex, among other brain areas. Rats were orally administered flurazepam for one week, a protocol which results in benzodiazepine anticonvulsant tolerance in vivo, and in the hippocampus in vitro, in the absence of behavioral signs of withdrawal. Autoradiographs of brain sections, hybridized with [ 35S]oligoprobes in situ, were examined immediately (day 0) or two days after drug treatment, when rats were tolerant, or seven days after treatment, when tolerance had reversed, and were compared to sections from pair-handled, vehicle-treated controls. α1 subunit messenger RNA level was significantly decreased in CA1 pyramidal cells and dentate granule cells at day 0, an effect which persisted only in CA1 neurons. Decreased “α1-specific” silver grain density over a subclass of interneurons at the pyramidal cell border suggested concomitant regulation of interneuron GABA A receptors. A reduction in β3 subunit messenger RNA levels was more widespread among hippocampal cell groups (CA1, CA2, CA3 and dentate gyrus), immediately and two days after treatment, and was also detected in the frontal and parieto-occipital cortices. Changes in β2 subunit messenger RNA levels in CA1, CA3 and dentate gyrus cells two days after ending flurazepam treatment suggested a concomitant up-regulation of β2 messenger RNA. There was a trend toward an increased level of α5, β3 and γ2 subunit messenger RNAs in CA1, CA3 and dentate gyrus cells, which was significant for the β3 and γ2 subunit messenger RNAs in the frontal cortex seven days after ending flurazepam treatment. There were no flurazepam treatment-induced changes in any other GABA A receptor subunit messenger RNAs. The messenger RNA levels of three (α1, β2 and β3) of nine GABA A receptor subunits were discretely regulated as a function of time after ending one-week flurazepam treatment related to the presence of anticonvulsant tolerance, but not dependence. The findings suggested that a localized switch in the subunit composition of GABA A receptor subtypes involving these specific subunits may represent a minimal requirement for the changes in GABA A receptor-mediated function recorded previously at hippocampal CA1 GABAergic synapses, associated with benzodiazepine anticonvulsant tolerance.

Differential distribution of N-methyl- d-aspartate receptor-2 subunit messenger RNA in the rat superior olivary complex

The distribution of N-methyl- D-aspartate receptor subunit messenger RNA was examined in the superior olivary complex of the rat using in situ hybridization methods. Non-radioactive methods showed N-methyl- D-aspartate receptor-1 and N-methyl- D-aspartate receptor-2A, -2B and -2C subunit expression to be abundant in principal cells of all major superior olivary complex regions, with N-methyl- D-aspartate receptor-1 expression higher than N-methyl- D-aspartate receptor-2. After autoradiographic in situ hybridization, counting silver grains over cells was used to quantitate and compare levels of expression of the N-methyl- D-aspartate receptor-2 subunits. N-methyl- D-aspartate receptor-2A, -2B and -2C expression was detected over cells in the lateral and medial superior olive, the medial, lateral, and ventral nucleus of the trapezoid body, and the superior paraolivary nucleus. N-methyl- D-aspartate receptor-2D expression was only observed in the lateral superior olive, lateral nucleus of the trapezoid body and ventral nucleus of the trapezoid body. Three distinct patterns of labeling were observed. The lateral superior olive and ventral nucleus of the trapezoid body showed higher expression of N-methyl- D-aspartate receptor-2A and -2C than other subunits; the medial nucleus of the trapezoid body showed higher N-methyl- D-aspartate receptor-2B expression than other subunits; and the medial superior olive, superior paraolivary nucleus and ventral nucleus of the trapezoid body showed relatively equivalent expression of N-methyl- D-aspartate receptor-2A, -2B and -2C subunits. N-methyl- D-aspartate receptor-2D had the lowest expression, with levels greater than background in only the lateral superior olive, ventral nucleus of the trapezoid body and lateral nucleus of the trapezoid body. Immunocytochemistry using antibodies to N-methyl- D-aspartate receptor-2A, -2B and -2C showed immunolabeling consistent with in situ hybridization results.

Dopamine D 1 receptor modulation of glutamate receptor messenger RNA levels in the neocortex and neostriatum of unilaterally 6-hydroxydopamine-lesioned rats

The effect of treatment with the D 1 dopamine receptor agonist SKF 38393 on the expression of metabotropic glutamate receptor 1, 3, 4 and 5 receptor subtypes and of the glutamate N-methyl- d-aspartate ionotropic receptor subunits NR1, NR2A and NR2B was analysed using in situ hybridization. We studied the neocortex and neostriatum of normal rats and of rats treated with 6-hydroxydopamine, a neurotoxin that, after intracerebral injection into the ventral tegmental area, causes selective degeneration of the ascending dopamine pathway. In the 6-hydroxydopamine-lesioned rats, metabotropic glutamate receptor subtype 3 messenger RNA levels were ipsilaterally increased in the neocortex and neostriatum, while the levels of metabotropic glutamate receptor subtype 4 messenger RNA were bilaterally increased in both regions. When administered to the 6-hydroxydopamine-lesioned rats, the D 1 receptor agonist SKF 38393 (3×20 mg/kg, s.c.) produced a bilateral decrease in the expression of the metabotropic glutamate receptor subtype 1 and 5 receptor messenger RNA levels in the neocortex and neostriatum. In the neostriatum, SKF 38393 attenuated the ipsilateral increase in the expression of striatal metabotropic glutamate receptor subtype 3 messenger RNA produced by the 6-hydroxydopamine lesion. Furthermore, SKF 38393 produced a bilateral decrease in the levels of NR1 receptor subunit messenger RNA and, in contrast, an increase in the striatal NR2B messenger RNA levels. All these effects were abolished by the D 1 receptor antagonist SCH 23360. These results indicate a differential D 1 receptor-mediated modulation of the expression of some glutamate receptor subtypes in the neostriatum and neocortex, in agreement with the idea of a functional coupling between dopamine and excitatory amino acid systems in both regions. Thus, pharmacological targeting of excitatory amino acid systems could provide alternative or complementary treatment strategies for diseases involving dopaminergic systems in the striatum (e.g., Parkinson's disease) and cortex (e.g., schizophrenia).

Expression of α 3, β 3 and γ 1 GABA A receptor subunit messenger RNAs in visual cortex and lateral geniculate nucleus of normal and monocularly deprived monkeys

Complementary RNA probes derived from complementary DNA specifically subcloned from monkey tissue were used to localize, by in situ hybridization histochemistry, the relatively rare α 3, β 3 and γ 1 subunit transcripts of the GABA A receptor in visual cortex and lateral geniculate nucleus of normal monkeys and in monkeys that had been deprived of vision in one eye. Overall, levels of α 3, β 3 and γ 1 subunit transcripts were very low. In the primary visual cortex (area 17) they were concentrated in layers II and VI and in a stratum of white matter subjacent to layer VI. The localization and density of the three messenger RNAs closely resembled those of other rare (α 2, α 5 and β 1) transcripts but their distribution also overlapped that of the predominant α 1, β 2 and γ 2 subunit transcripts. In area 18, α 3 and β 3 transcript distribution resembled that in area 17, with the addition of a third band of hybridization in layer IV for β 3. γ 1 subunit transcript localization in area 18 differed significantly from that in area 17, with increased expression restricted to layer IV. In the dorsal lateral geniculate nucleus, β 3 and γ 1 transcripts were expressed at low levels across all layers while α 3 transcripts were restricted to the magnocellular layers. Following 15 and 18 day periods of monocular deprivation, induced by intravitreal injections of tetrodotoxin, levels of α 3 receptor subunit transcripts showed modest reductions in layer VI of area 17 and in deprived geniculate laminae of adult animals. Reductions in α 3 transcript levels were much more pronounced in layer IVCβ of a five-month-old monkey deprived for the same time. Levels of β 3 and γ 1 transcripts were unaffected by monocular deprivation in cortex and geniculate at any age. Taken together with studies of other GABA A receptor transcripts, these results demonstrate the heterogeneity of GABA A receptor messenger RNA expression in the monkey geniculo-striate pathway and the varied response to reduced neuronal activity.

Developmental expression of NMDA receptor subunits and the emergence of glutamate neurotoxicity in primary cultures of murine cerebral cortical neurons.

Using primary cultures of murine cerebral cortices, we investigated the developmental expression of N-methyl-D-aspartate (NMDA) receptor subunits in relation to the appearance of NMDA receptor-mediated glutamate neurotoxicity. The cultures were not affected by glutamate exposure on culture days 7-9, but became sensitive to glutamate neurotoxicity on day 11. The expression of NMDA receptor subunit messenger RNAs (mRNAs) was investigated by means of reverse transcription polymerase chain reaction (RT-PCR). The epsilon 3-NR2C and epsilon 4-NR2D transcripts could not be detected in the culture. The epsilon 2-NR2B and zeta 1-NR1 subunit mRNAs, on the other hand, could be detected clearly and continuously from the culture initiation, and the epsilon 1-NR2A subunit mRNA became clearly detectable on culture day 4. The expression of these three subunits' proteins in the glutamate-insensitive stage (culture day 8) and the sensitive stage (day 11) were studied by means of Western blotting. The epsilon 2-NR2B and zeta 1-NR1 subunit proteins were clearly expressed on culture days 8 and 11, but the epsilon 1-NR2A subunit protein could hardly be detected on either day 8 or day 11. These results suggest that the glutamate neurotoxicity in the primary culture was mediated mainly by epsilon 2/zeta 1 NMDA receptors. The time lag between the protein expression of the epsilon 2-NR2B and zeta 1-NR1 subunits and the emergence of glutamate neurotoxicity may be necessary for the maturation of functional NMDA receptor systems, including heteromeric receptor formation, increase in receptor density and maturation of the postreceptor signal transduction system.

Expression of 10 GABA A receptor subunit messenger RNAs in the motor-related thalamic nuclei and basal ganglia of Macaca mulatta studied with in situ hybridization histochemistry

In situ hybridization histochemistry technique with [ 35S]UTP-labelled riboprobes was used to study the expression pattern of 10 GABA A receptor subunit messenger RNAs in the basal ganglia and motor thalamic nuclei of rhesus monkey. Human transcripts were used for the synthesis of α 2, α 4, β 2, β 3, γ 1 and δ subunit messenger RNA probes. Rat complementary DNAs were used for generating α 1, α 3, β 1 and γ 2 subunit messenger RNA probes. Nigral, pallidal and cerebellar afferent territories in the ventral tier thalamic nuclei all expressed α 1, α 2, α 3, α 4, β 1, β 2, β 3, δ and γ 2 subunit messenger RNAs but at different levels. Each intralaminar nucleus displayed its own unique expression pattern. In the thalamus, γ 1 subunit messenger RNA was detected only in the parafascicular nucleus. Comparison of the expression patterns with the known organization of GABA A connections in thalamic nuclei suggests that (i) the composition of the receptor associated with reticulothalamic synapses, except for those in the intralaminar nuclei, may be α 1 α 4 β 2 δ, (ii) receptors of various other subunit compositions may operate in the local GABAergic circuits, and (iii) the composition of receptors at nigro- and pallidothalamic synapses may differ, with those at nigrothalamic probably containing β 1 and γ 2 subunits. In the medial and lateral parts of the globus pallidus, the subthalamic nucleus and the substantia nigra pars reticularis, the α 1, β 2 and γ 2 messenger RNAs were co-expressed at a high level suggesting that this subunit composition was associated with all GABAergic synapses in the direct and indirect striatal output pathways. Various other subunit messenger RNAs were also expressed but at a lower level. In the substantia nigra pars compacta the most highly expressed messenger RNAs were α 3, α 4 and β 3; all other subunit messenger RNAs studied, except for γ 1, α 1 and α 2, were expressed at a moderate to high level. In the striatum, the following subunit messenger RNAs were expressed (listed in order of decreasing signal intensity): α 4, β 3, α 2, α 3, β 2, δ, γ 2, α 1. The expression patterns found in the monkey were similar to those described in comparable nuclei in the rat by Wisden et al. [ J. Neurosci. (1992) 12, p. 1040]; however, the monkey nuclei displayed a much greater variety of GABA A receptor subunit messenger RNAs.

Regulation of NMDA receptor subunit messenger RNA levels in the rat brain following acute and chronic exposure to antipsychotic drugs

Based on anatomical and biochemical observations a role of glutamate in schizophrenia has been postulated. In the present work we have investigated the gene expression for two families of NMDA receptor subunits (NR-1 and NR-2) following acute and chronic treatment with typical (haloperidol) and atypical (clozapine) antipsychotic drug (APD) in rats. A single injection of the two drugs elicited a significant increase in the mRNA levels of NR-2B in the nucleus accumbens, whereas only haloperidol was able to elevate NR-2A and NR-2B in the hippocampus. Following a 21 day treatment, significant differences in the regulatory pattern of NMDA-R subunits were observed. Haloperidol increased their mRNA levels in striatum whereas clozapine, consistent with its relatively weaker influence on nigro-striatal dopamine function, did not change the expression of NR subunits in this region. Both APD's were able to decrease the expression of NR-2 subunits in the hypothalamus, but only clozapine was capable of reducing NR-2C in frontal cortex and accumbens. The regulation of NMDA-R subunits in specific brain regions may represent a novel and important mechanism through which APD's exert some of their effects on brain function.

The projection from the striatum to the nucleus basalis in the rat: an electron microscopic study

Previous studies have shown that the striatum provides synaptic inputs to the globus pallidus and entopeduncular nucleus in which GABA is co-localized with the peptides enkephalin and substance P. The aim of this study in the rat was to determine whether the striatal projections also make synaptic contact with the cholinergic neurons of the nucleus basalis, which lie near to the pallidal areas in the rat brain. The anterograde tracer biocytin was injected into different parts of the striatum, and brain sections were stained for biocytin and choline acetyltransferase immunoreactivity by using a dual colour method. Terminals labelled with biocytin by anterograde transport and which made synaptic contact with choline acetyltransferase-positive soma and dendrites were identified by light–electron microscopic correlation methods. In the cases where the biocytin injections had been made in the dorsal or lateral striatum, biocytin-labelled terminals made synaptic contact with cholinergic cells in the region between the main termination zones in the globus pallidus and the entopeduncular nucleus. In the cases where the injections had been made in the ventromedial and posterior striatum, there was greater overlap between choline acetyltransferase-positive structures and biocytin-labelled terminals in the main termination zones in the globus pallidus or entopeduncular nucleus, but relatively few of these terminals made synaptic contacts on to the cholinergic neurons. The results therefore indicate that the cholinergic nucleus basalis cells receive a relatively sparse synaptic input from all parts of the striatum. It has recently been shown that the cholinergic cells of the nucleus basalis selectively express high levels of substance P and opioid receptor messenger RNAs, while the non-cholinergic pallidal cells have much higher levels of GABA A receptor subunit messenger RNAs. It is concluded that the cholinergic neurons of the nucleus basalis in the rat may be selectively responsive to the peptidergic components of the striatal outputs, and that they are most likely to be influenced by both the limbic and sensorimotor parts of the striatum.

N-methyl- d-aspartate receptors containing the NR2D subunit in the retina are selectively expressed in rod bipolar cells

N-methyl- d-aspartate receptor subunit messenger RNAs are widely expressed in the retina and several types of second and third order neurons are responsive to N-methyl- d-aspartate. Functional N-methyl- d-aspartate receptors are assembled from the NR1 subunit with at least one of the four NR2 subunit variants (NR2A–2D). We have analysed immunohistochemically the cellular distribution of N-methyl- d-aspartate receptors containing the NR2D subunit in the rat and rabbit retina. Using a subunit-specific NR2D antiserum, exclusively bipolar cells with somata localized close to the outer plexiform layer were labelled in both species. The axons were immunoreactive and arborized in the innermost inner plexiform layer. The morphology and localization of these cells, which were much more numerous in rat than in rabbit, suggested that they are rod bipolar cells. This was confirmed in both species by co-localization of the NR2D subunit immunoreactivity with protein kinase C- α, a selective marker for rod bipolar cells. At the subcellular level, a distinct polarization in the distribution of NR2D immunoreactivity was demonstrated by confocal laser scanning microscopy: staining was moderate in dendrites arborizing within the outer plexiform layer, intense at that pole of the soma facing the outer plexiform layer, and low in the portion of the soma embedded in the inner nuclear layer. Proximal axonal segments and axonal end-feet in the inner plexiform layer displayed the strongest NR2D subunit immunoreactivity. The axonal staining suggests that neurotransmission of the rod bipolar cells is modulated within the inner plexiform layer by N-methyl- d-aspartate receptors containing the NR2D subunit.