KA2 mRNA Research Articles

Expression of amino acid receptors and neural peptides in the weaver mouse brain

In the present study, we conducted: (i) in situ hybridization in order to investigate the expression of kainate and GABA A receptor subunits and the pre-proenkephalin and prodynorphin peptides in the brain of weaver mouse (a genetic model of dopamine deficiency) and (ii) immunocytochemistry in order to study the somatostatin-positive cells in weaver striatum. Our results indicated: (i) increases in mRNA levels of KA2 and GluR6 kainate receptor subunits, of α 4 and β 3 GABA A receptor subunits and of pre-proenkephalin and prodynorphin in 6-month-old weaver striatum; (ii) a decrease in α 1 and β 2 GABA A subunit mRNAs in 6-month-old weaver globus pallidus; (iii) increases in KA2, α 4 and β 3 and decreases in α 2 and β 2 mRNAs in the 6-month-old weaver somatosensory cortex; and (iv) an increase in somatostatin-immunopositive cells in 3-month-old weaver striatum. We suggest that: (i) in striatum, the alterations are induced by the induction of the transcription factor ΔfosB (for GluR6, pre-proenkephalin and prodynorphin mRNAs) and the suppression of transcription factors like NGF-IB (nerve growth factor inducible B; for the KA2 mRNA), in response to dopamine depletion; (ii) in striatum and cortex, the alterations in the expression of the GABA A subunits indicate an increase of extrasynaptic versus a decrease of synaptic GABA A receptors; and (iii) in globus pallidus, the increased striatopallidal GABAergic transmission leads to a decrease in the number of GABA A receptors. Our results further clarify the regulatory role of dopamine in the expression of amino acid receptors and striatal neuropeptides.

Thyroid hormone regulation of N-methyl-D-aspartic acid receptor subunit mRNA expression in adult brain.

Thyroid hormone is an essential modulator of brain development, but little is known about its actions in the adult brain. Hypothyroidism is associated with gene expression changes in both central and peripheral nervous tissue. Functional consequences of adult-onset hypothyroidism include an inability to produce long-term potentiation in rat hippocampus and impaired learning and memory in both rats and man. Long-term potentiation is a form of learning that is dependent on functional N-methyl-d-aspartic acid (NMDA)-preferring ionotropic glutamate receptors. This work examines the expression of ionotropic glutamate receptor subunit mRNA following surgical thyroidectomy with or without thyroid hormone replacement. In situ hybridization histochemistry was used to determine the mRNA levels of the NMDA receptor subunits NR1, NR2A, NR2B, the AMPA receptor subunit GluR1, and the kainate receptor subunit KA2. Reducing circulating concentrations of thyroid hormone by surgical removal of the thyroid gland 2 weeks before sacrifice decreased the expression of NR1 mRNA exclusively in the hippocampus. Conversely, hyperthyroidism selectively reduced NR2B mRNA expression in the dorsal hippocampus. Altering thyroid hormone status had no effect on the expression of KA2 or GluR1 subunit mRNA. The regulation of expression of NR1 and NR2B mRNA by thyroid hormone is a novel mechanism for explaining the relationship between thyroid hormone and cognitive function.

Quantitative changes in gene expression of glutamate receptor subunits/subtypes in the vestibular nucleus, inferior olive and flocculus before and following unilateral labyrinthectomy in the rat: real-time quantitative PCR method.

Spontaneous recovery from the oculomotor and postural symptoms of unilateral labyrinthectomy (UL) is known as vestibular compensation, which is a useful model for investigation of the mechanisms of lesion-induced CNS plasticity. In the present study, to elucidate the molecular biological basis of vestibular compensation, we investigated changes in the mRNA expression of glutamate receptor subunit/subtypes in the rat central vestibular system, including the vestibular nucleus complex (VNC), inferior olive (IO), and cerebellar flocculus following UL, using a real-time quantitative polymerase chain reaction (PCR) method. In normal control animals, regional differences in the expression of several glutamate receptor subunit/subtypes, e.g., NR1 and NR2A subunits of the N-methyl-D-aspartic acid (NMDA) receptor, GluR2 and KA2 subtypes of non-NMDA receptors, and mGluR1 and mGluR7 metabotropic glutamate receptors, were consistent with previous results from studies using in situ hybridization histochemistry, suggesting that the real-time quantitative PCR method was a reliable procedure for evaluation of changes in mRNA expression. In the vestibular nucleus complex, NR2A, GluR2 and mGluR7 mRNA were ipsilaterally downregulated by 6 h following UL (P<0.05, P<0.05 and P<0.01, respectively). In the inferior olive, no changes in gene expression were observed. In the ipsilateral flocculus, KA2 mRNA expression was increased by 50 h post-UL (P<0.05). However, in the contralateral flocculus, mGluR1 mRNA was downregulated by 6 h post-UL (P<0.005). Both the increase in KA2 mRNA expression in the ipsilateral flocculus and the decrease in mGluR1 mRNA expression in the contralateral flocculus may have had the effect of reducing Purkinje cell inhibition of ipsilateral VNC neurons, thereby contributing to the rebalancing of spontaneous resting activity between the ipsilateral and contralateral VNCs. It is suggested that such changes in the activities of the floccular-VNC pathways may be important to the vestibular compensation process.

Abnormal kainate receptor expression in prefrontal cortex in schizophrenia.

Abnormalities of molecules associated with the glutamate synapse have been implicated in the pathophysiology of schizophrenia. Of the many glutamate receptors, those most commonly suggested to be involved in schizophrenia are the ionotropic subtypes, the NMDA, AMPA, and kainate receptors. Both the NMDA and AMPA subtypes have been extensively studied in postmortem brains of individuals with schizophrenia, but relatively little is known about the expression of the kainate subtype of glutamate receptor. In this study, we have determined cortical and striatal kainate receptor expression in brains from persons with schizophrenia and a comparison group, using both in situ hybridization and receptor autoradiography. At the level of subunit mRNA expression, a shift in subunit stoichiometry was evident in multiple regions of the prefrontal cortex, with increased expression of gluR7 mRNA and decreased expression of KA2 mRNA. Decreased kainate receptor binding was also observed in the subjects with schizophrenia, but was restricted to infragranular laminae of the prefrontal cortex. No differences in kainate receptor binding or subunit mRNA levels were found in striatum or occipital cortex, suggesting that these findings may be restricted to association cortex. These data add to the growing literature implicating ionotropic glutamate receptor disturbances in schizophrenia, and indicate that in addition to AMPA and NMDA receptors, the kainate receptors are also abnormally expressed in this illness.

Altered Hippocampal Kainate-Receptor mRNA Levels in Temporal Lobe Epilepsy Patients

This study determined whether hippocampal kainate (KA) receptor mRNA levels were increased or decreased in temporal lobe epilepsy patients compared with nonseizure autopsies. Hippocampal sclerosis (HS;n= 17), nonsclerosis (non-HS;n= 11), and autopsy hippocampi (n= 9) were studied for KA1–2 and GluR5–7 mRNA levels using semiquantitativein situhybridization techniques, along with neuron densities. Compared with autopsy hippocampi, HS and non-HS cases showed decreased GluR5 and GluR6 hybridization densities per CA2 and/or CA3 pyramid. Furthermore, HS patients demonstrated increased KA2 and GluR5 hybridization densities per granule cell compared with autopsy hippocampi. These findings indicate that chronic temporal lobe seizures were associated with differential changes in hippocampal KA1–2 and GluR5–7 hybridization densities that vary by subfield and pathology group. In temporal lobe epilepsy patients, these results support the hypothesis that pyramidal cell GluR5 and GluR6 mRNA levels are decreased as a consequence of seizures, and in HS patients granule cell KA2 and GluR5 mRNA levels are increased in association with aberrant fascia dentata mossy fiber sprouting and/or hippocampal neuronal loss.

Differential expression of kainate receptors in the basal ganglia of the developing and adult rat brain

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.

Clozapine and haloperidol differentially affect AMPA and kainate receptor subunit mRNA levels in rat cortex and striatum

Dopamine is the neurotransmitter most often implicated in the pathogenesis of schizophrenia. However, glutamatergic antagonists can cause psychotic symptoms in otherwise normal humans, and exacerbate these symptoms in schizophrenics. These findings have led to a model of dopamine–glutamate interactions in limbic cortex and striatum as a potential substrate for symptom production in schizophrenia. From this model, we might expect that cortical and striatal expression of non-NMDA ionotropic glutamate receptors would be differentially regulated by antipsychotic treatment. To begin to address this question, we examined the regulation of mRNA levels of the AMPA (gluR1-gluR4), low affinity kainate (gluR5-gluR7), and high affinity kainate (KA1-KA2) receptor subunits by clozapine (20 mg/kg/day) and haloperidol (2 mg/kg/day) treatment for 2 weeks. Both clozapine and haloperidol caused region-specific alterations in the mRNA levels of these subunits, but there was no differential regulation in the cortex vs. the striatum. Haloperidol caused a decrease in gluR2 and gluR4 mRNA levels in both cortex and striatum and an increase in KA2 mRNA levels in the striatum only. However, clozapine treatment caused an increase in gluR7 mRNA expression, and a decrease in gluR3 mRNA expression, in both cortex and striatum while causing an increase in KA2 mRNA levels, and a decrease in gluR4 mRNA levels, in the striatum only. These dissimilarities may represent an interesting mechanism for some of the differential therapeutic or toxic effects of clozapine and haloperidol, and also may be relevant to our understanding of dopamine–glutamate interactions in schizophrenia.

Distribution of kainate receptor subunit mRNAs in human hippocampus, neocortex and cerebellum, and bilateral reduction of hippocampal GluR6 and KA2 transcripts in schizophrenia

The mRNAs encoding kainic acid (KA) preferring glutamate receptor subunits (GluR5-7, KA1 and KA2) are differentially expressed in rat brain. We have used regional and cellular in situ hybridization histochemistry with subunit-specific 35S-labelled oligodeoxyribonucleotides to examine these mRNAs in adult human hippocampus, neocortex and cerebellum. GluR5 mRNA was detected only in Purkinje cells and a few scattered hippocampal neurons. GluR6 mRNA was relatively abundant in all areas, notably in dentate gyrus, pyramidal neurons of CA3, and cerebellar granule cells, as well as being present in superficial and deep laminae of the neocortex. Moderate signal for GluR7 mRNA was seen in deep laminae of the neocortex with a weak signal in the dentate gyrus; in dipped sections GluR7 mRNA was also apparent over some pyramidal and non-pyramidal cells in hippocampus and over putative cerebellar stellate/basket cells. KA1 mRNA was detected in the dentate gyrus but not reliably elsewhere. The expression profile and abundance of KA2 mRNA was similar to that of GluR6 mRNA. For all five transcripts, concurrent hybridization of rat brain sections produced the anticipated distribution of signal. The data indicate that the regional and cellular distribution of KA receptor subunit mRNAs in human hippocampus, neocortex and cerebellum largely parallels that in the corresponding areas of rat brain, albeit at lower levels, especially with regard to GluR5 and KA1 transcripts. In schizophrenia there is a partial loss of hippocampal non-NMDA receptors, but there are no data concerning KA receptor subunit expression. KA2 and GluR6 mRNAs were sufficiently abundant for a comparison in the left and right hippocampus between 11 schizophrenics and 13 controls. Using film autoradiography, both mRNAs were significantly reduced in the schizophrenics, having controlled for the effects of brain pH, post mortem interval and age. GluR6 mRNA was also quantitated in cerebellum, wherein no differences were found between cases and controls. In conjunction with earlier findings of reduced hippocampal GluR1 and GluR2 expression and a loss of [ 3H]KA binding sites, these data show that schizophrenia is associated with impaired expression of both AMPA- and KA-preferring ionotropic glutamate receptors. These deficits are likely to contribute to the glutamatergic component of the disease pathophysiology. © 1997 Elsevier Science B.V. All rights reserved.

Expression of glutamate receptor subunit mRNAs in gonadotropin-releasing hormone neurons during the sexual maturation of the female rat.

Excitatory amino acids, particularly glutamate, are thought to be important for the maturation of the brain-pituitary-gonadal axis and the induction of puberty in the rat. We have previously shown that, in the female rat, GnRH neurons preferentially express the KA2 and NMDAR2A receptor subunit mRNAs, but not AMPA or NMDAR1 mRNA. The aim of the present study was to determine whether the onset or rate of KA2 and NMDAR2A receptor expression in GnRH neurons is correlated with the onset of puberty. Dual in situ hybridization using digoxigenin-labeled GnRH cRNA probes and 35S-labeled glutamate receptor subunit probes, followed by autoradiography and image analysis were used to measure the KA2 or NMDAR2A mRNA content in GnRH neurons in 20- to 50-day-old female rats which were sacrificed at 08.00 or 17.00 h. The results show that: (a) the KA2 mRNA content of GnRH neurons and the number of GnRH neurons expressing KA2 mRNA increase progressively in the morning hours between postnatal days 20 and 40; (b) the diurnal pattern of KA2 mRNA levels in GnRH neurons changes between days 40 and 50 from high KA2 levels in the morning hours before day 40 to high KA2 mRNA levels in the afternoon in 45- and 50-day-old animals; (c) while the high levels of KA2 mRNA in GnRH neurons in the morning hours of 20- to 40-day-old animals are paralleled by an overall increase in KA2 expression in the preoptic area, the rise in KA2 mRNA in GnRH neurons in the afternoon of 45- and 50-day-old animals appears to be specific for the GnRH neurons, and (d) no significant differences were detected for the NMDAR2A mRNA content in GnRH neurons among the different age groups and the morning and afternoon values. Since the gradual increase in the KA2 mRNA content in GnRH neurons of animals reaching puberty as well as the reversal of diurnal rhythmicity in KA2 receptor mRNA content of GnRH neurons coincide with the times of vaginal opening and first ovulation, it is suggested that glutamate, acting through KA2 receptors directly on GnRH neurons is, at least in part, an important factor in the excitatory regulation of the postnatal sexual development of the female rat. In contrast, expression of the NMDA-preferring receptor, NMDAR2A, in GnRH neurons appeared to be unchanged during this development.

The effect of chronic haloperidol treatment on glutamate receptor subunit (GluR1, GluR2, KA1, KA2, NR1) mRNAs and glutamate binding protein mRNA in rat forebrain

Antipsychotic (neuroleptic) drugs have effects on the glutamatergic system which include changes in the expression of glutamate receptor subunits. There are, however, no long-term studies. We have investigated the influence of 16 weeks' treatment with haloperidol on eight glutamate receptor mRNAs in dorsolateral striatum, frontoparietal cortex and hippocampus using in situ hybridization histochemistry. The mRNAs targetted were the flip and flop isoforms of GluR1 and GluR2, KA1 and KA2, NR1, and the glutamate binding protein (GBP). The flip isoform of GluR2 was elevated in striatum and cortex, leading to an increase in the GluR2 flip/flop ratio. KA2 mRNA was increased in hippocampus and cortex. GBP mRNA was increased in striatum. The other mRNAs were unaffected. The data show that the profile of glutamate receptor subunit mRNA expression is altered in a molecularly and anatomically selective way following chronic haloperidol administration. They provide another indication of glutamatergic involvement in the biochemical response to antipsychotic medication.

Contrasting effects of electroconvulsive shock on rnRNAs encoding the high affinity kainate receptor subunits (KA1 and KA2) and cyclophilin in the rat

Kainate-preferring glutamate receptors may contribute to the glutamatergic responses to seizures. The cloning of their encoding genes overcomes limitations of the receptor ligands available for their investigation. We have examined the expression of the high affinity kainate receptor subunits KA1 and KA2 mRNAs in the rat hippocampus, using electroconvulsive shock (ECS) as a seizure paradigm not confounded by neurotoxicity. A single shock reduced the levels of KAI mRNA in the CA3c region, while increasing the expression of KA2 mRNA in the dentate gyrus. Following repeated ECS (5 shocks over 10 days), KAI mRNA was reduced in CA3c and in CA3a-b but was unchanged in dentate gyrus. KA2 mRNA, on the other hand, significantly increased in dentate gyrus, and to a lesser extent in CA3c and CA1. All changes in KAI and KA2 mRNAs had returned to baseline 3 weeks after the last shock. We also measured the expression of cyclophilin mRNA, and found it to be reduced in all hippocampul subfields, and in the parietal cortex, after a single ECS. It returned to control levels after repeated ECS but was again reduced following 3 weeks recovery from repeated ECS. These results indicate that the expression of KA1 and KA2 not only change in opposite directions in the rat hippocampus after ECS, but that the alterations are anatomically and temporally regulated. In the respect that cyclophilin is regarded as a housekeeping gene, the reduction in its mRNA suggests that ECS may have more persistent and widespread effects on brain gene expression than previously suspected.

A complex mosaic of high-affinity kainate receptors in rat brain

The significance for CNS function of glutamate-gated cation channels that exhibit high-affinity kainate sites is not understood. Such receptors, which on dorsal root ganglia and in recombinant systems exhibit currents that rapidly desensitize to kainate application, have not been detected electrophysiologically in the brain. However, a comparison of the distribution of mRNAs encoding five glutamate receptor subunits exhibiting high-affinity kainate sites (GluR-5-GluR-7, KA-1, and KA-2) indicates that high-affinity kainate receptors are most likely involved in all central neuronal circuits of the rat brain. The KA-1 mRNA occurs mainly in the CA3 field of the hippocampus and dentate gyrus, with much lower amounts being found in inner cortical layers, cerebellar Purkinje cells, and white matter (e.g., corpus callosum and anterior commissure). The KA-2 gene is widely expressed in many neuronal nuclei including layers II-VI of neocortex, hippocampal pyramidal (CA1-CA3) and dentate granule cells, septal nuclei such as the bed nucleus of the stria terminalis, medial preoptic, suprachiasmatic, and ventral medial hypothalamic nuclei, dorsal raphe, locus coeruleus, and cerebellar granule cells. KA-2 mRNA is also found in the pineal gland. GluR-5 transcripts are in the cingulate and piriform cortex, the subiculum, lateral septal nuclei, anteroventral thalamus, suprachiasmatic nucleus, the tegmental nuclei, pontine nuclei, and Purkinje cells. GluR-6 mRNA is most abundant in cerebellar granule cells, with lower levels in caudate-putamen and the pyramidal cell layers and dentate granule cells of hippocampus. The GluR-7 gene is prominently expressed in the inner neocortical layers and some cells in layer II, subiculum, caudate-putamen, reticular thalamus, ventral medial hypothalamic nucleus, pontine nuclei, and in putative stellate/basket cells in the cerebellum. These findings suggest that a complex mosaic of receptor variants underlies the high-affinity kainate receptor in the vertebrate brain.

The KA-2 subunit of excitatory amino acid receptors shows widespread expression in brain and forms ion channels with distantly related subunits

A new ionotropic glutamate receptor subunit termed KA-2, cloned from rat brain cDNA, exhibits high affinity for [ 3H]kainate (K D ≈ 15 nM). KA-2 mRNA is widely expressed in embryonic and adult brain. Homomeric KA-2 expression does not generate agonist-sensitive channels, but currents are observed when KA-2 is coexpressed with GluR5 or GIuR6 subunits. Specifically, coexpression of GluR5(R) and KA-2 produces channel activity, whereas homomeric expression of either subunit does not. Currents through heteromeric GluR5(Q)/KA-2 channels show more rapid desensitization and different current-voltage relations when compared with GIuR5(Q) currents. GluR6/KA-2 channels are gated by AMPA, which fails to gate homomeric GIuR6 receptor channels. These results suggest possible in vivo partnership relations for high affinity kainate receptors.