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Recent studies suggest a functional diversity of native alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type glutamate receptor channels (AMPARs). In several types of interneurons, AMPARs are characterized by higher Ca2+ permeability and faster kinetics than AMPARs in principal cells. We studied the expression profile of AMPAR subunits in the hippocampal parvalbumin (PV)- and calretinin (CR)-positive cells, which represent different populations of non-principal cells. To this end, non-radioactive in situ hybridization with AMPAR subunit specific cRNAs was combined with immunocytochemistry for PV or CR. Double-immunolabelling using antibodies against AMPAR subunits and PV or CR was also performed. PV-containing neurons represent a fairly homogeneous population of cells expressing high levels of GluR-A and GluR-D mRNAs, moderate levels of GluR-C and low levels of GluR-B mRNAs in all the examined regions of hippocampus. The vast majority of CR-containing cells have a much lower expression of GluR-A, -C and -D mRNA than PV-positive neurons, although similarly featuring low levels of GluR-B mRNA. Only a subpopulation of CR-containing cells, the spiny neurons of the dentate gyrus and CA3 region of the hippocampus were characterized by a strong expression of GluR-A and -D subunit mRNAs. The differential pattern found for the AMPAR subunit mRNA expression was confirmed by immunocytochemistry at protein level. Despite the common feature of low GluR-B subunit expression, PV- and CR-containing interneurons differ with respect to the density and combination of their expressed AMPAR subunits. The different combination of subunits might subserve different properties of the AMPA channels featured by these cell types, with implications for the functioning of the hippocampal network.

Chemical lesion of olfactory neuroepitheium induced an up-regulation of the mGlu1a metabotropic glutamate receptor protein in the olfactory bulb, as shown by Western blot analysis. At 2 days after the lesion, the increase in the receptor protein was associated with an increase in mGlu1a mRNA levels; in contrast, at longer times after the lesion (16 days), mRNA levels were reduced in spite of the high expression of the receptor protein, perhaps as a result of product-inhibition of mGlu1 gene expression. Immunohistochemical analysis showed that the increase in mGlu1a induced by olfactory denervation was confined to the glomeruli, which occupy the external portion of the olfactory bulb. Within these structures, mGlu1a receptors are mainly localized on the distal dendrites of mitral cells, which are innervated by the glutamatergic axons of the olfactory nerve. These results demonstrate that the expression of mGlu1a receptors is up-regulated in response to glutamatergic deafferentation, supporting a role for this particular receptor subtype in the physiology of synaptic transmission.

A recently cloned rat brain cDNA derives from a novel gene, termed dendrin (DEN), expressed exclusively in forebrain structures, particularly in neocortex, olfactory bulb, hippocampus, caudate-putamen, and limbic system. In these structures, the cognate mRNA is present in neuronal cell bodies and their dendrites, whereas near exclusive dendritic localization is observed for the polypeptide product. In the hippocamus, DEN mRNA is highly expressed in the cell laminae and dendritic layers of the dentate gyrus and CA1 field, but expression is markedly reduced in the CA3 and CA4 areas. The predicted primary structure of the hydrophilic, highly basic 653-amino-acid polypeptide does not suggest a function. The restricted expression and dendritic location are compatible with a role for DEN in synaptic plasticity of central neocortical forebrain neurons.

The physiological properties of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptor (GluR) channels are determined by their subunit composition. In particular, the expression of the GluR-B subunit determines the divalent ion selectivity of the channel. We studied the distribution of GluR-B mRNA and protein in adult rat hippocampal GABAergic neurons combining non-radioactive in situ hybridization with immunocytochemistry. The majority of GABAergic hippocampal neurons were GluR-B mRNA-positive, but overall the levels of GluR-B transcript were lower than in pyramidal and granule cells, which showed the highest hybridization signal. The different GluR-B mRNA expression levels in GABAergic versus principal neurons were also observed at the protein level. There was a paucity of GluR-B/C immunoreactivity in the vast majority of somata of the GABAergic neurons studied, which contrasted with the strong expression of GluR-B/C proteins in the hippocampal principal neurons. Our results demonstrate the general low expression of the GluR-B subunit mRNA and GluR-B/C proteins in GABAergic hippocampal neurons. Considering the dominant role of the GluR-B subunit in determining the divalent ion permeability of the receptor, it is likely that most GABAergic neurons express AMPA receptor channels with different calcium permeabilities.

AMPA receptor (AMPAR) subunits expression was studied in nitric oxide synthase (NOS)-positive neurons of the adult rat cortex, striatum, and hippocampus, by a double-labeling approach, combining nonradioactive in situ hybridization and immunocytochemistry. The majority of cortical and hippocampal NOS-immunopositive neurons were characterized by a predominant expression of GluR-A and -D mRNA and low or undetectable expression of GluR-B and -C mRNA. In the striatum, the expression profile of AMPAR subunits in NOS-positive neurons differed from that in the other two regions. This is reflected in the overall low expression of all AMPA receptor subunits and the paucity of GluR-D subunit expression that contrasts with the high expression of this subunit in NOS-positive cells in the hippocampus. Double-labeling experiments revealed a substantial correspondence between mRNA and protein levels of AMPAR subunits. Further evidence for the regional diversity of NOS-positive neurons is derived from the expression analysis of glutamate decarboxylase (GAD)-65 and -67 mRNAs. NOS-positive neurons expressed high levels of GAD-65, but not -67 in the cortex, high levels of both forms in the hippocampus, and low or undetectable levels of both mRNAs in the striatum. Despite of these differences, NOS-positive neurons share the common feature of low GluR-B subunit expression, suggesting the presence of AMPAR channels with high Ca2+ permeability, regardless of the regional location. The relative resistance of NOS-positive interneurons in neurodegenerative diseases suggests that glutamate receptor-mediated Ca2+ influx alone does not suffice to explain neuronal vulnerability, and additional factors have thus to be considered.

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

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

Metabotropic receptor subtypes have been proposed based on pharmacological, signal transduction and cDNA sequence data. We assessed potential metabotropic binding site subtypes with in vitro quantitative [3H]glutamate autoradiography in adult rat brains in the presence of saturating concentrations of N-methyl-D-aspartate (NMDA) and (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA). Quisqualate (QUIS) competition curves resolved two differentially distributed binding sites (KIhigh = 17 nM; KIlow = 62 microM). Trans-1-amino-cyclopentane- 1,3-dicarboxylic acid (t-ACPD) and 1S,3R-ACPD displaced [3H]glutamate binding both in the absence and presence of a quisqualate concentration (2.5 microM) that saturates the high affinity sites, suggesting that both sites are linked to metabotropic receptors. We conclude that two metabotropic binding sites with different distributions and pharmacological profiles can be detected with selective [3H]glutamate binding assays.

We have studied the "trophic" action of thyrotropin releasing hormone (TRH) in cultured cerebellar granule cells, a pure and homogeneous population of glutamatergic neurons. As an index of neuronal maturation, we have measured the uptake of D-[3H]aspartate (a non-metabolizable analog of glutamate) at different days of maturation in vitro (DIV). In control cultures, D-[3H]Aspartate increased linearly during maturation reaching plateau values between 7 and 9 DIV; daily addition of TRH tartrate (TRH-t) or RGH-2202 (a TRH analog) accelerated in a concentration-dependent manner the maturation profile of D-[3H]aspartate uptake. This effect was more pronounced for RGH-2202: in cultures treated daily with RGH-2202, D-[3H]aspartate uptake was fully expressed after 3 DIV. Neither TRH-t nor RGH-2202 significantly increased D-[3H]aspartate uptake in mature cells, excluding a direct action on the glutamate transport system. Both compounds specifically potentiated the increase in [3H]inositol monophosphate formation (but not the stimulation of 45Ca2+ influx) induced by N-methyl-D-aspartate (NMDA) receptor agonists, without affecting the stimulation of inositol phospholipid hydrolysis by quisqualate or carbamylcholine. We suggest that, in cultured cerebellar granule cells, TRH and RGH-2202 enhance the trophic action of endogenous glutamate by amplifying some of the intracellular events that follow the influx of extracellular Ca2+ through NMDA-gated ion channels.

Melittin, a potent activator of phospholipase A2, enhanced both spontaneous and depolarization-induced release of D-[3H]aspartate in primary cultures of cerebellar granule cells. The action of melittin was concentration-dependent (EC50 value = 300 ng/ml) and did not require the presence of extracellular Ca2+. Melittin also stimulated the release of glutamate and aspartate, in addition to other endogenous amino acids (taurine, alanine and gamma-aminobutyric acid). These effects were accompanied by an enhanced influx of 45Ca2+, which was in part mediated by the activation of excitatory amino acid receptors by endogenous agonists. Low concentrations of melittin (50 ng/ml) potentiated the efficacy of AMPA in stimulating 45Ca2+ influx without affecting stimulation by kainate or by glutamate added in the absence of extracellular Mg2+ (a condition that favors the activation of NMDA receptors). These results indicate that activation of phospholipase A2 evokes both an enhanced glutamate release and an increased sensitivity of AMPA receptors, two events that may support synaptic facilitation and LTP formation.

beta-N-Methylamino-L-alanine (BMAA) stimulated the hydrolysis of polyphosphoinositides (PPI) in hippocampal slices prepared from 8-day old rats. The action of BMAA was antagonized by D,L-2-amino-3-phosphonopropionate (an antagonist of metabotropic receptors) and was largely reduced after lowering the concentration of bicarbonate ions from 25 to 1 mM. In cultured cerebellar neurons, stimulation of PPI hydrolysis by BMAA was mediated by the activation of both metabotropic and N-methyl-D-aspartate (NMDA) receptors. However, BMAA exhibited low activity as an NMDA receptor agonist, as reflected by its low efficacy in increasing cGMP formation in cultures incubated in the absence of extracellular Mg2+. A preferential interaction of BMAA with non-NMDA receptors was confirmed by binding studies on crude synaptic membranes from rat brain. Accordingly, BMAA was more potent in displacing specifically bound [3H]glutamate than 3-(2-carboxypiperazin-4-yl)[1,23H]propyl-1-phosphonic acid (CPP) (a selective NMDA receptor ligand). As expected, the affinity of BMAA for [3H]glutamate or [3H]CPP binding sites was greater in the presence of 25 mM bicarbonate. BMAA weakly displaced specifically bound [3H]glycine in the absence of bicarbonate and, in cultured neurons incubated with buffer containing 1 mM bicarbonate, mimicked glycine in reversing the inhibitory action of kynurenic acid on glutamate-stimulated 45Ca2+ influx. Taken collectively, these results suggest that BMAA acts as a mixed agonist of 'metabotropic' and NMDA receptors.

Addition of adenosine deaminase to cultured cerebellar neurones, led to large increases in the influx of 45Ca2+ and hydrolysis of polyphosphoinositide. These effects were inhibited or attenuated by glutamate receptor antagonists (AP5 or MK-801) and were not observed in cells stimulated by maximum concentrations of glutamate or quisqualate. Stimulation of the influx of 45Ca2+ and hydrolysis of phosphoinositide by adenosine deaminase may be secondary to an enhanced release of endogenous glutamate that in turn activates specific excitatory amino acid receptors. Accordingly, adenosine deaminase potently increased release of D-[3H]aspartate, an effect that requires the presence of extracellular Na+ and is insensitive to inhibition by MK-801. None of the effects of adenosine deaminase may be simply related to a fall in endogenous adenosine. In fact, the action of adenosine deaminase was neither reversed by agonists (L-PIA or NECA), nor mimicked by antagonists (IBMX or theophylline) of adenosine receptors. It is speculated that adenosine deaminase stimulates release of neurotransmitter through a mechanism independent of depletion of adenosine. A possible direct action of adenosine deaminase should be taken into account when the enzyme is used to unmask the effects of endogenous adenosine.

Single administration of PCA-Mg2+ (200 mg/kg, s.c.) increased the latency and shortened the duration of seizures produced by systemic injection of N-methyl-D-aspartate (NMDA) in mice. Mortality was reduced in mice pretreated with PCA-Mg2+. Single or repeated (twice a day for 5 days) injections of PCA-Mg2+ (250 mg/kg, i.p.) also attenuated kainate-induced seizures in rats. However, PCA-Mg2+ had no effect on kainate-induced automatisms but reduced the frequency of generalized seizures. These results indicate that pharmacological doses of Mg2+ protect experimental animals against excitatory amino acid-induced seizures.

The diagnostic accuracy of sonography in pelvic pathology is assessed in the case in which the operator is aware of the clinical report. Results attribute in absolute terms more effectiveness to sonography than to the clinical examination but it must, in any case, be considered as an extension of the gynaecological examination and not a replacement of the anamnestic history and physical examination.

Release of both D-[3H]aspartate and endogenous amino acids was measured in primary cultures of cerebellar granule cells. Two hour-pretreatment with the glycosphingolipids, GM1 or GT1b, attenuated the stimulation of excitatory amino acid release induced by depolarizing concentrations of K+ (50 mM). Gangliosides inhibited the phencyclidine (PCP)-sensitive component of depolarization-induced release, i.e. the amplification of release that follows activation of NMDA receptors by the endogenous glutamate.