Marcello Melone

Expression of NR1 and NR2A/B subunits of the NMDA receptor in cortical astrocytes.

Ionotropic glutamate (Glu) receptors of the N‐methyl‐D‐aspartate type (NMDA) play a fundamental role in many cortical functions. Native NMDA receptors are composed of a heteromeric assembly of different subunits belonging to two classes: NMDAR1 (NR1) and NMDAR2 (NR2). To date, NMDA receptors are believed to be expressed only in neurons, although electrophysiological and in situ hybridization studies have suggested that this class of Glu receptors might be also expressed by some astrocytes. In this study, we have investigated in the cerebral cortex of adult rats the presence of astrocytes expressing NR1 and NR2A/B subunits by immunocytochemistry with specific antibodies, and we show that some distal astrocytic processes, but only rarely astrocytic cell bodies, contain immunoreaction product indicative of NR1 and NR2A/B expression. These findings suggest that at least part of the role NMDA has in cortical functions might depend on the activation of astrocytic NMDA receptors; the subcellular localization of NR1 and NR2A/B subunits in distal processes suggests that NMDA receptors contribute to monitoring Glu levels in the extracellular space.

Neuronal and glial localization of GAT-1, a high-affinity gamma-aminobutyric acid plasma membrane transporter, in human cerebral cortex: with a note on its distribution in monkey cortex

High‐affinity γ‐aminobutyric (GABA) plasma membrane transporters (GATs) influence the action of GABA, the main inhibitory neurotransmitter in the human cerebral cortex. In this study, the cellular expression of GAT‐1, the main cortical GABA transporter, was investigated in the human cerebral cortex by using immunocytochemistry with affinity‐purified polyclonal antibodies directed to the C‐terminus of rat GAT‐1.

Increased expression of the astrocytic glutamate transporter GLT-1 in the prefrontal cortex of schizophrenics.

To verify whether altered glial glutamate uptake contributes to the reduced efficacy of glutamatergic transmission reported in the prefrontal cortex of schizophrenics, we studied the expression of GLT‐1, the transporter responsible for most glutamate transport, in autoptic samples of prefrontal cortex using real time quantitative RT‐PCR, immunocytochemistry, and functional assays. GLT‐1 mRNA levels in medication‐free patients were 2.5‐fold higher than in controls, whereas they were normal or reduced in patients treated with antipsychotics. We also observed a 4‐fold increase in L‐[3H]‐Glu uptake in Xenopus oocytes injected with mRNA from the prefrontal cortex of a medication‐free schizophrenic and a 2‐fold increase in GLT‐1 protein in the same cortical area of another medication‐free patient. Results suggest that GLT‐1 mRNA, protein and function are increased in prefrontal cortex of schizophrenics.

The expression of glutamate transporter GLT-1 in the rat cerebral cortex is down-regulated by the antipsychotic drug clozapine

We show here that clozapine, a beneficial antipsychotic, down-regulates the expression of the glutamate transporter GLT-1 in the rat cerebral cortex, thereby reducing glutamate transport and raising extracellular glutamate levels. Clozapine treatment (25–35u2009mgu2009kg−1 day−1 orally) reduced GLT-1 immunoreactivity in several brain regions after 3 weeks; this effect was most prominent after 9 weeks and most evident in the frontal cortex. GLT-1 protein levels were reduced in the cerebral cortex of treated rats compared with controls and were more severely affected in the anterior (71.9u2009±u20094.5%) than in the posterior (53.2u2009±u200915.4%) cortex. L-[3H]-glutamate uptake in Xenopus laevis oocytes injected with mRNA extracted from the anterior cerebral cortex of rats treated for 9 weeks was remarkably reduced (to 30.6u2009±u20098.6%) as compared to controls. In addition, electrophysiological recordings from oocytes following application of glutamate revealed a strong reduction in glutamate uptake currents (46.3u2009±u200910.2%) as compared to controls. Finally, clozapine treatment led to increases in both the mean basal (8.1u2009±u20090.7u2009μM) and the KCl-evoked (28.7u2009±u20097.7u2009μM) output of glutamate that were 3.1 and 3.5, respectively, higher than in control rats. These findings indicate that clozapine may potentiate glutamatergic synaptic transmission by regulating glutamate transport.

Neuronal, glial, and epithelial localization of γ-aminobutyric acid transporter 2, a high-affinity γ-aminobutyric acid plasma membrane transporter, in the cerebral cortex and neighboring structures

Neuronal and glial high‐affinity Na+/Cl −‐dependent plasma membrane γ‐aminobutyric acid (GABA) transporters (GATs) contribute to regulating neuronal function. We investigated in the cerebral cortex and neighboring regions of adult rats the distribution and cellular localization of the GABA transporter GAT‐2 by immunocytochemistry with affinity‐purified polyclonal antibodies that react monospecifically with a protein of 82 kDa. Conventional and confocal laser‐scanning light microscopic studies revealed intense GAT‐2 immunoreactivity (ir) in the leptomeninges, choroid plexus, and ependyma. Weak GAT‐2 immunoreactivity also was observed in the cortical parenchyma, where it was localized to puncta of different sizes scattered throughout the radial extension of the neocortex and to few cell bodies. In sections double‐labeled with GAT‐2 and glial fibrillary acidic protein (GFAP) antibodies, some GAT‐2‐positive profiles also were GFAP positive. Ultrastructural studies showed GAT‐2 immunoreactivity mostly in patches of varying sizes scattered in the cytoplasm of neuronal and nonneuronal elements: GAT‐2‐positive neuronal elements included perikarya, dendrites, and axon terminals forming both symmetric and asymmetric synapses; nonneuronal elements expressing GAT‐2 were cells forming the pia and arachnoid mater; astrocytic processes, including glia limitans and perivascular end feet; ependymal cells; and epithelial cells of the choroid plexuses. The widespread cellular expression of GAT‐2 suggests that it may have several functional roles in the overall regulation of GABA levels in the brain. J. Comp. Neurol. 409:482–494, 1999.

Presynaptic NMDA receptors in the neocortex are both auto- and heteroreceptors.

We used electron microscopic immunocytochemistry with antibodies against NR1 and NR2A and B subunits to study the distribution of N-methyl-D-aspartate (NMDA) receptors in presynaptic axon terminals in the rat cerebral cortex. In all sections examined, NR1 and NR2A/B immunoreactivities were observed in axon terminals: NR1- and NR2A/B-positive axon terminals made both symmetrical and asymmetrical synapses on unlabelled dendritic profiles. Combined pre- and postembedding studies showed that all NR1 and NR2A/B-positive axon terminals making symmetrical synapses were gamma-aminobutyric acid (GABA)-positive. These observations show that both auto- and hetero- NMDA receptors do exist in the cerebral cortex, and indicate that part of the effects of NMDA receptor activation might be determined by modulating glutamate and GABA release.

Neuronal and glial localization of NMDA receptors in the cerebral cortex.

AbstractThe crucial role of glutamate receptors of theN-methyl-d-aspartate (NMDA) type in many fundamental cortical functions has been firmly established, as has its involvement in several neuropsychiatric diseases, but until recently, very little was known of the anatomical localization of NMDA receptors in the cerebral cortex of mammals. The recent application of molecular biological techniques to the study of NMDA receptors has allowed the production of specific tools, the use of which has much increased our understanding of the localization of NMDA receptors in the cerebral cortex. In particular, immunocytochemical studies on the distribution of cortical NMDA receptors have:1.Demonstrated the preferential localization of NMDA receptors in dendritic spines, in line with previous work;2.Disclosed a thus far unknown fraction of presynaptic NMDA receptors on both excitatory and inhibitory axon terminals; and3.Shown that cortical astrocytes express NMDA receptors.n These studies indicate that the effects of cortical NMDA receptor activation are not caused exclusively by the opening of NMDA channels on neuronal postsynaptic membranes, as previously assumed, and that the activation of presynaptic and glial NMDA receptors can contribute significantly to these effects.

Synaptic localization of GLT-1a in the rat somatic sensory cortex

GLT‐1a, the major glutamate transporter, plays an important role in both physiological and pathological conditions. Uncertainty regarding its localization in the cerebral cortex prompted us to re‐examine its cellular and subcellular localization in the rat somatic sensory cortex. GLT‐1a detection was sensitive to fixation; in optimal conditions ∼25% of GLT‐1a+ profiles were axon terminals. GLT‐1a/VGLUT1 double‐labeling and pre‐embedding electron microscopy studies showed that ∼50% of GLT‐1a+ profiles were in the vicinity of asymmetric synapses. Using pre‐embedding electron microscopy, we found that ∼70% of GLT‐1a located in the vicinity of asymmetric synapses was astrocytic and ∼30% was neuronal. Post‐embedding immunogold studies showed that the density of gold particles coding for GLT‐1a was much higher in astrocytic processes than in axon terminals, and that in the latter they were never at the active zone. In both astrocytic processes and axon terminals most gold particles were localized in a membrane region extending for about 250 nm from active zone margin, with a peak at 140 nm for astrocytic processes and at 80 for axon terminals. We conclude that, although GLT‐1a is expressed by both astrocytes and axon terminals, astrocytic GLT‐1a predominates at asymmetric synapses, and that the perisynaptic localization of GLT‐1a in cortex is well‐suited to modulate Glu concentrations at the cleft and also to restrict Glu spillover.

Up-regulation of GLT-1 severely impairs LTD at mossy fibre–CA3 synapses

Glutamate transporters are responsible for clearing synaptically released glutamate from the extracellular space. By this action, they maintain low levels of ambient glutamate, thus preventing excitotoxic damage, and contribute to shaping synaptic currents. We show that up‐regulation of the glutamate transporter GLT‐1 by ceftriaxone severely impaired mGluR‐dependent long‐term depression (LTD), induced at rat mossy fibre (MF)–CA3 synapses by repetitive stimulation of afferent fibres. This effect involved GLT‐1, since LTD was rescued by the selective GLT‐1 antagonist dihydrokainate (DHK). DHK per se produced a modest decrease in fEPSP amplitude that rapidly regained control levels after DHK wash out. Moreover, the degree of fEPSP inhibition induced by the low‐affinity glutamate receptor antagonist γ‐DGG was similar during basal synaptic transmission but not during LTD, indicating that in ceftriaxone‐treated rats LTD induction did not alter synaptic glutamate transient concentration. Furthermore, ceftriaxone‐induced GLT‐1 up‐regulation significantly reduced the magnitude of LTP at MF–CA3 synapses but not at Schaffer collateral–CA1 synapses. Postembedding immunogold studies in rats showed an increased density of gold particles coding for GLT‐1a in astrocytic processes and in mossy fibre terminals; in the latter, gold particles were located near and within the active zones. In both CEF‐treated and untreated GLT‐1 KO mice used for verifying the specificity of immunostaining, the density of gold particles in MF terminals was comparable to background levels. The enhanced expression of GLT‐1 at release sites may prevent activation of presynaptic receptors, thus revealing a novel mechanism by which GLT‐1 regulates synaptic plasticity in the hippocampus.

The glutamine commute: lost in the tube?

The glutamate-glutamine cycle appears to have an important, albeit not exclusive role, in the recycling of glutamate (Glu) between neurons and astrocytes. Recent studies show that the efflux of glutamine (Gln) from astrocytes is mediated by SNAT3 (formerly SN1), a system N amino acid transporter localized to perisynaptic astrocytes, whereas its influx into neurons is thought to be mediated by transporters of the system A family, specifically SNAT1 and SNAT2. However, the results of our confocal and electron microscopy immunocytochemical studies of the localization of these transporters in the cerebral cortex show that SNAT1 and SNAT2 are robustly expressed in the somatodendritic domain of cortical neurons, but rarely to axon terminals. To rule out a possible influence of fixation and procedural variables on detection of SNAT1 and SNAT2 immunoreactivity in axon terminals, we used non-conventional immunocytochemical methods, which, in certain cases, improve antigen detection. Though evidencing a slightly increased percentage of axon terminals expressing the two transporters, these techniques demonstrated that SNAT1 and SNAT2 are indeed rarely localized to axon terminals. Our data thus suggest that neither SNAT1 nor SNAT2 meet the criteria for their postulated role in the glutamate-glutamine cycle, and indicate that other Gln transporters (either orphan or yet to be identified) must be expressed at axon terminals and sustain the Glu (and gamma-aminobutyric acid) neurotransmitter pool (s).