Fragile X mental retardation protein (FMRP) and metabotropic glutamate receptor subtype 5 (mGlu5) control stress granule formation in astrocytes.

Fragile X syndrome (FXS) is a common form of intellectual disability and autism caused by the lack of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein involved in RNA transport and protein synthesis. Upon cellular stress, global protein synthesis is blocked and mRNAs are recruited into stress granules (SGs), together with RNA-binding proteins including FMRP. Activation of group-I metabotropic glutamate (mGlu) receptors stimulates FMRP-mediated mRNA transport and protein synthesis, but their role in SGs formation is unexplored. To this aim, we pre-treated wild type (WT) and Fmr1 knockout (KO) cultured astrocytes with the group-I-mGlu receptor agonist (S)-3,5-Dihydroxyphenylglycine (DHPG) and exposed them to sodium arsenite (NaAsO2), a widely used inducer of SGs formation. In WT cultures the activation of group-I mGlu receptors reduced SGs formation and recruitment of FMRP into SGs, and also attenuated phosphorylation of eIF2α, a key event crucially involved in SGs formation and inhibition of protein synthesis. In contrast, Fmr1 KO astrocytes, which exhibited a lower number of SGs than WT astrocytes, did not respond to agonist stimulation. Interestingly, the mGlu5 receptor negative allosteric modulator (NAM) 2-methyl-6-(phenylethynyl)pyridine (MPEP) antagonized DHPG-mediated SGs reduction in WT and reversed SGs formation in Fmr1 KO cultures. Our findings reveal a novel function of mGlu5 receptor as modulator of SGs formation and open new perspectives for understanding cellular response to stress in FXS pathophysiology.

Role of fragile X messenger ribonucleoprotein 1 in the pathophysiology of brain disorders: a glia perspective.

Fragile X messenger ribonucleoprotein 1 (FMRP) is a widely expressed RNA binding protein involved in several steps of mRNA metabolism. Mutations in the FMR1 gene encoding FMRP are responsible for fragile X syndrome (FXS), a leading genetic cause of intellectual disability and autism spectrum disorder, and fragile X-associated tremor-ataxia syndrome (FXTAS), a neurodegenerative disorder in aging men. Although FMRP is mainly expressed in neurons, it is also present in glial cells and its deficiency or altered expression can affect functions of glial cells with implications for the pathophysiology of brain disorders. The present review focuses on recent advances on the role of glial subtypes, astrocytes, oligodendrocytes and microglia, in the pathophysiology of FXS and FXTAS, and describes how the absence or reduced expression of FMRP in these cells can impact on glial and neuronal functions. We will also briefly address the role of FMRP in radial glial cells and its effects on neural development, and gliomas and will speculate on the role of glial FMRP in other brain disorders.

5-HT(1A) and 5-HT(7) receptors differently modulate AMPA receptor-mediated hippocampal synaptic transmission.

We have studied the effects of 5-HT(1A) and 5-HT(7) serotonin receptor activation in hippocampal CA3-CA1 synaptic transmission using patch clamp on mouse brain slices. Application of either 5-HT or 8-OH DPAT, a mixed 5-HT(1A)/5-HT(7) receptor agonist, inhibited AMPA receptor-mediated excitatory post synaptic currents (EPSCs); this effect was mimicked by the 5-HT(1A) receptor agonist 8-OH PIPAT and blocked by the 5-HT(1A) antagonist NAN-190. 8-OH DPAT increased paired-pulse facilitation and reduced the frequency of mEPSCs, indicating a presynaptic reduction of glutamate release probability. In another group of neurons, 8-OH DPAT enhanced EPSC amplitude but did not alter paired-pulse facilitation, suggesting a postsynaptic action; this effect persisted in the presence of NAN-190 and was blocked by the 5-HT(7) receptor antagonist SB-269970. To confirm that EPSC enhancement was mediated by 5-HT(7) receptors, we used the compound LP-44, which is considered a selective 5-HT(7) agonist. However, LP-44 reduced EPSC amplitude in most cells and instead increased EPSC amplitude in a subset of neurons, similarly to 8-OH DPAT. These effects were respectively antagonized by NAN-190 and by SB-269970, indicating that under our experimental condition LP-44 behaved as a mixed agonist. 8-OH DPAT also modulated the current evoked by exogenously applied AMPA, inducing either a reduction or an increase of amplitude in distinct neurons; these effects were respectively blocked by 5-HT(1A) and 5-HT(7) receptor antagonists, indicating that both receptors exert a postsynaptic action. Our results show that 5-HT(1A) receptors inhibit CA3-CA1 synaptic transmission acting both pre- and postsynaptically, whereas 5-HT(7) receptors enhance CA3-CA1 synaptic transmission acting exclusively at a postsynaptic site. We suggest that a selective pharmacological targeting of either subtype may be envisaged in pathological loss of hippocampal-dependent cognitive functions. In this respect, we underline the need for new selective agonists of 5-HT(7) receptors.

Endothelin-1 Induces Degeneration of Cultured Motor Neurons Through a Mechanism Mediated by Nitric Oxide and PI3K/Akt Pathway.

Endothelin-1 (ET-1) is a vasoactive peptide produced by activated astrocytes and microglia and is implicated in initiating and sustaining reactive gliosis in neurodegenerative diseases. We have previously suggested that ET-1 can play a role in the pathophysiology of amyotrophic lateral sclerosis (ALS). Indeed, we reported that this peptide is abundantly expressed in reactive astrocytes in the spinal cord of SOD1-G93A mice and ALS patients and exerts a toxic effect on motor neurons (MNs) in an in vitro model of mixed spinal cord cultures enriched with reactive astrocytes. Here, we explored the possible mechanisms underlying the toxic effect of ET-1 on cultured MNs. We show that ET-1 toxicity is not directly caused by oxidative stress or activation of cyclooxygenase-2 but requires the synthesis of nitric oxide and is mediated by a reduced activation of the phosphoinositide 3-kinase pathway. Furthermore, we observed that ET-1 is also toxic for microglia, although its effect on MNs is independent of the presence of this type of glial cells. Our study confirms that ET-1 may contribute to MN death and corroborates the view that the modulation of ET-1 signaling might be a therapeutic strategy to slow down MN degeneration in ALS.

Expression of pannexin1 in the CNS of adult mouse: cellular localization and effect of 4-aminopyridine-induced seizures.

The expression pattern of pannexin1, a gene coding for a protein that forms gap junction channels, was studied as both mRNA and protein in the CNS of adult mouse. Pannexin1 was widely expressed in the CNS by neuronal cell types but not glial cells, except for Bergmann glial cells of the cerebellar cortex. Cells positive to Ca-binding proteins, principally parvalbumin, but also calbindin and calretinin, as well as glutamate decarboxylase 67 kDa isoform, were pannexin1-positive. Pannexin1 labeling was found in cells which are known to exhibit spontaneous and synchronous discharge, such as neurons of the inferior olivary complex and the reticular thalamic nucleus, and also in neurons whose electrical activity is not coupled with neighboring cells, such as motoneurons of the spinal cord. The analysis of cellular localization showed puncta that surrounded cell bodies (e.g. the pyramidal cells of hippocampus) or restricted areas inside the cell bodies (e.g. the spinal motoneurons). In Bergmann glial cells the staining was present as fine grains that covered a large part of the cellular surface. Pannexin1 stained cells that previous studies have reported as expressing connexin36, another protein forming gap junction channels. Thus, it was possible that these two proteins could be integrated in the same functions. Since connexin36 expression levels change after seizures, we examined the expression of both pannexin1 and connexin36 in cerebral cortex, hippocampus, cerebellum and brain stem at different time intervals (2, 4 and 8 h) after i.p. injection of 4-aminopyridine, which resulted in systemic seizures. The only modification of the expression levels observed in this study concerned the progressive decrement of the connexin36 in the hippocampus, while pannexin1 expression was unchanged. This finding suggested that pannexin1 and connexin36 are involved in different functional roles or that they are expressed in different cell types and that only those expressing the Cx36 are induced to apoptosis by epileptic seizures.

Increased expression of neuronal nitric oxide synthase spliced variants in reactive astrocytes of amyotrophic lateral sclerosis human spinal cord.

The expression of three different neuronal nitric oxide synthase (nNOS) spliced variants, named nNOSalpha, nNOSbeta, and nNOSgamma, was investigated in the spinal cord of control and both familiar and sporadic amyotrophic lateral sclerosis (FALS and SALS) patients. Western blot analysis showed a consistent increase in nNOS expression in six SALS patients compared with controls when antibodies recognizing both nNOSalpha and nNOSbeta, or nNOSalpha, nNOSbeta, and nNOSgamma were used, whereas no change was observed when a selective anti-nNOSalpha antibody was used. Immunoreactivity signal for nNOSalpha-beta-gamma and nNOSalpha-beta was equally present in ventral horn neurons of control and ALS spinal cord but was dramatically increased in reactive astrocytes of the ventral horn and white matter in both FALS and SALS. nNOSalpha signal was equally expressed in motor neurons of normal and ALS spinal cord but was not evident in astrocytes. This finding indicates that nNOSbeta and nNOSgamma spliced variants are upregulated in reactive astrocytes in ALS. This may contribute to the peroxynitrite-mediated oxidative damage involved in the pathogenesis of both FALS and SALS.

Endogenous activation of group-I metabotropic glutamate receptors is required for differentiation and survival of cerebellar Purkinje cells.

We have applied subtype-selective antagonists of metabotropic glutamate (mGlu) receptors mGlu1 or mGlu5 [7-(hydroxy-imino) cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) or 2-methyl-6-(phenylethynyl)pyridine (MPEP)] to mixed rat cerebellar cultures containing both Purkinje and granule cells. The action of these two drugs on neuronal survival was cell specific. Although CPCCOEt (1, 10, 30 microm) reduced the survival of Purkinje cells, MPEP (3 or 30 microm) selectively reduced the survival of granule cells. Both effects required an early exposure of cultures to antagonists [from 3 to 6 d in vitro (DIV) for CPCCOEt, and from 3 to 6 or 6 to 9 DIV for MPEP]. Addition of MPEP from 6 to 9, 9 to 13, or 13 to 17 DIV also induced profound morphological changes in the dendritic tree and dendritic spines of Purkinje cells, suggesting that endogenous activation of mGlu5 receptors is required for the age-dependent refinement of Purkinje cell phenotype. In in vivo studies, an early blockade of mGlu1 receptors induced in rats by local injections of LY367385 (20 nmol/2 microl), local injections of mGlu1 antisense oligonucleotides (12 nmol/2 microl), or systemic administration of CPCCOEt (5 mg/kg, s.c.) from postnatal day (P) 3 to P9 reduced the number and dramatically altered the morphology of cerebellar Purkinje cells. In contrast, mGlu5 receptor blockade induced by local injections of antisense oligonucleotides reduced the number of granule cells but also produced substantial morphological changes in the dendritic tree of Purkinje cells. These results provide the first evidence that the development of cerebellar neurons is under the control of mGlu1 and mGlu5 receptors, i.e., the two mGlu receptor subtypes coupled to polyphosphoinositide hydrolysis.

Fragile X mental retardation protein (FMRP) interacting proteins exhibit different expression patterns during development.

Fragile X syndrome is caused by the lack of expression of fragile X mental retardation protein (FMRP), an RNA-binding protein involved in mRNA transport and translation. FMRP is a component of mRNA ribonucleoprotein complexes and it can interact with a range of proteins either directly or indirectly, as demonstrated by two-hybrid selection and co-immunoprecipitation, respectively. Most of FMRP-interacting proteins are RNA-binding proteins such as FXR1P, FXR2P and 82-FIP. Interestingly, FMRP can also interact directly with the cytoplasmic proteins CYFIP1 and CYFIP2, which do not bind RNA and link FMRP to the RhoGTPase pathway. The interaction with these different proteins may modulate the functions of FMRP by influencing its affinity to RNA and by affecting the FMRP ability of cytoskeleton remodeling through Rho/Rac GTPases. To better define the relationship of FMRP with its interacting proteins during brain development, we have analyzed the expression pattern of FMRP and its interacting proteins in the cortex, striatum, hippocampus and cerebellum at different ages in wild type (WT) mice. FMRP and FXR2P were strongly expressed during the first week and gradually decreased thereafter, more rapidly in the cerebellum than in the cortex. FXR1P was also expressed early and showed a reduction at later stages of development with a similar developmental pattern in these two regions. CYFIP1 was expressed at all ages and peaked in the third post-natal week. In contrast, CYFIP2 and 82-FIP (only in forebrain regions) were moderately expressed at P3 and gradually increased after P7. In general, the expression pattern of each protein was similar in the regions examined, except for 82-FIP, which exhibited a strong expression at P3 and low levels at later developmental stages in the cerebellum. Our data indicate that FMRP and its interacting proteins have distinct developmental patterns of expression and suggest that FMRP may be preferentially associated to certain proteins in early and late developmental periods. In particular, the RNA-binding and cytoskeleton remodeling functions of FMRP may be differently modulated during development.

Gangliosides attenuate NMDA receptor-mediated excitatory amino acid release in cultured cerebellar neurons.

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.

Adenosine deaminase increases release of excitatory amino acids through a mechanism independent of adenosine depletion.

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.

Neuroprotective activity of the potent and selective mGlu1a metabotropic glutamate receptor antagonist, (+)-2-methyl-4 carboxyphenylglycine (LY367385): comparison with LY357366, a broader spectrum antagonist with equal affinity for mGlu1a and mGlu5 receptors.

(+)-2-Methyl-4-carboxyphenylglycine (LY367385), a potent and selective antagonist of mGlu1a metabotropic glutamate receptors, was neuroprotective in the following in vitro and in vivo models of excitotoxic death: (i) mixed cultures of murine cortical cells transiently exposed to N-methyl-D-aspartate (NMDA); (ii) rats monolaterally infused with NMDA into the caudate nucleus; and (iii) gerbils subjected to transient global ischemia. We have compared the activity of LY367385 with that of the novel compound (+/-)-alpha-thioxantylmethyl-4-carboxyphenylglycine (LY367366), which antagonizes both mGlu1a and -5 receptors at low micromolar concentrations, but also recruits other subtypes at higher concentrations. Although LY367366 was neuroprotective, it was in general less efficacious than LY357385, suggesting that inhibition of mGlu1 receptors is sufficient to confer significant neuroprotection. We conclude that endogenous activation of mGlu1a receptors (or perhaps other mGlu1 receptor splice variants) contributes to the development of neuronal degeneration of excitotoxic origin.

Group-I metabotropic glutamate receptors: hypotheses to explain their dual role in neurotoxicity and neuroprotection.

The role of group-I metabotropic glutamate receptors (mGlu1 and 5) in neurodegeneration is still controversial. While antagonists of these receptors are consistently neuroprotective, agonists have been found to either amplify or attenuate excitotoxic neuronal death. At least three variables affect responses to agonists: (i) the presence of the NR2C subunit in the NMDA receptor complex; (ii) the existence of an activity-dependent functional switch of group-I mGlu receptors, similar to that described for the regulation of glutamate release; and (iii) the presence of astrocytes expressing mGlu5 receptors. Thus, a number of factors, including the heteromeric composition of NMDA receptors, the exposure time to drugs or to ambient glutamate, and the function of astrocytes clearing extracellular glutamate and producing neurotoxic or neuroprotective factors, must be taken into account when examining the role of group-I mGlu receptors in neurodegeneration/neuroprotection.

An increased expression of the mGlu5 receptor protein following LTP induction at the perforant path-dentate gyrus synapse in freely moving rats.

The involvement of metabotropic glutamate (mGlu) receptors in the induction of long-term potentiation (LTP) in vivo has been consistently documented. We have investigated whether LTP induction in the dentate gyrus of rats leads to changes in expression of mGlu2/3 or -5 receptor subtypes in the hippocampus. LTP was induced at the medial perforant path-dentate gyrus synapses, and mGlu receptor expression was examined by Western blot or in situ hybridization. An up-regulation of mGlu5 receptors was observed in the hippocampus both 24 and 48 h following LTP induction. This effect was restricted to the dentate gyrus and CA1 region, whereas no changes in mGlu5 receptor protein (but an increase in mRNA levels) were observed in the CA3 region. The increased expression of mGlu5 receptors was directly related to the induction of LTP, because it was not observed when tetanic stimulation was carried out in animals treated with the NMDA receptor antagonist, 2-amino-5-phosphonopentanoate (AP5). Western blot analysis also showed a reduced expression of mGlu2/3 receptors in the whole hippocampus 24 h after LTP induction, indicating that the increased expression of mGlu5 receptors was specific. These data suggest that an up-regulation of mGlu5 receptors is a component of the plastic changes that follow the induction of LTP at the perforant path-dentate gyrus synapse.

Immunohistochemical localization of group I and II metabotropic glutamate receptors in control and amyotrophic lateral sclerosis human spinal cord: upregulation in reactive astrocytes.

Excitotoxicity, which is mediated by the excessive activation of glutamate receptors, has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). There is substantial information about the distribution and function of ionotropic glutamate receptors in the spinal cord, although the role of metabotropic glutamate receptors (mGluRs) is poorly understood in this region of the brain, particularly under pathological conditions. We used immunocytochemistry to study the general distribution of group I and group II mGluR immunoreactivity in the human spinal cord, as well as the cell-specific expression of these receptors. We also investigated whether mGluR expression was altered in the spinal cord of patients with sporadic and familial ALS. Immunocytochemical analysis of control human spinal cord demonstrated that mGluR1alpha and mGluR5 (group I mGluRs) were highly represented in neuronal cells throughout the spinal cord. mGluR1alpha showed the highest relative level of expression in ventral horn neurons (laminae VIII and IX), whereas intense mGluR5 immunoreactivity was observed within the dorsal horn (superficial laminae I and II). Group II mGluRs (mGluR2/3) immunoreactivity was mainly concentrated in the inner part of the lamina II. With respect to specific neuronal populations, mGluR2/3 and mGluR5 appeared to be most frequently expressed in calbindin-containing and calretinin-containing cells, respectively. In control spinal cord only sparse astrocytes showed a weak to moderate mGluR immunoreactivity. Regional differences in immunoreactivity were apparent in ALS compared to control. In particular, mGluR expression was increased in reactive glial cells in both gray (ventral horn) and white matter of ALS spinal cord. Upregulation of mGluRs in reactive astrocytes may represent a critical mechanism for modulation of glial function and changes in glial-neuronal communication in the course of neurodegenerative diseases.

An enhanced expression of the immediate early gene, Egr-1, is associated with neuronal apoptosis in culture.

Cultured cerebellar granule cells grown in medium containing 10 mM K+ (K10) underwent apoptosis after four to five days in vitro, unless they were rescued by the addition of insulin-like growth factor-I. The few GABAergic neurons present in the cultures were more resistant to apoptotic degeneration, as indicated by double fluorescent staining with the chromatin dye Hoechst 33258 and with glutamate decarboxylase-67 antibodies. As compared with sister cultures grown in 25 mM K+, K10 cultures showed an increased expression of the Egr-1 protein and a reduced expression of the Fos protein. The increase in Egr-1 was more substantial in granule cells than in GABAergic neurons, and was not observed in K10 cultures chronically exposed to insulin-like growth factor-I. To examine the temporal relationship between the increase in Egr-1 and the development of programmed cell death, we induced apoptosis in K25 cultures at six days in vitro by replacing their medium with serum-free K10 medium. A substantial, but transient, increase in Egr- expression was observed in granule cells 6 h after switching the medium, a time that preceded the appearance of the phenotypical markers of apoptotic death. An early reduction in the Fos protein was observed after switching the medium from K25 into serum-free K10, but also after switching the medium into serum-free K25, a condition which was not associated with the development of apoptosis nor with the increase in Egr-1. We suggest that a transient induction of Egr-1 contributes to the chain of events leading to the execution phase of neuronal apoptosis in culture.

Metabotropic glutamate receptors are differentially regulated during development.

The postnatal expression of metabotropic glutamate receptors was studied in rat brain by in situ hybridization and autoradiographic binding techniques. The messenger RNAs encoding five metabotropic glutamate receptor subtypes named mGluR1-5 had distinct regional and temporal expression profiles. mGluR1, mGluR2 and mGluR4 messenger RNA expression was low at birth and increased during postnatal development. In contrast, mGluR3 and mGluR5 were highly expressed at birth and decreased during maturation to adult levels of expression. [3H]Glutamate binding competition studies in developing brain disclosed the presence of two types of binding sites with the pharmacological properties of metabotropic glutamate receptors, having high (metabotropic type-1 binding sites; K1 = 8 nM) and low affinity (metabotropic type-2 binding sites; K1 = 50 microM) for quisqualic acid, as in adult rat brain. The densities of metabotropic binding sites changed during development in a complex, regionally specific fashion. Metabotropic type-1 binding sites were present at low levels at birth and gradually increased during the second postnatal week. In the striatum, globus pallidus and cerebellar granule layer, the increase in density of metabotropic type-1 binding sites was transient but persisted in the cerebellar molecular layer. In contrast, metabotropic type-2 binding sites were present at high densities in most regions in the first postnatal week and decreased during the second and third week, particularly in the thalamic reticular nucleus and globus pallidus. Only in the external cortex did both metabotropic type-1 and metabotropic type-2 binding sites increase during development. A striking correspondence between the temporal pattern of expression of specific metabotropic glutamate receptor transcripts and metabotropic binding sites was observed in the reticular nucleus of the thalamus (mGluR3; metabotropic type-2 binding sites) and cerebellum (mGluR1; metabotropic type-1 binding sites) suggesting early translation of these metabotropic glutamate receptor messenger RNAs into receptor proteins. In other regions the relationship between messenger RNA expression and binding sites was less direct: comparison between expression of metabotropic glutamate receptor messenger RNA and binding sites suggests both a pre- and postsynaptic location of some receptor subtypes. These data imply a functional role of mGluR3 and mGluR5 during synaptogenesis and maintenance of adult synapses and of mGluR1, mGluR2 and mGluR4 in mature synaptic transmission.

Quisqualate resolves two distinct metabotropic [3H]glutamate binding sites.

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.

Glutamate receptors in striatum and substantia nigra: effects of medial forebrain bundle lesions.

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.

Melittin enhances excitatory amino acid release and AMPA-stimulated 45Ca2+ influx in cultured neurons.

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.

Glutamate receptor expression in rat striatum: effect of deafferentation.

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.