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Immunocytochemistry was used to study the distribution of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subtypes in the inferior colliculus (IC) of genetically epilepsy-prone rats (GEPR-9s) and normal Sprague-Dawley (SD) rats. The analysis was conducted using 3 antibodies specific for glutamate receptor subtypes, GluR 1, GluR 2/3, and GluR 4. Light microscopy showed that immunostaining of the IC was most dense with the GluR 2/3 antibody for both strains of animals. The amount of GluR 2/3 immunolabeling was similar for sound-stimulated GEPR-9s, seizure-naive GEPR-9s, and SD rats. The electron microscopy of GluR 2/3 in the IC revealed immunoreaction products associated with the postsynaptic densities of asymmetric synapses. The thin sections had comparable amounts of reaction product in dendrites or dendritic spines for both strains. Since the distribution and quantity of AMPA receptors in the IC of GEPR-9s and SD rats are similar, our results indicate that altered AMPA receptors are probably not the primary cause of seizure initiation in GEPR-9s.

Olfaxin, which is a BNIP2 and Cdc42GAP homology (BCH) domain-containing protein, is predominantly expressed in mitral and tufted (M/T) cells in the olfactory bulb (OB). Olfaxin and Caytaxin, which share 56.3% amino acid identity, are similar in their glutamatergic terminal localization, kidney-type glutaminase (KGA) interaction, and caspase-3 substrate. Although the deletion of Caytaxin protein causes human Cayman ataxia and ataxia in the mutant mouse, the function of Olfaxin is largely unknown. In this study, we generated Prune2 gene mutant mice ( Prune2 Ex16−/− ; knock out [KO] mice) using the CRISPR/Cas9 system, during which the exon 16 containing start codon of Olfaxin mRNA was deleted. Exon 16 has 80 nucleotides and is contained in four of five Prune2 isoforms, including PRUNE2, BMCC1, BNIPXL, and Olfaxin/BMCC1s. The levels of Olfaxin mRNA and Olfaxin protein in the OB and piriform cortex of KO mice significantly decreased. Although Prune2 mRNA also significantly decreased in the spinal cord, the gross anatomy of the spinal cord and dorsal root ganglion (DRG) was intact. Further, disturbance of the sensory and motor system was not observed in KO mice. Therefore, in the current study, we examined the role of Olfaxin in the olfactory system where PRUNE2, BMCC1, and BNIPXL are scarcely expressed. Odor preference was impaired in KO mice using opposite-sex urinary scents as well as a non-social odor stimulus (almond). Results of the odor-aversion test demonstrated that odor-associative learning was disrupted in KO mice. Moreover, the NMDAR2A/NMDAR2B subunits switch in the piriform cortex was not observed in KO mice. These results indicated that Olfaxin may play a critical role in odor preference and olfactory memory. [ABSTRACT FROM AUTHOR]

Abstract: For more than a century, the hippocampal sub-fields have been recognized as being differentially vulnerable to injury. While the cause remains unknown, the explanations generally considered have involved either vascular differences, or innate variability among cells. To examine the latter possibility, we prepared acute hippocampal slices from Sprague-Dawley rats, applied a brief period of oxygen–glucose deprivation (OGD; an in vitro model of ischemia), and assessed the viability of dissected sub-fields (CA1, CA3, dentate gyrus) by measuring mitochondrial 2,3,5-triphenyltetrazolium chloride (TTC) metabolism. In slices from young animals (15 weeks of age), post-OGD TTC metabolism was significantly reduced in the CA sub-fields relative to the dentate gyrus. Since previous studies found increasing age may worsen ischemic injury, we completed the same experiment using tissue from animals at 52 weeks of age, and found no differences in TTC metabolism across sub-fields. Given the established role of glutamate receptors in ischemic cell death, we examined two key subunit proteins (GluN1, found in all NMDA receptors, and GluA2, found in most AMPA receptors) across sub-fields and age to determine whether their expression complemented our viability data. We found that, relative to the CA1, the DG displayed greater GluN1 expression and lower GluA2 expression in both young and old animals. Our results confirm that regional vulnerability can be shown in a slice model, that the property is not intransigent, and that these features are likely not attributable to the expression pattern of key glutamate receptor subunits, but another molecular variable that changes over the lifespan. [Copyright &y& Elsevier]

The immunomodulatory role of medial septum (MS) has been explored so far only in MS lesioned rats. But in MS lesioned rats, all the nerve cells and fibres of the lesioned area are damaged and the specific role of the neural circuits of MS on immunomodulation cannot be assessed from the lesion of MS. Considering the presence of a large number of glutamate receptors in MS, the specific role of glutamate receptors stimulation on some immune responses has been investigated in the present study. Hyperreactive behaviour, TC and DC of WBC, phagocytic activity of peripheral leukocytes, adhesibility and cytotoxicity of splenic mononuclear cells (MNC), delayed type of hypersensitivity (DTH) responses and the serum corticosterone (CORT) were measured after microinfusion of glutamate into MS of rats. To ascertain the specific role of those glutamate receptors, the parameters were also measured after microinfusion of glutamate receptor blocker 6, 7-dinitroquinoxaline-2, 3-dione (DNQX). The hyperreactive behaviour, TC and DC of WBC remained unaltered after stimulation or blocking of glutamate receptors. The phagocytic activity, adhesibility and cytotoxicity of splenic MNC, and DTH responses were increased after infusion of 0.25 and 0.5µM glutamate. But after infusion of higher dose of glutamate (1µM), the phagocytic activity and the adhesibility of splenic MNC were decreased and other parameters remained unaltered in that condition. After infusion of 4 and 8mM DNQX all the observed immunological parameters were decreased. The CORT concentration was decreased after infusion of 0.25 and 0.5µM of glutamate but it was increased after infusion of 1µM glutamate or 4 and 8mM DNQX. Results indicate that the medial septal glutamate receptors play an important role in the modulation of some immune responses. [ABSTRACT FROM AUTHOR]

Abstract: The sensorimotor striatum is critical for the acquisition and consolidation of skilled learning-related motor sequences. Excitatory corticostriatal synapses undergo neuroplastic changes that impact signal transmission efficacy. Modification of N-methyl d-aspartate (NMDA) and α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subunit composition and phosphorylation is critical for bidirectional experience-driven plasticity observed at these synapses. Metaplastic regulation of the ratio of NR2A to NR2B subunits of the NMDA receptor controls the threshold for the induction of subsequent plasticity. However, little is known about how repeated practice effects the differential regulation of glutamate receptors during the acquisition of a unilateral motor skill. Using immunoblot analysis, we assessed changes in NMDA and AMPA receptors during the associative stage of skill acquisition in synaptoneurosome preparations from the rat sensorimotor striatum. We found that the NR2A/B subunit ratio in the striatum contralateral to the trained limb decreased during skill acquisition optimizing the threshold for inducing subsequent synaptic plasticity during learning of the lateralized motor skill. In contrast, there was a significant increase in the NR2A/B subunit ratio in the ipsilateral striatum making the induction of subsequent plasticity more difficult. In addition, there was a selective decrease in AMPAR phosphorylation levels at serine site 831 but not 845 on the GluR1 subunit ipsilaterally with a trend toward a decrease contralaterally. These findings suggest that the successful acquisition of a lateralized motor skill necessitates the integration of motor programs in both striata, each of which reflects unique changes in the NR2A/B ratio that modulate the different task demands on the associated limb. [Copyright &y& Elsevier]

Abstract: Fluoxetine is a widely used antidepressant with an action that is primarily attributed to the inhibition of serotonin re-uptake into the synaptic terminals of the central nervous system. Fluoxetine also has blocking effects on various ion channels, including Ca2+ channels. It remains unclear, however, how fluoxetine may affect synaptically induced [Ca2+] i spikes. We investigated the effects of fluoxetine on [Ca2+] i spikes, along with the subsequent neurotoxicity that is synaptically evoked by lowering extracellular Mg2+ in cultured rat hippocampal neurons. Fluoxetine inhibited the synaptically induced [Ca2+] i spikes in p-chloroamphetamine-treated and non-treated neurons, in a concentration-dependent manner. However, other selective serotonin reuptake inhibitors, such as paroxetine and citalopram, did not significantly affect the spikes. Pretreatment with fluoxetine for 5min inhibited [Ca2+] i increases induced by glutamate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and N-methyl-d-aspartate. Fluoxetine also inhibited α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced currents. In addition, fluoxetine decreased the [Ca2+] i responses induced by the metabotrophic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine or the ryanodine receptor agonist caffeine. Fluoxetine inhibited [Ca2+] i responses induced by 20mM KCl. Fluoxetine decreased the release of FM1-43 induced by electric field stimulation. Furthermore, fluoxetine inhibited 0.1mM [Mg2+] o -induced cell death. Collectively, our results suggest that fluoxetine suppresses the spikes and protects neurons against excitotoxicity, particularly in cultured rat hippocampal neurons, presumably due to both direct inhibition of presynaptic glutamate release and postsynaptic glutamate receptor-mediated [Ca2+] i signaling. In addition to an indirect inhibitory effect via 5-HT levels, these data suggest a new, possibly direct inhibitory action of fluoxetine on synaptically induced [Ca2+] i spikes and neuronal cell death. [Copyright &y& Elsevier]

Abstract: The cervical facet joint and its capsule are a common source of neck pain from whiplash. Mechanical hyperalgesia elicited by painful facet joint distraction is associated with spinal neuronal hyperexcitability that can be induced by transmitter/receptor systems that potentiate the synaptic activation of neurons. This study investigated the temporal response of a glutamate receptor and transporters in the dorsal root ganglia (DRG) and spinal cord. Bilateral C6/C7 facet joint distractions were imposed in the rat either to produce behavioral sensitivity or without inducing any sensitivity. Neuronal metabotropic glutamate receptor-5 (mGluR5) and protein kinase C-epsilon (PKCε) expression in the DRG and spinal cord were evaluated on days 1 and 7. Spinal expression of a glutamate transporter, excitatory amino acid carrier 1 (EAAC1), was also quantified at both time points. Painful distraction produced immediate behavioral hypersensitivity that was sustained for 7days. Increased expression of mGluR5 and PKCε in the DRG was not evident until day 7 and only following painful distraction; this increase was observed in small-diameter neurons. Only painful facet joint distraction produced a significant increase (p<0.001) in neuronal mGluR5 over time, and this increase also was significantly elevated (p≤0.05) over responses in the other groups at day 7. However, there were no differences in spinal PKCε expression on either day or between groups. Spinal EAAC1 expression was significantly increased (p<0.03) only in the nonpainful groups on day 7. Results from this study suggest that spinal glutamatergic plasticity is selectively modulated in association with facet-mediated pain. [Copyright &y& Elsevier]

Abstract: In the vestibular nuclei, acute hypotension induces excitation of electrical activity and expression of c-Fos protein and phosphorylated extracellular signal-regulated kinase (pERK). Expression of c-Fos protein and pERK is mediated by the excitatory neurotransmitter, glutamate. We investigated the signaling pathway of glutamate and its receptors in the vestibular nuclei following acute hypotension in conscious rats. Glutamate release and the expression of c-Fos protein in the medial vestibular nuclei (MVN) were measured by microdialysis and immunohistochemical analysis, respectively. We compared the responses of rats with unilateral labyrinthectomy to unaltered controls. Acute hypotension was induced by infusing sodium nitroprusside (SNP) into the femoral vein. In the control group, glutamate release and the expression of c-Fos protein increased in the bilateral MVN following acute hypotension. In the unilateral labyrinthectomy group, glutamate release and the expression of c-Fos protein increased in the MVN contralateral to the lesion, but did not change in the ipsilateral MVN following acute hypotension. Microinjection of NMDA or AMPA into the lateral ventricle increased the expression of c-Fos protein in the bilateral MVN of conscious intact labyrinthine rats. However, after intracerebroventricular microinjection of MK-801 or CNQX little c-Fos protein was expressed in the bilateral MVN of these rats following acute hypotension. These results suggest that in response to acute hypotension, excitatory afferent signals from the peripheral vestibular receptors release glutamate into postsynaptic neurons in the vestibular nuclei. These excitatory signals are transmitted through the NMDA receptors and AMPA receptors of glutamate in the vestibular system. [Copyright &y& Elsevier]

Abstract: Receptor subunit composition is believed to play a major role in the synaptic trafficking of AMPA receptors (AMPARs), and thus in activity-dependent synaptic plasticity. To isolate a physiological role of GluA1-containing AMPARs in area CA3 of the hippocampus, pair recordings were performed in organotypic hippocampal slices taken from genetically modified mice lacking the GluA1 subunit. We report here that long-term potentiation (LTP) is impaired not only at active but also at silent synapses when the GluA1 subunit is absent. The GluA1 knockout mice also exhibited reduced AMPAR-mediated evoked currents between pairs of CA3 pyramidal neurons under baseline conditions suggesting a significant role for GluA1-containing AMPARs in regulating basal synaptic transmission. In two independent measures, however, long-term depression (LTD) was unaffected in tissue from these mice. These data provide a further demonstration of the fundamental role that GluA1-containing AMPARs play in activity-dependent increases in synaptic strength but do not support a GluA1-dependent mechanism for reductions in synaptic strength. [Copyright &y& Elsevier]

Abstract: Insulin modulates N-methyl-d-aspartate (NMDA) receptors in the CNS and potentiates recombinant NMDA receptor currents in Xenopus oocytes. We have previously found that insulin''s potentiation of NMDA receptor currents in oocytes occurs in a subunit specific manner and via phosphorylation of specific C-terminal sites by protein tyrosine kinases (PTKs) and C-type protein kinases (PKCs). Insulin-mediated current potentiation of receptors containing the NR2A subunit occurs solely through the activation of PKCs. Activation of phosphoinositide 3-kinase (PI 3-kinase) is known to trigger many insulin-stimulated signaling pathways, and we show here that it lies at a critical step in the insulin-mediated potentiation of NMDA receptor currents. Incubation with the PI 3-kinase inhibitor wortmannin eliminates insulin potentiation of NMDA receptor currents in the oocytes. Atypical isoforms of PKC are known to be activated downstream in the insulin signaling pathway via activation of PI 3-kinase. We demonstrate that the atypical isoform PKC zeta (PKCζ) has a role in insulin-stimulated current potentiation of NR2A-containing NMDA receptors using an isoform-specific pseudosubstrate inhibitor of PKCζ. [Copyright &y& Elsevier]

Abstract: Corticostriatal and thalamostriatal projections utilize glutamate as their neurotransmitter. Their influence on striatum is mediated, in part, by ionotropic AMPA-type glutamate receptors, which are heteromers composed of GluR1–4 subunits. While the cellular localization of AMPA-type subunits in the basal ganglia has been well characterized in rodents, the cellular localization of AMPA subunits in primate basal ganglia is not. We thus carried out immunohistochemical studies of GluR1–4 distribution in rhesus monkey basal ganglia in conjunction with characterization of each major neuron type. In striatum, about 65% of striatal neurons immunolabeled for GluR1, 75%–79% immunolabeled for GluR2 or GluR2/3, and only 2.5% possessed GluR4. All neurons the large size of cholinergic interneurons (mean diameter 26.1μm) were moderately labeled for GluR1, while all neurons in the size range of parvalbuminergic interneurons (mean diameter 13.8μm) were intensely rich in GluR1. Additionally, somewhat more than half of the neurons in the size range of projection neurons (mean diameter 11.6μm) immunolabeled for GluR1, and about one third of these were very rich in GluR1. About half of the neurons the size of cholinergic interneurons were immunolabeled for GluR2, and the remainder of the neurons that were immunolabeled for GluR2 coincided with projection neurons in size and shape (GluR2 diameter=10.7μm), indicating that the vast majority of striatal projection neurons possess immunodectible GluR2. Similar results were observed with GluR2/3 immunolabeling. Half of the neurons the size of cholinergic interneurons immunolabeled for GluR4 and seemingly all neurons in the size range of parvalbuminergic interneurons possessed GluR4. These results indicate that AMPA receptor subunit combinations for striatal projection neurons in rhesus monkey are similar to those for the corresponding neuron types in rodents, and thus their AMPA responses to glutamate are likely to be similar to those demonstrated in rodents. [Copyright &y& Elsevier]

Abstract: Synaptic loss underlies the memory deficit of Alzheimer''s disease (AD). The molecular mechanism is elusive; however, excitatory synapses organized by the postsynaptic density (PSD) have been used as targets for AD treatment. To identify pathological entities at the synapse in AD, synaptic proteins were screened by quantitative proteomic profiling. The critical proteins were then selected for immunoblot analysis. The glutamate receptors N-methyl-d-aspartate (NMDA) receptor 1 and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor 2 (GluR2) were substantially lost; specifically, the loss of GluR2 was up to 40% at PSD in AD. Shank proteins, the organizers of these glutamate receptors at excitatory synapses, were dramatically altered in AD. The level of Shank2 was increased, whereas the protein level of Shank3 was decreased. Further, the Shank3 protein was modified by ubiquitin, indicating that abnormal activity of the ubiquitin–proteasome system may lead to Shank3 degradation in AD. Our findings suggest that disruption of glutamate receptors at the Shank-postsynaptic platform could contribute to destruction of the PSD which underlies the synaptic dysfunction and loss in AD. [Copyright &y& Elsevier]

Abstract: Status epilepticus is a life-threatening form of seizure activity that represents a major medical emergency associated with significant morbidity and mortality. Protein Kinase A is an important regulator of synaptic strength that may play an important role in the development of status epilepticus-induced neuronal pathology. This study demonstrated an increase in PKA activity against exogenous and endogenous substrates during later stages of SE. As SE progressed, a significant increase in PKA-mediated phosphorylation of an exogenous peptide substrate was demonstrated in cortical structures. The increased activity was not due to altered expression of either regulatory or catalytic subunits of the enzyme. Through the use of phospho-specific antibodies, this study also investigated the effects of SE on the phosphorylation of the GluR1 subunit of the AMPA subtype of glutamate receptor. After the onset of continuous seizure activity, an increase in phosphorylation of the PKA site on the GluR1 subunit of the AMPA receptor was observed. These data suggest a potential mechanism by which SE may increase neuronal excitability in the cortex, potentially leading to maintenance of seizure activity or long-term neuronal pathology. [Copyright &y& Elsevier]

Abstract: Glutamate receptor-mediated neurotoxicity is a major mechanism contributing to hypoxic–ischemic brain injury (HIBI). Memantine is a safe non-competitive NMDA receptor blocker characterized by its low affinity and fast unblocking kinetics. Topiramate is an AMPA/KA receptor blocker and use-dependent sodium channel blocker with several other neuroprotective actions and little neurotoxicity. We hypothesized that the coadministration of memantine and topiramate would be highly effective to attenuate HIBI in neonatal rats. Seven-day-old Sprague–Dawley rat pups were subjected to right common carotid artery ligation and hypoxia for 2 h, and then were randomly and blindly assigned to one of four groups: vehicle, memantine, topiramate and combination group. Brain injury was evaluated by gross damage and weight deficit of the right hemisphere at 22d after hypoxic-ischemia (HI) and by neurofunctional assessment (foot-fault test) at 21d post-HI. Acute neuronal injury was also evaluated by microscopic damage grading at 72 h post-HI. Results showed the combination of memantine and topiramate improved both pathological outcome and performance significantly. The drug-induced apoptotic neurodegeneration was assessed by TUNEL staining at 48 h post-HI and the result showed no elevated apoptosis in all observed areas. The result of the experiment indicates the combination therapy is safe and highly effective to reduce brain damage after HIBI. [Copyright &y& Elsevier]

Abstract: Brain oscillations provide a rich source of potentially useful endophenotypes (intermediate phenotypes) for psychiatric genetics, as they represent important correlates of human information processing and are associated with fundamental processes from perception to cognition. These oscillations are highly heritable, are modulated by genes controlling neurotransmitters in the brain, and provide links to associative and integrative brain functions. These endophenotypes represent traits that are less complex and more proximal to gene function than either diagnostic labels or traditional cognitive measures, providing a powerful strategy in searching for genes in psychiatric disorders. These intermediate phenotypes identify both affected and unaffected members of an affected family, including offspring at risk, providing a more direct connection with underlying biological vulnerability. Our group has utilized heritable neurophysiological features (i.e., brain oscillations) as endophenotypes, making it possible to identify susceptibility genes that may be difficult to detect with diagnosis alone. We have discussed our findings of significant linkage and association between brain oscillations and genes in GABAergic, cholinergic and glutamatergic systems (GABRA2, CHRM2, and GRM8). We have also shown that some oscillatory indices from both resting and active cognitive states have revealed a common subset of genetic foci that are shared with the diagnosis of alcoholism and related disorders. Implications of our findings have been discussed in the context of physiological and pharmacological studies on receptor function. These findings underscore the utility of quantitative neurophysiological endophenotypes in the study of the genetics of brain function and the genetic diathesis underlying complex psychiatric disorders. [Copyright &y& Elsevier]

Abstract: Electrical synapses play an important role in signaling between neurons and the synaptic connections between many neurons possess both electrical and chemical components. Although modulation of electrical synapses is frequently observed, the cellular processes that mediate such changes have not been studied as thoroughly as plasticity in chemical synapses. In the leech (Hirudo sp), the competitive AMPA receptor antagonist CNQX inhibited transmission at the rectifying electrical synapse of a mixed glutamatergic/electrical synaptic connection. This CNQX-mediated inhibition of the electrical synapse was blocked by concanavalin A (Con A) and dynamin inhibitory peptide (DIP), both of which are known to inhibit endocytosis of neurotransmitter receptors. CNQX-mediated inhibition was also blocked by pep2-SVKI (SVKI), a synthetic peptide that prevents internalization of AMPA-type glutamate receptor. AMPA itself also inhibited electrical synaptic transmission and this AMPA-mediated inhibition was partially blocked by Con A, DIP and SVKI. Low frequency stimulation induced long-term depression (LTD) in both the electrical and glutamatergic components of these synapses and this LTD was blocked by SVKI. GYKI 52466, a selective non-competitive antagonist of AMPA receptors, did not affect the electrical EPSP, although it did block the glutamatergic component of these synapses. CNQX did not affect non-rectifying electrical synapses in two different pairs of neurons. These results suggest an interaction between AMPA-type glutamate receptors and the gap junction proteins that mediate electrical synaptic transmission. This putative interaction between glutamate receptors and gap junction proteins represents a novel mechanism for regulating the strength of synaptic transmission. [Copyright &y& Elsevier]

Abstract: Abnormalities in glutamate neurotransmission are thought to be among the major contributing factors to the pathophysiology of schizophrenia. Although schizophrenia has been regarded mostly as a disorder of higher cortical function, the cortex and thalamus work as a functional unit. Existing data regarding alterations of glutamate receptor subunit expression in the thalamus in schizophrenia remain equivocal. This postmortem study examined mRNA expression of ionotropic glutamate receptor (iGluR) subunits and PSD95 in 5 precisely defined and dissected thalamic subdivisions (medial and lateral sectors of the mediodorsal nucleus; and the ventral lateral posterior, ventral posterior, and centromedian nuclei) of persons with schizophrenia and matched controls using quantitative PCR with normalization to multiple endogenous controls. Among 15 genes examined (NR1 and NR2A-D subunits of the NMDA receptor; GluR1-4 subunits of the AMPA receptor; GluR5-7 and KA1-2 subunits of the kainate receptor; PSD95), all but two (GluR4 and KA1) were expressed at quantifiable levels. Differences in iGluR gene expression were seen between different thalamic nuclei but not between diagnostic groups. The relative abundance of transcripts was: NR1≫NR2A>NR2B>NR2D>NR2C for NMDA, GluR2>GluR1>GluR3 for AMPA, and KA2>GluR5>GluR7>GluR6 for kainate receptors. The expression of PSD95 correlated with the expression of NR1, NR2A, NR2B, NR2D and GluR6 in all nuclei. These results provide detailed and quantitative information on iGluR subunit expression in multiple nuclei of the human thalamus but suggest that alterations in their expression are not a prominent feature of schizophrenia. [Copyright &y& Elsevier]

Abstract: AMPA-type glutamate receptors in the nucleus tractus solitarii (NTS) are necessary for the baroreceptor reflex, a primary mechanism for homeostatic regulation of blood pressure. Within NTS, the GluR1 subunit of the AMPA receptor is found primarily in dendritic spines. We previously showed that both GluR1 and dendritic spine density are increased in NTS of spontaneously hypertensive rats (SHRs). We hypothesize that both receptor and synaptic plasticity are induced by a sustained elevation in arterial pressure. To test the general nature of this hypothesis, we examined whether similar changes in GluR1 density are found in a renovascular model of hypertension, the DOCA–salt rat, and if these changes are preventable by normalizing blood pressure with hydralazine, a peripherally acting vasodilator. Using immunoperoxidase detection, GluR1 appears as small puncta at the light microscopic level, and is found in dendritic spines at the ultrastructural level. Following the development of hypertension, GluR1 spine and puncta counts were significantly greater in DOCA–salt rats than controls. Hydralazine treatment (4–5 weeks) prevented the development of hypertension in DOCA–salt rats and reduced blood pressure of SHRs to normotensive levels. The density of GluR1 puncta in the NTS was significantly reduced by hydralazine treatment in the SHR model. These results show that hypertension alters dendritic spines containing AMPA-type glutamate receptors within NTS, suggesting that adjustments in GluR1 expression within NTS are part of the synaptic adaptations to the hypertensive state. [Copyright &y& Elsevier]

Abstract: Kainate receptors (KARs) are involved in the modulation and transmission of nociceptive information from peripheral afferents to neurons in the spinal cord and trigeminal dorsal horns. KARs are found at both pre- and postsynaptic sites in the dorsal horn. We hypothesized that KARs and Substance P (SP), a modulatory neuropeptide that is used as a marker of nociceptive afferents, have a complex interactive relationship. To determine the cellular relationship and connectivity between KARs and SP afferents, we used electron microscopic dual immunocytochemical analysis to examine the ultrastructural localization of KAR subunits GluR5, 6 and 7 (GluR5,6,7) in relation to SP within laminae I and II in the rat trigeminal dorsal horn. KARs were distributed both postsynaptically in dendrites and somata (51% of GluR5,6,7 immunoreactive (-ir) profiles) and presynaptically in axons and axon terminals (45%). We also found GluR5,6,7-ir glial profiles (5%). The majority of SP-ir profiles were presynaptic axons and axon terminals. SP-ir dendritic profiles were rare, yet 23% contained GluR5,6,7 immunoreactivity. GluR5,6,7 and SP were also colocalized at presynaptic sites (18% of GluR5,6,7-ir axons and axon terminals contained SP; while 11% of SP-ir axons and axon terminals contained GluR5,6,7). The most common interaction between KARs and SP we observed was GluR5,6,7-ir dendrites contacted by SP-ir axon terminals; 54% of the dendritic targets of SP-ir axon terminals were GluR5,6,7-ir. These results provide anatomical evidence that KARs primarily mediate nociceptive transmission postsynaptic to SP-containing afferents and may also modulate the presynaptic release of SP and glutamate in trigeminal dorsal horn. [Copyright &y& Elsevier]

Abstract: The locus coeruleus (LC) is a major noradrenergic brain nucleus that regulates states of arousal, optimizes task-oriented decision making, and may also play an important role in modulating the activity of the reproductive neuroendocrine axis. Rodent studies have shown that the LC is responsive to glutamate receptor agonists, and that it expresses various glutamate receptor subunits. However, glutamate receptor subunit expression has not been extensively examined in the primate LC. We previously demonstrated expression of the NR1 NMDA glutamate receptor subunit in the rhesus macaque LC and now extend this work by also examining the expression of non-NMDA (AMPA and kainate) ionotropic glutamate receptor subunits. Using in situ hybridization histochemistry and immunohistochemistry, we confirmed the presence of the obligatory NR1 subunit in the LC. In addition, we demonstrated expression of the AMPA glutamate receptor subunits GluR1, GluR2, and GluR3. More extensive receptor profiling, using rhesus monkey gene microarrays (Affymetrix GeneChip®), further corroborated the histological findings and showed expression of mRNA encoding ionotropic glutamate receptor subunits NR2A, NR2D, GluR4, and GluR6, as well as the metabotropic glutamate receptor subunits mGluR1, mGluR3, mGluR4, mGluR5, and mGluR7. These data provide a foundation for future examination of how changes in glutamate receptor composition contribute to the control of primate physiology. [Copyright &y& Elsevier]

Abstract: Neuritic dystrophy, loss of synapses and neuronal death in the cerebral cortex and hippocampus are hallmarks of Alzheimer''s disease. The aim of the present study was to investigate the differential susceptibility of cortical and hippocampal neurons to amyloid-beta (Aβ)-induced toxicity. For that, we have used primary neuronal cultures prepared from rat brain cortex and hippocampus which were treated with the synthetic peptides Aβ25-35 or Aβ1-40. Aβ-induced apoptotic cell death was analyzed by determining caspase-3-like activity. Neuritic dystrophy was evaluated by cobalt staining and MAP2 immunoreactivity. Perturbation of Ca2+ homeostasis caused by exposure to Aβ was evaluated by determining basal cytosolic calcium levels in the whole neuronal population and by single cell calcium imaging under basal and KCl-depolarization conditions. Finally, levels of GluR2 subunit of glutamate AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-proprionate) receptors were quantified by western blotting. Our results demonstrated that hippocampal neurons in culture are more susceptible than cortical neurons to Aβ-induced apoptosis and also that this mechanism involves the perturbation of Ca2+ homeostasis. Accordingly, the exposure of hippocampal neurons to Aβ peptides decreases the protein levels of the GluR2 subunit of glutamate AMPA receptors that may be associated with a significant rise of cytosolic Ca2+ concentration, leading to dendritic dystrophy and activation of apoptotic neuronal death. [Copyright &y& Elsevier]

Abstract: The Lurcher mouse was first discovered in 1954 as a spontaneously occurring autosomal dominant mutation that caused the degeneration of virtually all cerebellar Purkinje cells and most olivary neurons and granule cells. More recent molecular studies revealed that Lurcher is a gain of function mutation in the δ2 glutamate receptor (GluRδ2) that converts an alanine to threonine in the highly conserved third hydrophobic segment of GluRδ2. The mutation converts the receptor into a constitutively leaky cation channel. The GluRδ2 receptor is predominantly expressed in cerebellar Purkinje cells and in the heterozygous Lurcher mutant (+/Lc). Purkinje cells die due to the mutation in the GluRδ2 receptor, while olivary neurons and granule cells degenerate due to the loss of their Purkinje cell targets. The purpose of the review is to provide highlights from 5 decades of research on the Lurcher mutant that have provided insights into the developmental mechanisms that regulate cell number during development, cerebellar pattern formation, cerebellar physiology, and the role of the cerebellum in CNS function. [Copyright &y& Elsevier]

Abstract: In order to understand whether glutamatergic excitatory presynaptic input is an absolute requirement for the adult regulation of postsynaptic glutamate receptors we analyzed if a period of 11 days of excitatory deprivation affects the expression, distribution and Ca2+ permeability of AMPA receptor subunits in the ventral cochlear nucleus of the rat. Bilateral cochlear ablations were performed in 30-day-old rats. After 11 days of survival, immunohistochemistry for GluR1, GluR2/3 and GluR4 AMPA receptor subunits showed no changes in the normal pattern of distribution, with GluR2/3 and GluR4 immunoreactivity predominating, and little GluR1. No changes in the amount of these AMPA receptor subunits were found between normal and cochleotomized rats in Western blots. AMPA receptors lacking the GluR2 subunit are Ca2+ permeable. Kainate-induced Co2+ uptake histochemistry, which labels AMPA Ca2+ permeable receptors, demonstrated no changes in somatic labeling intensity for Co2+, 11 days after cochleotomy. Therefore, our data indicate that excitatory input is not an absolute requirement to maintain AMPA receptor subunit expression, distribution and functional properties such as Ca2+ permeability in VCN neurons. Nevertheless, subtle changes in AMPA receptors through regulatory post-transductional mechanisms cannot be ruled out. [Copyright &y& Elsevier]

Abstract: Neuronal excitability is inhibited by somatostatin, which might play important roles in seizure and neuroprotection. The possibility of whether the effect of somatostatin on neurotransmission is susceptible to desensitization was investigated. We tested the effects of prolonged exposure to somatostatin on 0.1 mM extracellular Mg2+ concentration ([Mg2+]o)-induced intracellular free Ca2+ concentration ([Ca2+]i) spikes in cultured rat hippocampal neurons using fura-2-based microfluorimetry. Reducing [Mg2+]o to 0.1 mM elicited repetitive [Ca2+]i spikes. These [Ca2+]i spikes were inhibited by exposure to somatostatin-14. The inhibitory effects of somatostatin were blocked by pretreatment with pertussis toxin (PTX, 100 ng/ml) for 18–24 h. Prolonged exposure to somatostatin induced a desensitization of the somatostatin-induced inhibition of [Ca2+]i spikes in a concentration-dependent manner. The somatostatin-induced desensitization was retarded by the nonspecific protein kinase C (PKC) inhibitor staurosporin (100 nM) or chronic treatment with phorbol dibutyrate (1 μM) for 24 h, but not by the protein kinase A inhibitor KT5720. The desensitization was significantly retarded by the novel PKCε  translocation inhibitor peptide (1 μM). In addition, suramin (3 μM), an inhibitor of G-protein-coupled receptor kinase 2 (GRK2), caused a reduction in the desensitization. After tetrodotoxin (TTX, 1 μM) completely blocked the low [Mg2+]o-induced [Ca2+]i spikes, glutamate-induced [Ca2+]i transients were slightly inhibited by somatostatin and the inhibition was desensitized by prolonged exposure to somatostatin. These results indicate that the prolonged activation of somatostatin receptors induces the desensitization of somatostatin-induced inhibition on low [Mg2+]o-induced [Ca2+]i spikes through the activation of GRK2 and partly a novel PKCε in cultured rat hippocampal neurons. [Copyright &y& Elsevier]

Abstract: Several molecules, involved in cellular communication in the mature nervous system, appear to play important roles during neural development. These roles include neuronal growth, morphological changes of neurites, and neuronal survival. Such plasticity processes seem to be in part the result of activation of different receptor subtypes, which could cause Ca2+ influx, a major candidate to be an outgrowth promoter. In this context, we performed immunohistochemical and in situ hybridization experiments to examine the following aspects of the development of chick cerebellum Purkinje cells: (i) expression of AMPA-type glutamate receptor GluR2/3 proteins; (ii) the levels of mRNAs coding for the GluR2 and GluR3 flip/flop isoforms; and (iii) expression of calbindin (CB) and parvalbumin (PV). Expression of GluR2/3 proteins, CB, PV, and the mRNAs coding for GluR2 and GluR3 splice variants all revealed a differential expression during development in chick Purkinje cells. GluR2/3 proteins and the GluR3 flop variant start to be expressed at E10, while the expression of CB, PV, the GluR3 flip isoform and the splice variants of GluR2 all started around E12–E14. All proteins showed an increasing expression from embryonic stages into the posthatching period. These results reveal a developmentally regulated expression of GluR2/3 proteins, including their splice variants, and of CB and PV in Purkinje cells. These findings may suggest a relationship between these proteins and specific cerebellar developmental processes. [Copyright &y& Elsevier]

Abstract: This study was designed to evaluate the role of spinal glutamatergic receptors in the antinociception elicited by agmatine in mice. Intraperitoneal (i.p.) administration of agmatine (1.0–100.0 mg/kg) dose dependently inhibited the nociceptive response induced by intrathecal (i.t.) injection of glutamate, N-methy-d-aspartate (NMDA) and (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), with mean ID50 values of 16.7, 6.8 and 27.0 mg/kg, respectively. However, agmatine completely failed to affect the nociception induced by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainic acid (kainate). Agmatine injected by i.t. route (10–100 μg/site) also produced dose-related inhibition of NMDA- and trans-ACPD-induced biting response with mean ID50 values of 29.6 and 36.0 μg/site, respectively. The nitric oxide synthase inhibitor N ω-nitro-l-arginine (l-NOARG) (75.0 mg/kg, i.p.) also consistently inhibited glutamate-, NMDA- and trans-ACPD-induced nociception (41 ± 13, 100 and 83 ± 6%, of inhibition, respectively) but had no effect on the same response caused by AMPA and kainate agonists. The selective NMDA receptor antagonist (5S,10R)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d] (MK-801) at a low dose (0.05 mg/kg, i.p.) inhibited the nociceptive response caused by both glutamate and NMDA agonists (inhibitions of 35 ± 1 and 72 ± 2%, respectively). At a high dose, MK-801 (0.5 mg/kg, i.p.) significantly inhibited the biting response induced by i.t. administration of all the glutamatergic agonists tested: glutamate, AMPA, NMDA, kainate and trans-ACPD, with inhibitions of 49 ± 8, 84 ± 16, 84 ± 3, 76 ± 8 and 97 ± 2%, respectively. Together, these results provide experimental evidence indicating that agmatine given systemically and spinally produce marked antinociception at spinal sites in mice. Furthermore, an interaction with glutamate receptors, namely NMDA and trans-ACPD, metabotropic and NMDA-ionotropic origin, by a mechanism similar to that of nitric oxide (NO) inhibitors, seems to account for the agmatine antinociceptive action. [Copyright &y& Elsevier]

Abstract: We have studied the effects of AMPA/kainate receptor agonists on GABAA receptor subunit mRNA expression in vitro in cultured rat cerebellar granule cells (CGCs). Kainate (KA) (100 μM) and high K+ (25 mM) dramatically up-regulated δ subunit mRNA expression to 500–700% of that in control cells grown in low K+ (5 mM). KA or high K+ had no effect on the expression of the other major GABAA receptor subunits α1, α6, β2, β3 or γ2. Up-regulation of δ mRNA was also detected with the AMPA receptor-selective agonist CPW-399 and to a lesser extent with the KA receptor-selective agonist ATPA. AMPA/kainate receptor-selective antagonist DNQX completely inhibited KA-, CPW-399- and ATPA-induced δ mRNA up-regulation indicating that the effects were mediated via AMPA and KA receptor activation. NMDA receptor-selective antagonist MK-801 inhibited 76% of the KA- and 57% of the CPW-399-induced δ up-regulation suggesting that KA and CPW-399 treatments may induce glutamate release resulting in NMDA receptor activation, and subsequently to δ mRNA up-regulation. In CGCs, δ subunit is a component of extrasynaptic α6βδ receptors that mediate tonic inhibition. Up-regulation of δ during prolonged glutamate receptor activation or cell membrane depolarization may be a mechanism to increase tonic inhibition to counteract excessive excitation. [Copyright &y& Elsevier]

Abstract: Physical activity has been proposed as a behavior intervention that promotes mental health and some of the benefits induced by exercise have been related to the glutamatergic system. Indeed, glutamate is the most abundant excitatory neurotransmitter in brain. Thus, we evaluated if voluntary exercise in mice could modulate glutamatergic synapses at level of postsynaptic density (PSD). Through Western blot, we found that exercise during 1 month increased glutamatergic-related protein content in PSD from cortex of mice. Exercise increased the immunocontent of GluR1 (129%), SAP-97 (179%), GRIP-1 (129%), and in less extent, GluR2/3 (118%) and PSD-95 (112%) proteins. The overall content of NMDA subunits R1, R2A and R2B were not altered in mice that had exercised, however, the phosphorylated NMDA subunits, phospho-NMDAR1 (150%), and phospho-NMDAR2B (183%) showed a strong increase. Because exercise increased the content of phosphorylated forms of NMDA receptors, we evaluated the binding of MK-801, a specific ligand that binds to open NMDA channel. Exercise increased the binding of MK-801 in cortical cellular membranes in 51%. Altogether, our results point to a modulation of glutamatergic synapses by exercise with likely implications in the exercise-induced mental health. [Copyright &y& Elsevier]

This study was aimed to investigate the expression of glutamate receptors and calcium-binding proteins in 1- and 4-month/s (mo) streptozotocin (STZ)-induced diabetic rats. Upregulation of glutamate receptors'' [N-methyl-d-aspartate receptor (NMDAR)1 and GluR2/3] immunoreactivities was observed in the ganglion, amacrine and bipolar cells as well as in the inner and outer plexiform layers (IPL and OPL) in 1 mo diabetes and was further enhanced at 4 mo. Immunoreactivity of calcium-binding proteins (calbindin and parvalbumin) was also concomitantly increased. The present results suggest that upregulation of glutamate receptors and calcium-binding proteins may reflect changes of the glutamate and calcium metabolism in the diabetic retina. It is speculated that the above changes in the IPL and OPL may be linked to alteration of synaptic transmission in the diabetic retina. [Copyright &y& Elsevier]

Carbon monoxide (CO) poisoning caused by CO exposure at 3000 ppm for 40 min resulted in stimulation of hydroxyl radical (⋅OH) generation (estimated by measuring 2,3-dihydroxybenzoic acid (2,3-DHBA) production from salicylic acid) in the striatum of free-moving rats, as determined by means of brain microdialysis. Pretreatment with a voltage-dependent Na+ channel blocker, tetrodotoxin (TTX), lowered the basal level of 2,3-DHBA and strongly suppressed the increase in 2,3-DHBA induced by CO poisoning. CO poisoning significantly, though only slightly, increased extracellular glutamate in the striatum, and glutamate (Glu) receptor antagonists, such as MK-801 (dizocilpine) and NBQX, failed to suppress the CO-induced increase in 2,3-DHBA. These findings suggest that CO poisoning may induce Na+ influx via the voltage-dependent Na+ channels, resulting in stimulation of ⋅OH generation in rat striatum. This effect may be independent of Glu receptor activation by increased extracellular Glu. [Copyright &y& Elsevier]