Search

Previous studies have indicated that presynaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) contribute to the regulation of neurotransmitter release. In hippocampal synapses, the presynaptic surface expression of several AMPAR subunits, including GluA2, is regulated in a ligand-dependent manner. However, the molecular mechanisms underlying the presynaptic trafficking of AMPARs are still unknown. Here, using bright-field immunocytochemistry, western blots, and quantitative immunogold electron microscopy of the hippocampal CA1 area from intact adult rat brain, we demonstrate the association of AMPA receptors with the presynaptic active zone and with small presynaptic vesicles, in Schaffer collateral synapses in CA1 of the hippocampus. Furthermore, we show that GluA2 and protein interacting with C kinase 1 (PICK1) are colocalized at presynaptic vesicles. Similar to postsynaptic mechanisms, overexpression of either PICK1 or pep2m, which inhibit the N-ethylmaleimide sensitive fusion protein (NSF)-GluA2 interaction, decreases the concentration of GluA2 in the presynaptic active zone membrane. These data suggest that the interacting proteins PICK1 and NSF act as regulators of presynaptic GluA2-containing AMPAR trafficking between the active zone and a vesicle pool that may provide the basis of presynaptic components of synaptic plasticity. [ABSTRACT FROM AUTHOR]

The neuropeptide PACAP modulates synaptic transmission in the hippocampus exerting multiple effects through different receptor subtypes: the underlying mechanisms have not yet been completely elucidated. The neurotransmitter acetylcholine (ACh) also exerts a well-documented modulation of hippocampal synaptic transmission and plasticity. Since PACAP was shown to stimulate ACh release in the hippocampus, we tested whether PACAP acting through ACh might indirectly modulate glutamate-mediated synaptic transmission at a pre- and/or at a post-synaptic level. Using patch clamp on rat hippocampal slices, we tested PACAP effects on stimulation-evoked AMPA receptor-mediated excitatory post-synaptic currents (EPSCs AMPA ) in the CA3-CA1 synapse and on spontaneous miniature EPSCs (mEPSCs) in CA1 pyramidal neurons. A subnanomolar dose of PACAP (0.5 nM) decreased EPSCs AMPA amplitude, enhanced EPSC paired-pulse facilitation (PPF) and reduced mEPSC frequency, indicating a pre-synaptic decrease of glutamate release probability: these effects were abolished by simultaneous blockade of muscarinic and nicotinic ACh receptors, indicating the involvement of endogenous ACh. The effect of subnanomolar PACAP was abolished by a PAC1 receptor antagonist but not by a VPAC receptor blocker. At a higher concentration (10 nM), PACAP inhibited EPSCs AMPA : this effect persisted in the presence of ACh receptor antagonists and did not involve any change in PPF or in mEPSC frequency, thus was not mediated by ACh and was exerted post- synaptically on CA1 pyramidal neurons. We suggest that a high-affinity PAC1 receptor pre-synaptically modulates hippocampal glutamatergic transmission acting through ACh. Therefore, administration of PACAP at very low doses might be envisaged in cognitive diseases with reduced cholinergic transmission. [ABSTRACT FROM AUTHOR]

Previous studies investigating the pathophysiology of neuropathic pain caused by injury to the spinal cord suggest that pain may result, at least in part, from maladaptive plasticity in the somatosensory cortex and associated pain networks. However, little is known about the molecular and cellular mechanisms leading to maladaptive plasticity in the cortex and how they contribute to the development of neuropathic pain. AMPA-type glutamate receptors (GluARs) mediate fast excitatory synaptic transmission in the mammalian brain and play an important role in pain processing. Here we used an electrolytic lesion model of spinal cord injury in animals to study the expression and phosphorylation of GluA1 and 2 in the primary somatosensory cortex (S1). Experiments in rats and mice revealed that maladaptive plasticity and hypersensitivity after spinal cord lesion (SCL) are associated with a reduction in the fraction of GluA1 subunits that are phosphorylated at serine 831 (S831) in the hindlimb representation of S1 (S1HL). Manipulations that reduce the fraction of phosphorylated S831 in S1HL of non-lesioned animals, including low-frequency electrical stimulation and viral-mediated gene transfer of mutant S831, were associated with the development of hypersensitivity. Taken together, these findings suggest that phosphorylation of GluA1 at S831 plays an important role in the development of hypersensitivity after SCL. [ABSTRACT FROM AUTHOR]

Meloxicam is a non-steroidal anti-inflammatory drug which has been reported to lessen the ischemic transcriptional effects in some of the glutamatergic system genes as well as to decrease the infarct volume in in vivo assays. In this study, we show how the presence of meloxicam decreases cell mortality in assays of oxygen–glucose deprivation (OGD) in rat organotypic hippocampal slices culture. Mortality was measured using propidium iodide. Transcript levels of some glutamatergic system genes, including vesicular and membrane glutamate transporters (VGLUT1, VGLUT2, GLAST-1A, GLT-1, and EAAC-1) and some glutamatergic receptor subunits (NMDA receptor, GluN1, GluN2A and GluN2B subunits and AMPA receptor, GluA1 and GluA2 subunits) were measured by real-time PCR (qPCR). The transcription of vesicular glutamate transporters and glutamatergic receptor subunits, but not membrane glutamate transporters, was modified by the presence of meloxicam. The study demonstrates the neuroprotective role of meloxicam in organotypic hippocampal slice cultures and shows how meloxicam is able to selectively increase or decrease the OGD-induced changes in the expression of the different glutamatergic system genes studied here. We suggest that the neuroprotective role of meloxicam could be due to a modification in the balance of the expression of some glutamatergic receptor subunits, leading to a different stoichiometry of receptors such as NMDA or AMPA. Thus, meloxicam would decrease the excitotoxicity induced by OGD. [ABSTRACT FROM AUTHOR]

Modulation of dopamine (DA) released by serotonin-2 (5-HT2) receptors has been implicated in the mechanism of action of antipsychotic drugs. The mesocortical DA system has been implicated particularly in the cognitive deficits observed in schizophrenia. Agonism at 5-HT2A receptors in the prefrontal cortex (PFC) is associated with increases in cortical DA release. Evidence indicates that 5-HT2A receptors in the cortex regulate mesocortical DA release through stimulation of a “long-loop” feedback system from the PFC to the ventral tegmental area (VTA) and back. However, a causal role for VTA glutamate in the 5-HT2-induced increases in PFC DA has not been established. The present study does so by measuring 5-HT2 agonist-induced DA release in the cortex after infusions of glutamate antagonists into the VTA of the rat. Infusions of a combination of a N -methyl- d -aspartic acid (NMDA) (AP-5: 2-amino-5-phosphopentanoic acid) and an AMPA/kainate (CNQX: 6-cyano-7-nitroquinoxaline-2,3-dione) receptor antagonist into the VTA blocked the increases in cortical DA produced by administration of the 5-HT2 agonist DOI [(±)-2,5-dimethoxy-4-iodoamphetamine] (2.5 mg/kg s.c.). These results demonstrate that stimulation of glutamate receptors in the VTA is necessary for 5-HT2 agonist-induced increases in cortical DA. [ABSTRACT FROM AUTHOR]

Highlights: [•] Stress can result in highly adaptive responses but is also an important risk factor for disease. [•] Acute stress rapidly enhances excitatory synaptic transmission and promotes learning and memory processes. [•] Chronic stress reduces excitatory synaptic transmission and learning and memory. [•] Early life experience has long-lasting effects on excitatory synaptic transmission and dendritic complexity. [ABSTRACT FROM AUTHOR]

Highlights: [•] Distinct mouse OFF bipolar cell types express different ionotropic glutamate receptor types. [•] Some OFF bipolar cell types express either AMPA or kainate receptors. [•] Some OFF bipolar cell types express both AMPA and kainate receptors. [•] AMPA and kainate receptors are expressed at separate synaptic sites. [ABSTRACT FROM AUTHOR]

Abstract: In hippocampal Cornu Ammonis 1 (CA1) neurons, a prolonged depolarization evokes a train of action potentials followed by a prominent afterhyperpolarizing potential (AHP), which critically dampens neuronal excitability. Because it is not known whether epileptiform activity alters the AHP and whether any alteration of the AHP is independent of inhibition, we acutely induced epileptiform activity by bath application of the GABAA receptor blocker gabazine (5 μM) in the rat hippocampal slice preparation and studied its impact on the AHP using intracellular recordings. Following 10 min of gabazine wash-in, slices started to develop spontaneous epileptiform discharges. This disinhibition was accompanied by a significant shift of the resting membrane potential of CA1 neurons to more depolarized values. Prolonged depolarizations (600 ms) elicited a train of action potentials, the number of which was not different between baseline and gabazine treatment. However, the AHP following the train of action potentials was significantly reduced after 20 min of gabazine treatment. When the induction of epileptiform activity was prevented by co-application of 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX, 10 μM) and d-(−)-2-amino-5-phosphonopentanoic acid (d-AP5, 50 μM) to block α-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) and N-methyl-d-aspartate (NMDA) receptors, respectively, the AHP was preserved despite of GABAA receptor inhibition suggesting that the epileptiform activity was required to suppress the AHP. Moreover, the AHP was also preserved when the slices were treated with the protein kinase blockers H-9 (100 μM) and H-89 (1 μM). These results demonstrate that the AHP following a train of action potentials is rapidly suppressed by acutely induced epileptiform activity due to a phosphorylation process—presumably involving protein kinase A. [Copyright &y& Elsevier]

Abstract: Interactions between dopamine and glutamate receptors are essential for the prefrontal cortical (PFC) and hippocampal cognitive functions. In order to understand the molecular basis of dopamine/glutamate interactions in rat PFC and hippocampus, we investigated (a) the effect of in vitro dopamine D1 receptor stimulation on glutamate N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits'' phosphorylation and (b) the signal transduction pathway underlying these interactions, by examining the involvement of D1–extracellular regulated kinase 1/2 (ERK1/2) and D1/protein kinase A (PKA)/dopamine- and cyclic AMP-regulated phosphoprotein-32 (DARPP-32) signaling pathways. Furthermore, we compared the D1/NMDA/AMPA receptor interactions seen in PFC and hippocampus with those appearing in striatum, in which the D1 receptors'' density is the highest within the mammalian brain. Our results showed that stimulation of D1 receptor by the specific agonist SKF38393 (10 μM) in PFC and hippocampal slices significantly increased the phosphorylation state of NR1ser897 and NR2Bser1303 subunits of NMDA receptor and of the GLUR1 (ser831 and ser845) subunit of AMPA receptor, as well as of ERK1/2, but not of DARPP-32. Interestingly, co-stimulation of D1 and NMDA receptors with an ineffective dose of SKF38393 (2 μM) and NMDA (5 μM) respectively, elevated further the phosphorylation level of NMDA and AMPA receptor subunits, as well as of ERK1/2, but not of DARPP-32. The D1- and D1/NMDA-induced phosphorylations were totally inhibited by SL327 (specific ERK1/2 inhibitor). Conversely, in striatal slices our data confirm that the D1-mediated phosphorylation of NMDA and AMPA receptor subunits relies on D1/PKA/DARPP-32 signaling. In conclusion, in PFC and hippocampus: (a) a strong synergistic interaction of D1 and NMDA receptors exists, which results in a significant ERK1/2 pathway activation, (b) the D1 and the D1/NMDA receptor-induced phosphorylation of NMDA and AMPA receptor subunits seems to rely on ERK1/2 signaling and could to some extent underlie the enhancement of NMDA and AMPA receptor currents mediated by D1 receptor activation. [Copyright &y& Elsevier]

Abstract: Several lines of evidence strongly suggest that brain-derived neurotrophic factor (BDNF) is associated with the formation, storage and recall of memory in the hippocampus and that it is important to maintain a considerable level of hippocampal BDNF in order to keep normal functions. BDNF can be synthesized in an activity-dependent manner. In fact, kainic acid or AMPA enhances BDNF levels in hippocampal granule neurons. However, the mechanisms of BDNF production are largely unclear. Recently, we have found that riluzole, which blocks voltage-gated sodium channels and thereby reduces glutamate release, actually strengthens immunoreactivity of BDNF in hippocampal granule neurons of rats. Therefore, we examined the riluzole-activated signaling pathways for BDNF production. Riluzole increased levels of phospho-p38 mitogen-activated protein kinase (p38 MAPK), as well as BDNF levels. Inhibition of p38 MAPK by SB203580 reduced riluzole effects, while activation of p38 MAPK by anisomycin increased levels of BDNF, suggesting that p38 MAPK can mediate BDNF production. Riluzole-induced elevation of phospho-activating transcription factor-2, a transcription factor downstream of p38 MAPK, was also observed. A blocker of N-type voltage-gated calcium channels reduced the effects of riluzole on BDNF production and p38 MAPK activation. We also examined a possible involvement of the adenosine A1 receptor in BDNF production because riluzole can influence ecto-nucleotide levels. An A1 receptor agonist inhibited riluzole-induced elevation of BDNF levels, whereas an antagonist not only increased levels of BDNF and active p38 MAPK but also augmented riluzole effects. These results indicate that, in the rat hippocampus, there is an in vivo signaling pathway for BDNF synthesis mediated by p38 MAPK, and that N-type voltage-gated calcium channels and/or adenosine A1 receptors contribute to p38 MAPK activation. [Copyright &y& Elsevier]

Abstract: Tumor necrosis factor (TNF)-alpha is a proinflammatory cytokine acting on two distinct receptor subtypes, namely p55 and p75 receptors. TNF-alpha p55 and p75 receptor knockout mice were previously shown to display a decreased or enhanced susceptibility to seizures, respectively, suggesting intrinsic modifications in neuronal excitability. We investigated whether alterations in glutamate system function occur in these naive knockout mice with perturbed cytokine signaling that could explain their different propensity to develop seizures. Using Western blot analysis of hippocampal homogenates, we found that p55−/− mice have decreased levels of membrane GluR3 and NR1 glutamate receptor subunits while GluR1, GluR2, GluR6/7 and NR2A/B were unchanged as compared to wild-type mice. In p75−/− mice, GluR2, GluR3, GluR6/7 and NR2A/B glutamate receptor subunits were increased in the hippocampus while GluR1 and NR1 did not change. Extracellular single-cell recordings of the electrical activity of hippocampal neurons were carried out in anesthetized mice by standard electrophysiological techniques. Microiontophoretic application of glutamate increased the basal firing rate of hippocampal neurons in p75−/− mice versus wild-type mice, and this effect was blocked by 2-amino-5-phosphopentanoic acid and 6-nitro-7-sulfamoyl-benzo(f)quinoxaline-2,3-dione denoting the involvement of N-methyl-d-aspartic acid and AMPA receptors. In p55−/− mice, hippocampal neurons responses to glutamate were similar to wild-type mice. Spontaneous glutamate release measured by in vivo hippocampal microdialysis was significantly decreased only in p55−/− mice. No changes were observed in KCl-induced glutamate release in both receptor knockout mice strains versus wild-type mice. These findings highlight specific molecular and functional interactions between p55 and p75 receptor-mediated signaling and the glutamate system. These interactions may be relevant for controlling neuronal excitability in physiological and pathological conditions. [Copyright &y& Elsevier]

Abstract: Glutamatergic signaling plays an important role in the behavioral and molecular plasticity observed in behavioral sensitization to cocaine. Redistribution of the glutamate receptors in the synaptosomal membrane fraction was investigated in the nucleus accumbens, dorsolateral striatum, and ventral tegmental area at 1 or 21 days of withdrawal in behaviorally sensitized rats. At 1 day of withdrawal, there were no changes in either tissue level or redistribution of glutamate receptors in nucleus accumbens core and shell and ventral tegmental area. At 21 days of withdrawal, there was a decrease in the expression of mGluR2/3 protein in core and shell, an increase in GluR1 and a decrease in Homer1b/c proteins in the nucleus accumbens core tissue. In dorsolateral striatum, the tissue level of NMDAR2B protein was increased. Moreover, there was an augmented presence of AMPA (GluR1, GluR2), NMDA (NMDAR1, 2A, 2B), and group 1 metabotropic glutamate receptor (mGluR5) proteins in the synaptosomal fraction in core and shell of the nucleus accumbens. There was also an increase in synaptosomal mGluR2/3 protein in nucleus accumbens core. The redistribution of glutamate receptors was selective for nucleus accumbens since no changes were observed in the dorsolateral striatum and ventral tegmental area. While the tissue level of the Homer1b/c protein was selectively reduced in nucleus accumbens core, that of PSD95, PICK1, and actin was not changed in any of the brain regions examined. However, the synaptosomal membrane fraction level of Homer1b/c and PSD95 was increased in nucleus accumbens core and shell, with no changes in PICK1, and a decrease in actin protein. These observations suggest that significant redistribution of glutamate receptors and postsynaptic scaffolding proteins into synaptosomal membrane fraction is associated with withdrawal from behavioral sensitization to cocaine. [Copyright &y& Elsevier]

Abstract: Epidermal growth factor (EGF) has a neurotrophic activity on developing midbrain dopaminergic neurons. We investigated developmental effects of peripheral EGF administration on dopaminergic neurons in midbrain slice preparations containing ventral tegmental area (VTA). Subcutaneous EGF administration to mouse neonates triggered phosphorylation of EGF receptors (ErbB1 and ErbB2) in the midbrain region, suggesting its penetration through the blood–brain barrier. We repeated EGF administration in postnatal mice and examined synaptic transmission in the VTA with electrophysiological recordings. Subchronic EGF treatment increased the amplitude of field excitatory postsynaptic potentials evoked by stimulation of the anterior VTA. To analyze the EGF effect at a single cell level, dopaminergic neurons were identified by their characteristic hyperpolarizing activated currents in whole cell recording. In these dopaminergic neurons, EGF effects the amplitude of spontaneous miniature excitatory postsynaptic currents (mEPSCs) without affecting their frequency. In agreement, EGF also enhanced the AMPA/NMDA ratio of evoked EPSCs in the dopaminergic neurons. In contrast, EGF effects on mEPSCs of neighboring neurons not exhibiting hyperpolarizing activated currents were modest or insignificant. Thus, these results suggest that circulating EGF substantially influences the physiological properties of developing midbrain dopaminergic neurons in perinatal and postnatal mice. [Copyright &y& Elsevier]

Abstract: Substance P (SP) and glutamate are implicated in cardiovascular regulation by the nucleus tractus solitarii (NTS). Our earlier studies suggest that SP, which acts at neurokinin 1 (NK1) receptors, is not a baroreflex transmitter while glutamate is. On the other hand, our recent studies showed that loss of NTS neurons expressing NK1 receptors leads to loss of baroreflex responses and increased blood pressure lability. Furthermore, studies have suggested that SP may interact with glutamate in the NTS. In this study, we sought to test the hypothesis that NK1 receptors colocalize with glutamate receptors, either N-methyl-d-aspartate (NMDA) receptors or AMPA receptors or both in the NTS. We performed double-label immunofluorescent staining for NK1 receptors and either N-methyl-d-aspartate receptor subunit 1 (NMDAR1) or AMPA specific glutamate receptor subunit 2 (GluR2) in the rat NTS. Because vesicular glutamate transporter 2 (VGLUT2) containing fibers are prominent in portions of the NTS where cardiovascular afferent fibers terminate, we also performed double-label immunofluorescent staining for NK1 receptors and VGLUT2. Confocal microscopic images showed that NK1 receptors-immunoreactivity (IR) and NMDAR1-IR colocalized in the same neurons in many NTS subnuclei. Almost all NTS neurons positive for NK1 receptor-IR also contained NMDAR1-IR, but only 53.4% to 74.8% of NMDAR1-IR positive neurons contained NK1 receptors-IR. NK1 receptor-IR and GluR2-IR also colocalized in many neurons in NTS subnuclei. A majority of NK1 receptor-IR positive NTS neurons also contained GluR2-IR, but only 45.8% to 73.9% of GluR2-IR positive NTS neurons contained NK1 receptors-IR. Our results also showed that fibers labeled for VGLUT2-IR were in close apposition to fibers and neurons labeled for NK1 receptor-IR. The data support our hypothesis, provide an anatomical framework for glutamate and SP interactions, and may explain the loss of baroreflexes when NTS neurons, which could respond to glutamate as well as SP, are killed. [Copyright &y& Elsevier]

Abstract: The bed nucleus of the stria terminalis (BST) is a brain structure located at the interface of the cortex and the cerebrospinal trunk. The BST is a cluster of nuclei organized in a complex intrinsic network that receives inputs from cortical and subcortical sources, and that sends a widespread top-down projection. There is growing evidence that the BST is a key component in the neurobiological basis of substance abuse. In the present study, the regulation of excitatory inputs onto identified neurons in the BST was examined in rats treated chronically with morphine. Neurons projecting to the ventral tegmental area (VTA) were identified by retrograde transport of fluorescent microspheres and recorded in the whole-cell voltage clamp configuration in brain slices. Selective excitatory inputs to these neurons were electrically evoked with electrodes placed in the medial and lateral aspects of the dorsal BST. The chronic morphine treatment selectively increased AMPA-dependent excitatory postsynaptic currents in a subset of inputs activated by dorso-lateral stimulation in the BST. Inputs activated by medial stimulation were not affected by morphine. Likewise, the inputs to neurons that did not project to the VTA were not changed by morphine. Altogether, these results extend the understanding of neuronal circuits intrinsically sensitive to drugs of abuse within the BST. [Copyright &y& Elsevier]

Abstract: Development of the mammalian CNS requires formation and stabilization of neuronal circuits and synaptic connections. Sensory stimulation provided by the environment orchestrates neuronal circuit formation in the waking state. Endogenous sources of activation are also implicated in these processes. Accordingly we hypothesized that sleep, especially rapid eye movement sleep (REMS), the stage characterized by high neuronal activity that is more prominent in development than adulthood, provides endogenous stimulation, which, like sensory input, helps to stabilize and refine neuronal circuits during CNS development. Young (Y: postnatal day (PN) 16) and adolescent (A: PN44) rats were rapid eye movement sleep–deprived (REMSD) by gentle cage-shaking for only 4 h on 3 consecutive days (total 12 h). The effect of REMS deprivation in Y and A rats was tested 3–7 days after the last deprivation session (Y, PN21–25; A, PN49–53) and was compared with younger (immature, I, PN9–12) untreated, age-matched, treated and normal control groups. REMS deprivation negatively affected the stability of long-term potentiation (LTP) in Y but not A animals. LTP instability in Y-REMSD animals was similar to the instability in even the more immature, untreated animals. Utilizing immunoblots, we identified changes in molecular components of glutamatergic synapses known to participate in mechanisms of synaptic refinement and plasticity. Overall, N-methyl-d-aspartate receptor subunit 2B (NR2B), N-methyl-d-aspartate receptor subunit 2A, AMPA receptor subunit 1 (GluR1), postsynaptic density protein 95 (PSD-95), and calcium/calmodulin kinase II tended to be lower in Y REMSD animals (NR2B, GluR1 and PSD-95 were significantly lower) compared with controls, an effect not present in the A animals. Taken together, these data indicate that early-life REMS deprivation reduces stability of hippocampal neuronal circuits, possibly by hindering expression of mature glutamatergic synaptic components. The findings support a role for REMS in the maturation of hippocampal neuronal circuits. [Copyright &y& Elsevier]

Abstract: The mechanism by which the sedative and amnestic recreational drug gamma hydroxybutyric acid (GHB) acts is controversial. Some studies indicate that it acts at its unique receptor, while others demonstrate effects mediated through the GABAB receptor. We examined the effect of GHB on evoked GABAA receptor–mediated mono- and polysynaptic inhibitory postsynaptic currents (IPSCs) as well as on N-methyl-d-aspartate (NMDA) and AMPA-mediated excitatory postsynaptic currents (EPSCs) in layers II/III pyramidal cells of the frontal cortex of rat brain. One millimolar (mM) GHB suppressed monosynaptic IPSCs by 20%, whereas polysynaptic IPSCs were reduced by 56%. GHB (1 mM) also produced a significant suppression of NMDA-mediated EPSCs by 53% compared with 27% suppression of AMPA-mediated EPSCs. All effects of GHB on IPSCs and EPSCs were reversed by the specific GABAB antagonist CGP 62349, but not by the GHB receptor antagonist (2E)-5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen-6-ylidene ethanoic acid. Consistent with a presynaptic site of action, GHB reduced the frequency but not the amplitude of AMPA receptor–mediated mEPSCs and had no effect on postsynaptic currents evoked by direct application of NMDA. Finally, even though GHB appeared to be acting at presynaptic GABAB receptors, GHB and the GABAB agonist baclofen appeared to have opposite potencies for depression of NMDA- vs. AMPA-mediated EPSCs. GHB showed a preference for depressing NMDA responses while baclofen more potently suppressed AMPA responses. The suppression of NMDA more than AMPA responses by GHB at intoxicating doses may make it attractive as a recreational drug and may explain why GHB is abused and baclofen is not. [Copyright &y& Elsevier]

Abstract: Early endosome antigen 1 (EEA1), a peripheral membrane protein associated with the cytoplasmic face of early endosomes, controls vesicle fusion during endocytosis, as extensively studied in non-neuronal cells. In neurons, early endosomes are involved in recycling of synaptic vesicles and neurotransmitter receptors. Since certain patients bearing autoantibodies that target EEA1 develop neurological disease, we studied the subcellular distribution of EEA1 in neurons and the effect on neurotransmission of purified immunoglobulins from the serum of a patient bearing EEA1 autoantibodies. EEA1 was localized in the soma and in the postsynaptic nerve terminals. Electrophysiological recordings in hippocampal slices including purified EEA1 antibodies in the patch pipette solution, revealed a run-up of AMPA, N-methyl-d-aspartate and kainate receptor-mediated excitatory post-synaptic currents recorded from CA3 pyramidal neurons, which was absent in the recordings obtained in the presence of control human immunoglobulin G. Inclusion of human EEA1 antibodies had no effect on inhibitory post-synaptic responses. Recordings in the presence of a dominant-negative C-terminal EEA1 deletion mutant produced a similar effect as observed with human anti-EEA1 antibodies. This specific effect on the excitatory synaptic transmission may be due to the impairment of internalization of specific glutamate receptors and their subsequent accumulation in the synapse. These results may account for the neurological deficits observed in some patients developing EEA1 autoantibodies. [Copyright &y& Elsevier]

Abstract: Cholinergic projections from the nucleus basalis play a critical role in cortical plasticity. For instance, cholinergic deafferentation increases dendritic spine density and expression of the GluR1 subunit of the α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor in frontal cortex. Acetylcholine modulates glutamatergic activity in cortex, and the N-methyl-d-aspartate subtype of glutamate receptor plays a role in many forms of synaptic plasticity. To assess whether N-methyl-d-aspartate receptors mediate the increase in GluR1 and spine density resulting from cholinergic deafferentation, we examined the effect of N-methyl-d-aspartate receptor blockade on nucleus basalis lesion-induced upregulation of GluR1 and dendritic spines. Rats received unilateral sham or 192 IgG saporin lesions of the nucleus basalis. Half of the rats in each group were treated with the N-methyl-d-aspartate antagonist MK-801 or phosphate-buffered saline. Two weeks later, brains were processed for either immunohistochemical staining of the GluR1 subunit or Golgi histology. In layer II–III of frontal cortex, neuronal GluR1 expression was assessed using an unbiased stereological technique, and spine density was assessed on basilar branches of pyramidal neurons. GluR1 expression was increased after nucleus basalis lesion, but this increase was prevented with MK-801. Similarly, nucleus basalis-lesioned animals had significantly higher spine densities, and this effect was also prevented by treatment with MK-801. Thus, N-methyl-d-aspartate receptor blockade prevented both GluR1 and spine density upregulation following cholinergic deafferentation, suggesting that these effects are N-methyl-d-aspartate receptor-mediated. [Copyright &y& Elsevier]

Abstract: Ligand-gated ion channels (ionotropic receptors) link to the cortical cytoskeleton via specialized scaffold proteins and thereby to appropriate signal transduction pathways in the cell. We studied the role of filamentous actin in the regulation of Ca influx through glutamate receptor-activated channels in third-order neurons of salamander retina. Staining by Alexa-Fluor 488-phalloidin, to visualize polymerized actin, we show localization of filamentous actin in neurites, and the membrane surrounding the cell soma. With Ca2+ imaging we found that in dissociated neurons, depolymerization of filamentous actin by latrunculin A, or cytochalasin D significantly reduced glutamate-induced intracellular Ca2+ accumulation to 53±7% of control value. Jasplakinolide, a stabilizer of filamentous actin, by itself slightly increased the glutamate-induced Ca2+ signal and completely attenuated the inhibitory effect when applied in combination with actin depolymerizing agents. These results indicate that in salamander retinal neurons the actin cytoskeleton regulates Ca2+ influx through ionotropic glutamate receptor-activated channels, suggesting regulatory roles for filamentous actin in a number of Ca2+-dependent physiological and pathological processes. [Copyright &y& Elsevier]

Abstract: The corticothalamic feedback pathway provides excitatory synaptic input to both the thalamic reticular nucleus and the lateral geniculate nucleus. We studied excitatory postsynaptic currents elicited from corticothalamic stimulation in the visual sector of the thalamic reticular nucleus and the lateral geniculate nucleus to compare the response of these neurons to stimulation of their common input pathway. Using whole cell patch clamp recordings in ferret thalamic slices, we compared single excitatory postsynaptic current decay kinetics, presynaptic glutamate release dynamics through paired pulse facilitation and responses to corticothalamic train stimulation. We found that single thalamic reticular nucleus excitatory postsynaptic currents were significantly sharper than lateral geniculate nucleus responses. The mean thalamic reticular nucleus excitatory postsynaptic current decay constant (τ) was 4.9±0.5ms, while the mean lateral geniculate nucleus excitatory postsynaptic current τ value was 11.8±0.8ms. Presynaptic release dynamics as measured by responses to paired stimuli were conserved between the thalamic reticular nucleus and lateral geniculate nucleus. However, facilitating responses to train stimulation were markedly different between nuclei. Lateral geniculate nucleus responses showed proportionately larger facilitation (reaching 842.9±76.4% of excitatory postsynaptic current 1 amplitude) than thalamic reticular nucleus responses (reaching 223.1±44.0% of excitatory postsynaptic current 1 amplitude). These data indicate that while the corticothalamic pathway produces excitatory postsynaptic currents in both the thalamic reticular nucleus and lateral geniculate nucleus, other factors uniquely affect the functional integration of the inputs in each nucleus. [Copyright &y& Elsevier]

Abstract: The N-methyl-d-aspartic acid (NMDA) receptor-dependent activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) is necessary for induction of the long-term potentiation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated responses in the CA1 region of the hippocampus, a putative model for learning and memory. We analyzed the interplay among NMDA receptor, CaMKII and AMPA receptor during consolidation of the memory for an inhibitory avoidance learning task in the rat. Bilateral intra-CA1 infusion of the NMDA receptor antagonist d-(−)-2-amino-5-phosphonopentanoic acid (AP5) or of the CaMKII inhibitor 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) (KN-93) immediately after step-down inhibitory avoidance training hindered memory consolidation. Learning of the avoidance response induced the NMDA receptor-dependent translocation of αCaMKII to a postsynaptic density-enriched fraction isolated from dorsal CA1 and the autophosphorylation of this kinase at Thr-286. Step-down inhibitory avoidance training increased the quantity of GluR1 and GluR2/3 AMPA receptor subunits and the phosphorylation of GluR1 at Ser-831 but not at Ser-845 in CA1 postsynaptic densities. The intra-CA1 infusion of KN-93 and AP5 blocked the increases in GluR1 and GluR2/3 levels and the phosphorylation of GluR1 brought on by step-down inhibitory avoidance training. Our data suggest that step-down inhibitory avoidance learning promotes the learning-specific and NMDA receptor-dependent activation of CaMKII in the CA1 region of the dorsal hippocampus and that this activation is necessary for phosphorylation and translocation of AMPA receptor to the postsynaptic densities, similarly to what happens during long-term potentiation. [Copyright &y& Elsevier]

Abstract: In the external plexiform layer (EPL) of the main olfactory bulb, apical dendrites of inhibitory granule cells form large numbers of synapses with mitral and tufted (M/T) cells, which regulate the spread of activity along the M/T cell dendrites. The EPL also contains intrinsic interneurons, the functions of which are unknown. In the present study, recordings were obtained from cell bodies in the EPL of mouse olfactory bulb slices. Biocytin-filling confirmed that the recorded cells included interneurons, tufted cells, and astrocytes. The interneurons had fine, varicose dendrites, and those located superficially bridged the EPL space below several adjacent glomeruli. Interneuron activity was characterized by high frequency spontaneous excitatory postsynaptic potential/currents that were blocked by the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione and largely eliminated by the voltage-sensitive Na+ channel blocker, tetrodotoxin. Interneuron activity differed markedly from that of tufted cells, which usually exhibited spontaneous action potential bursts. The interneurons produced few action potentials spontaneously, but often produced them in response to depolarization and/or olfactory nerve (ON) stimulation. The responses to depolarization resembled responses of late- and fast-spiking interneurons found in other cortical regions. The latency and variability of the ON-evoked responses were indicative of polysynaptic input. Interneurons expressing green fluorescent protein under control of the mouse glutamic acid decarboxylase 65 promoter exhibited identical properties, providing evidence that the EPL interneurons are GABAergic. Together, these results suggest that EPL interneurons are excited by M/T cells via AMPA/kainate receptors and may in turn inhibit M/T cells within spatial domains that are topographically related to several adjacent glomeruli. [Copyright &y& Elsevier]

Abstract: The motor output of hypoglossal motoneurons to tongue muscles takes place in concert with the respiratory rhythm and is determined by the balance between excitatory glutamatergic transmission and inhibitory transmission mediated by glycine or GABA. The relative contribution by these transmitters is a phasic phenomenon modulated by other transmitters. We examined how metabotropic muscarinic receptors, widely expressed in the brainstem where they excite cranial motor nuclei, might influence synaptic activity mediated by GABA or glycine. For this purpose, using thin slices of the neonatal rat brainstem, we recorded (under whole-cell patch clamp) glycinergic or GABAergic responses from visually identified hypoglossal motoneurons after pharmacological block of glutamatergic transmission. Muscarine inhibited spontaneous and electrically induced events mediated by GABA or glycine. The amplitude of glycinergic miniature inhibitory postsynaptic currents was slightly reduced by muscarine, while GABAergic miniature inhibitory postsynaptic currents were unaffected. Motoneuron currents induced by focally applied GABA and glycine were depressed by muscarine with stronger reduction in glycine-mediated responses. Histochemical observations indicated the presence of M1, M2 and M5 subtypes of muscarinic receptors in the neonatal hypoglossal nucleus. These results suggest that muscarine potently depressed inhibitory neurotransmission on brainstem motoneurons, and that this action was exerted via preterminal and extrasynaptic receptors. Since the large reduction in inhibitory neurotransmission may contribute to overall excitation of brainstem motoneurons by muscarinic receptors, these data might help to understand the central components of action of antimuscarinic agents in preanesthetic medication or against motion sickness. [Copyright &y& Elsevier]

Abstract: Our understanding of the role played by neurotransmitter receptors in the developing brain has advanced in recent years. The major excitatory and inhibitory neurotransmitters in the brain, glutamate and GABA, activate both ionotropic (ligand-gated ion channels) and metabotropic (G protein-coupled) receptors, and are generally associated with neuronal communication in the mature brain. However, before the emergence of their role in neurotransmission in adulthood, they also act to influence earlier developmental events, some of which occur prior to synapse formation: such as proliferation, migration, differentiation or survival processes during neural development. To fulfill these actions in the constructing of the nervous system, different types of glutamate and GABA receptors need to be expressed both at the right time and at the right place. The identification by molecular cloning of 16 ionotropic glutamate receptor subunits, eight metabotropic glutamate receptor subtypes, 21 ionotropic and two metabotropic GABA receptor subunits, some of which exist in alternatively splice variants, has enriched our appreciation of how molecular diversity leads to functional diversity in the brain. It now appears that many different types of glutamate and GABA receptor subunits have prominent expression in the embryonic and/or postnatal brain, whereas others are mainly present in the adult brain. Although the significance of this differential expression of subunits is not fully understood, it appears that the change in subunit composition is essential for normal development in particular brain regions. This review focuses on emerging information relating to the expression and role of glutamatergic and GABAergic neurotransmitter receptors during prenatal and postnatal development. [Copyright &y& Elsevier]

Abstract: Toluene, a representative member of the large class of abused inhalants, decreases neuronal activity and depresses behavior in both animals and humans. The sites of action of toluene are not completely known but recent studies suggest that ion channels that regulate neuronal excitability may be particularly sensitive. Previous studies with recombinant receptors showed that toluene decreases currents carried by N-methyl-d-aspartate (NMDA)-glutamate receptors without affecting those gated by non-NMDA receptors. In addition, toluene increases currents generated by GABA and glycine receptors. In the present study, primary cultures of rat hippocampal neurons were used to investigate the effects of acute and chronic toluene exposure on native excitatory and inhibitory ligand-gated ion channels. Toluene dose-dependently inhibited NMDA-mediated currents (IC50 1.5 mM) but had no effect on responses evoked by the non-NMDA agonist kainic acid. Prolonged treatment of neurons with toluene (1 mM; 4 days) increased whole-cell responses to exogenously applied NMDA, reduced those evoked by GABA but did not alter responses generated by kainic acid. Immunoblot analysis revealed that prolonged toluene exposure increased levels of NR2A and NR2B NMDA receptor subunits with no change in NR1. Immunohistochemical analysis with confocal imaging showed that toluene-treated neurons had significant increases in the density of NR1 subunits as compared with control neurons. Toluene exposure increased the amplitude of synaptic NMDA currents and decreased those activated by GABA. The results from this study suggest that toluene induces compensatory responses in the functional expression of ion channels that regulate neuronal excitability. [Copyright &y& Elsevier]

The anterior piriform cortex (APC) functions as a chemosensor for indispensable amino acid deficiency and responds to this deficiency with increased activity, as indicated by observations including averaged evoked-potentials and c-fos expression in the APC. Little is known of the intracellular signaling mechanisms that mediate this deficiency-related increase in neuronal excitability, but previous studies have shown effects on intracellular Ca2+ in deficient APC slices in vitro. In the present study we hypothesized that indispensable amino acid deficiency increases intraneuronal Ca2+, resulting in autophosphorylation of calcium/calmodulin-dependent protein kinase type II (CaMKII) in vivo. Results demonstrated that phosphorylation levels of CaMKII (pCaMKII) in APC neurons increased at 20 and 40 min after a single meal of threonine-devoid diet. Phosphorylation of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit (GluR1) at the serine 831 (S831) site was modestly increased in the APC in response to a threonine-devoid meal. The GluR1 subunit also showed increased phosphorylation at the 845 (S845) site, suggesting additional signaling mechanisms. Although phosphorylation of CaMKII was sustained, phosphorylation of the GluR1 subunit returned to control levels by 40 min. These effects of amino acid deficiency did not occur throughout the brain as neither CaMKII nor GluR1 showed increased phosphorylation in the neocortex.These findings support the notion that calcium and glutamate signaling in the APC, but not throughout the brain, are triggered during early responses to amino acid deficiency. They also suggest that longer-term changes in APC neurons in response to such a deficiency may be mediated at least in part by CaMKII. [Copyright &y& Elsevier]

The ionotropic glutamate receptors N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are present peripherally in the primary sensory afferent neurons innervating the viscera. Multiple studies have reported roles of glutamate receptors in gastric functions. However, no study has previously shown the direct influence of ionotropic glutamate receptor antagonist on vagal sensory neurons. The objective of this study was to investigate the effects of NMDA and AMPA receptor antagonists on mechanotransduction properties of vagal afferent fibers innervating the rat stomach. Action potentials were recorded from the hyponodal vagus nerve innervating the antrum of the Long-Evans rats. For antral distension (AD), a small latex balloon was inserted into the stomach and positioned in the antrum. The antral contractions were recorded with solid-state probe inserted into the water-filled balloon. Antral units were identified to isovolumic (0.2–1 ml) or isobaric AD (5–60 mm Hg). NMDA and AMPA receptor antagonists were injected in a cumulative fashion (1–100 μmol/kg, i.v.). After the conclusion of experiment, the abdomen was opened and receptive field was mapped by probing the serosa of the stomach. Thirty-two fibers were identified to AD. The receptive fields of 26 fibers were located in the posterior part of the antrum. All fibers exhibited spontaneous firing (mean: 7.00±0.97 impulses/s). Twenty fibers exhibited a rhythmic firing that was in phase with antral contractions, whereas 12 fibers exhibited non-rhythmic spontaneous firing unrelated to spontaneous antral contraction. Both groups of fibers exhibited a linear increase in responses to graded isovolumic or isobaric distensions. NMDA (memantine HCl and dizocilpine (MK-801)) and AMPA/kainate (6-cyano-7-nitroquinoxaline 2,3-dione; CNQX) receptor antagonists dose-dependently attenuated the mechanotransduction properties of these fibers to AD. However, competitive NMDA antagonist dl-2-amino-5 phosphopentanoic acid (AP-5) had no effect. The study documents that glutamate receptor antagonists can attenuate responses of gastric vagal sensory afferent fibers innervating the distal stomach, offering insight to potential pharmacological agents in the treatment of gastric disorders. [Copyright &y& Elsevier]

To determine the sensitivity of basal forebrain cholinergic neurons to ionotropic glutamate receptor activation, acetylcholine was collected from the cerebral cortex of urethane-anesthetized rats using microdialysis while monitoring cortical electroencephalographic (EEG) activity. α-Amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA; 1, 10, or 100 μM), N-methyl-d-aspartate (NMDA; 100 or 1000 μM) or a combination of AMPA (10 μM) and NMDA (100 μM) was administered to the basal forebrain using reverse microdialysis. Both glutamate receptor agonists produced concentration-dependent, several-fold increases in acetylcholine release indicating that they activated basal forebrain cholinergic neurons; AMPA was more potent, increasing acetylcholine release at a lower concentration than NMDA. The combination of AMPA and NMDA did not produce any greater release than each drug alone, indicating that the effects of these two drugs on cholinergic neurons are not additive. EEG was analyzed by fast Fourier transforms to determine the extent of physiological activation of the cortex. The highest concentrations of AMPA and NMDA tested produced small (25%) but significant increases in high frequency activity. There was a positive correlation across animals between the increases in power in the β (14–30 Hz) and γ (30–58 Hz) ranges and increases in acetylcholine release. These results indicate that glutamate can activate cholinergic basal forebrain neurons via both AMPA and NMDA ionotropic receptors but has a more modest effect on EEG activation. [Copyright &y& Elsevier]

Rat neocortex that has been injured on the first or second postnatal day (P0–1) develops an epileptogenic, aberrantly layered malformation called a microgyrus. To investigate the effects of this developmental plasticity on inhibitory interneurons, we studied a sub-population of GABAergic cells that co-express the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR1 subunit and the calcium-binding protein, calbindin (CB). Both malformed and control cortex of adult (P40–60) animals contained numerous interneurons double-stained for CB and GluR1. Immunoreactivity (IR) for CB was up-regulated in perikarya of interneurons within supragranular layers of control cortex between P12 and P40. However, in malformed adult (P40) cortex, CB-IR levels were significantly lower than in adult controls, and fell midway between levels in immature and adult control animals. Between P12 and P40, GluR1-IR was down-regulated in perikarya of interneurons in control cortex. Somatic GluR1-IR levels in malformed adult (P40) cortex were not different from adult controls. These neurons formed a dense plexus of highly GluR1-positive spiny dendrites within layer II. The dendritic plexus in the malformation was more intensely GluR1-immunoreactive than that in layer II of control cortex. This was due to apparent changes in thickness and length of dendrites, rather than to significant changes in the number of interneuronal perikarya in the microgyral cortex. Results indicate that the population of GluR1/CB-containing interneurons is spared in malformed microgyral cortex, but that these cells sustain lasting decreases in their somatic expression of calbindin and alterations of dendritic structure. Potential functional implications of these findings are discussed. [Copyright &y& Elsevier]

This study was designed to determine the effects of opiate drugs on the electrical activity of afferent neurons and on the ionic currents of hair cells from semicircular canals. Experiments were done on larval axolotls (Ambystoma tigrinum). The multiunit spike activity of afferent neurons was recorded in the isolated inner ear under both resting conditions and mechanical stimulation. Ionic currents were recorded using voltage clamp of hair cells isolated from the semicircular canal. In the isolated inner-ear preparation, microperfusion of either non-specific opioid receptor antagonist naloxone (10 nM to 1 mM), μ receptor agonist [D-Ala2, N-Me-Phe4,Gly5-ol]-enkephalin (1 pM to 10 μM), or κ receptor antagonist nor-binaltorphimine (10 nM to 100 μM) elicited a dose-dependent long-lasting (>5 min) increase of the electrical discharge of afferent neurons. The μ receptor agonist funaltrexamine (1 nM to 100 μM) and the κ receptor agonist U-50488 (1 nM to 10 μM) diminished the basal spike discharge of vestibular afferents. The δ receptor agonist D-Pen2-D-Pen5-enkephalin (1 nM to 10 mM) and the antagonist naltrindole (1 nM to 10 mM) were without a significant effect. The only drug that displayed a significant action on hair-cell ionic currents was trans-(±)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]-cyclohexyl) benzeneacetamide methanesulfonate (U-50488) that reduced the Ca2+ current in a dose-dependent fashion. On its own, μ receptor agonist [D-Ala2, N-Me-Phe4,Gly5-ol]-enkephalin (0.01 and 10 μM) significantly potentiated the response of afferent neurons to the excitatory amino acid agonist (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (0.1 μM), while synaptic transmission was blocked by the use of high-Mg2+, low-Ca2+ solutions.Our data indicate that the activity of vestibular afferent neurons may be regulated in a complex fashion by opioid receptors: μ opioid receptors mediating an excitatory, postsynaptic modulatory input to afferent neurons, and κ receptors mediating an inhibitory, presynaptic input to hair cells. [Copyright &y& Elsevier]

The modulatory neurotransmitter dopamine induces concentration-dependent changes in synaptic transmission in the entorhinal cortex, in which high concentrations of dopamine suppress evoked excitatory postsynaptic potentials (EPSPs) and lower concentrations induce an acute synaptic facilitation. Whole-cell current-clamp recordings were used to investigate the dopaminergic facilitation of synaptic responses in layer II neurons of the rat lateral entorhinal cortex. A constant bath application of 1 μM dopamine resulted in a consistent facilitation of EPSPs evoked in layer II fan cells by layer I stimulation; the size of the facilitation was more variable in pyramidal neurons, and synaptic responses in a small group of multiform neurons were not modulated by dopamine. Isolated inhibitory synaptic responses were not affected by dopamine, and the facilitation of EPSPs was not associated with a change in paired-pulse facilitation ratio. Voltage-clamp recordings of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) glutamate receptor-mediated excitatory postsynaptic currents (EPSCs) were facilitated by dopamine, but N-methyl-D-aspartate receptor-mediated currents were not. Bath application of the dopamine D₁-like receptor blocker SCH23390 (50 μM), but not the D₂-like receptor blocker sulpiride (50 μM), prevented the facilitation, indicating that it is dependent upon D₁-like receptor activation. Dopamine D₁ receptors lead to activation of protein kinase A (PKA), and including the PKA inhibitor H-89 or KT 5720 in the recording pipette solution prevented the facilitation of EPSCs. PKA-dependent phosphorylation of inhibitor 1 or the dopamine- and cAMP-regulated protein phosphatase (DARPP-32) can lead to a facilitation of AMPA receptor responses by inhibiting the activity of protein phosphatase 1 (PP1) that reduces dephosphorylation of AMPA receptors, and we found here that inhibition of PP1 occluded the facilitatory effect of dopamine. The dopamine-induced facilitation of AMPA receptor-mediated synaptic responses in layer II neurons of the lateral entorhinal cortex is therefore likely mediated via a D₁ receptor-dependent increase in PKA activity and a resulting inhibition in PP1-dependent dephosphorylation of AMPA receptors., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Neonatal handling, an experimental model of early life experiences, is known to affect hypothalamic-pituitary-adrenal (HPA) axis function, thus increasing adaptability, coping with stress, cognitive abilities and in general brain plasticity-related processes. AMPA receptors (AMPARs) mediate fast synaptic transmission at excitatory glutamatergic synapses in the CNS and are crucial during neuronal development, synaptic plasticity and structural remodeling. AMPARs are composed of four types of subunits, designated as AMPA glutamate receptor subunits (GluA1, GluA2, GluA3 and GluA4), which combine to form tetramers. The present study addressed the question of whether neonatal handling (15min daily maternal separation from postnatal day 1 (PND1) to PND21) might have an effect on GluA1-4 mRNA levels in adult rat male and female brain using in situ hybridization. We have identified selective sexually dimorphic effects of neonatal handling on the mRNA expression levels of AMPAR subunits in adult rat hippocampus and nuclei of the amygdaloid complex. In the dorsal hippocampus GluA1 mRNA levels were increased in handled males, while they were decreased in handled female animals. In the ventral hippocampus and the amygdaloid complex GluA2 mRNA was lower in handled females, while no effect was observed in handled males. Furthermore, we observed that neonatal handling induced in both sexes decreases of GluA2 mRNA in the dorsal hippocampus, as well as in the somatosensory and occipital cortex, of GluA3 mRNA in most hippocampal areas, amygdaloid complex and cortical regions studied, and of GluA4 mRNA in the ventral hippocampus. These results show that glutamatergic transmission is markedly affected by an early experience. The neonatal handling-induced alterations in AMPAR subunit composition are in line with the increased brain plasticity, the more effective HPA axis function, and in general the more adaptive behavioral phenotype known to characterize the handled animals., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Light stimulates specialized retinal ganglion cells to release glutamate (Glu) onto circadian clock neurons of the suprachiasmatic nucleus (SCN). Glu resets the phase of the SCN circadian clock by activating N-methyl-d-aspartate receptors (NMDAR) causing either delays or advances in the clock phase, depending on early- or late-night stimulation, respectively. In addition, these Glu-induced phase shifts require tropomyosin receptor kinase B (TrkB) receptor activity. Previous studies show that copper (Cu) released at hippocampal synapses can inhibit NMDAR activity, and application of exogenous Cu likewise inhibits NMDAR activity. We investigated the effects of Cu in acute SCN brain slices prepared from C57BL/6Nhsd adult, male mice using treatments that decrease or increase available Cu levels in vitro and recorded neuronal activity on the following day. When bath-applied for 10 min at zeitgeber time (ZT) 16 (where ZT0=lights-on in the donor animal colony), the Cu-specific chelators tetrathiomolybdate (TTM) and bathocuproine disulfonate each induce ∼2.5-3-h phase delays in circadian neuronal activity rhythms, similarly to Glu-induced phase delays. Co-application of 10 μM CuCl2, but not 10 μM CoCl₂ blocks TTM-induced phase delays. Furthermore, TTM causes phase advances when applied at ZT23. At both application times, TTM-induced phase shifts are blocked by NMDA or TrkB receptor antagonists. Surprisingly, bath-application of 10 μM Cu alone also induces phase shifts in analogous experiments at ZT16 and ZT23. Inhibiting NMDAR does not block Cu-induced phase shifts. TrkB inhibition blocks Cu-induced phase delays but not phase advances. Thus, increasing and decreasing Cu availability appear to shift the SCN clock phase through different mechanisms, at least at the receptor level. We propose that Cu plays a role in the SCN circadian clock by modulating Glu signaling., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Activation of glutamate receptors within the ventral tegmental area (VTA) stimulates extrasynaptic (basal) dopamine release in terminal regions, including the nucleus accumbens (NAc). Hindbrain inputs from the laterodorsal tegmental nucleus (LDT) are critical for elicitation of phasic VTA dopamine cell activity and consequent transient dopamine release. This study investigated the role of VTA ionotropic glutamate receptor (iGluR) stimulation on both basal and LDT electrical stimulation-evoked dopamine efflux in the NAc using in vivo chronoamperometry and fixed potential amperometry in combination with stearate-graphite paste and carbon fiber electrodes, respectively. Intra-VTA infusion of the iGluR agonists (±)-α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA; 1 μg/μl) or N-methyl-d-aspartic acid (NMDA; 2 μg/μl) enhanced basal NAc dopamine efflux. This iGluR-mediated potentiation of basal dopamine efflux was paralleled by an attenuation of LDT-evoked transient NAc dopamine efflux, suggesting that excitation of basal activity effectively inhibited the capacity of hindbrain afferents to elicit transient dopamine efflux. In line with this, post-NMDA infusion of the dopamine D2 autoreceptor (D2R) agonist quinpirole (1 μg/μl; intra-VTA) partially recovered NMDA-mediated attenuation of LDT-evoked NAc dopamine, while concurrently attenuating NMDA-mediated potentiation of basal dopamine efflux. Post-NMDA infusion of quinpirole (1 μg/μl) alone attenuated basal and LDT-evoked dopamine efflux. Taken together, these data reveal that hyperstimulation of basal dopamine transmission can stunt hindbrain burst-like stimulation-evoked dopamine efflux. Inhibitory autoreceptor mechanisms within the VTA help to partially recover the magnitude of phasic dopamine efflux, highlighting the importance of both iGluRs and D2 autoreceptors in maintaining the functional balance of tonic and phasic dopamine neurotransmission. Dysregulation of this balance may have important implications for disorders of dopamine dysregulation such as attention deficit hyperactivity disorder., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Dysfunctional glutamatergic neurotransmission has been implicated in schizophrenia and mood disorders. As a putative model for these disorders, a mouse line lacking the GluA1 subunit (GluA1-KO) of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor displays a robust novelty-induced hyperlocomotion associated with excessive neuronal activation in the hippocampus. Agonists of metabotropic glutamate 2/3 receptors (mGluR2/3) inhibit glutamate release in various brain regions and they have been shown to inhibit neuronal activation in the hippocampus. Here, we tested a hypothesis that novelty-induced hyperlocomotion in the GluA1-KO mice is mediated via excessive hippocampal neuronal activation by analyzing whether an mGluR2/3 agonist inhibits this phenotypic feature. GluA1-KO mice and littermate wildtype (WT) controls were administered with (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740) (15 mg/kg, i.p.) 30 min before a 2-h exposure to novel arenas after which c-Fos immunopositive cells were analyzed in the hippocampus. LY354740 (15 mg/kg) decreased hyperactivity in male GluA1-KO mice, with only a minimal effect in WT controls. This was observed in two cohorts of animals, one naïve to handling and injections, another pre-handled and accustomed to injections. LY354740 (15 mg/kg) also reduced the excessive c-Fos expression in the dorsal hippocampal CA1 pyramidal cell layer in maleGluA1-KO mice, while not affecting c-Fos levels in WT mice. In female mice, no significant effect for LY354740 (15 mg/kg) on hyperactive behavior or hippocampal c-Fos was observed in either genotype or treatment cohort. A higher dose of LY354740 (30 mg/kg) alleviated hyperlocomotion of GluA1-KO males, but not that of GluA1-KO females. In conclusion, the excessive behavioral hyperactivity of GluA1-KO mice can be partly prevented by reducing neuronal excitability in the hippocampus with the mGluR2/3 agonist suggesting that the hippocampal reactivity is strongly involved in the behavioral phenotype of GluA1-KO mice., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Central neuropathic pain (CNP) in the spinal cord, such as chronic pain after spinal cord injury (SCI), is an incurable ailment. However, little is known about the spinal cord mechanisms underlying CNP. Recently, reactive oxygen species (ROS) have been recognized to play an important role in CNP of the spinal cord. However, it is unclear how ROS affect synaptic transmission in the dorsal horn of the spinal cord. To clarify how ROS impact on synaptic transmission, we investigated the effects of ROS on synaptic transmission in rat spinal cord substantia gelatinosa (SG) neurons using whole-cell patch-clamp recordings. Administration of tert-butyl hydroperoxide (t-BOOH), an ROS donor, into the spinal cord markedly increased the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in SG neurons. This t-BOOH-induced enhancement was not suppressed by the Na(+) channel blocker tetrodotoxin. However, in the presence of a non-N-methyl-D-aspartate glutamate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, t-BOOH did not generate any sEPSCs. Furthermore, in the presence of a transient receptor potential ankyrin 1 (TRPA1) channel antagonist (HC-030031) or a transient receptor potential vanilloid 1 (TRPV1) channel antagonist (capsazepine or AMG9810), the t-BOOH-induced increase in the frequency of sEPSCs was inhibited. These results indicate that ROS enhance the spontaneous release of glutamate from presynaptic terminals onto SG neurons through TRPA1 and TRPV1 channel activation. Excessive activation of these ion channels by ROS may induce central sensitization in the spinal cord and result in chronic pain such as that following SCI., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)