Striatal spiny neurons are selectively vulnerable in Huntington's disease (HD). No effective treatment is available to limit neuronal death in this pathological condition. In an experimental model of HD, a beneficial effect has recently been reported by the neuroprotective agent riluzole. We performed intracellular recordings in order to characterize the electrophysiological effects of this compound on striatal spiny neurons. Riluzole (0.1-100 microM) affected neither the resting membrane potential nor the input resistance/membrane conductance of the recorded cells. Bath application of this pharmacological agent produced a dose-dependent reduction of the number of spikes evoked by long-lasting depolarizing pulses. The EC50 value for this effect was 0.5 microM. Low doses of riluzole selectively reduced the firing frequency in the last part of the depolarizing pulse suggesting a use-dependent action at low concentrations of this compound. Riluzole produced a dose-dependent reduction of the amplitude of the corticostriatal glutamatergic excitatory post-synaptic potentials (EPSPs) with an extrapolated EC50 value of 6 microM. This effect was reversible and maximal at a concentration of 100 microM. Paired-pulse facilitation (PPF) was not affected by riluzole suggesting that the reduction of excitatory transmission was not only caused by a decrease of presynaptic release. Accordingly, riluzole also reduced the amplitude of membrane depolarization induced by exogenous glutamate. The modulatory action of riluzole on the activity of striatal spiny neurons might support the use of this drug in experimental models of excitotoxicity and in the neurodegenerative disorders involving the striatum.

1. We investigated the role of nitric oxide (NO) in the induction of long-term potentiation (LTP) in slices prepared from the rat auditory cortex. 2. Tetanic stimulation of layer IV elicited LTP of field potentials in layer II-III (LTPII-III) and in layer V (LTPV). The magnitude of LTPII-III measured at 30 min after tetanic stimulation was 171 +/- 9% (n = 15, mean +/- s.e.m.) of the control measured before tetanic stimulation, while that of LTPV was 138 +/- 3% (n = 17). 3. NO synthase (NOS) inhibitors had no apparent effect on LTPII-III, but LTPV was significantly suppressed (P < 0.001). This suppression of LTPV was significantly antagonized by a NO donor (P < 0.001) or a cGMP analogue (P < 0.001). 4. Small non-pyramidal neurones in the auditory cortex were stained with an anti-neuronal NOS antibody. More neurones were stained with the antibody in the deeper cortical layers. 5. We measured neocortical NO release with electrochemical NO probes. Layer IV stimulation elicited significantly more NO release in layer V than in layer II-III (P < 0.001). The amplitude of the increase in NO concentration elicited by stimulation at 20 Hz for 5 s was 380 +/- 14 pM (n = 55) in layer V and 55 +/- 8 pM (n = 5) in layer II-III. 6. NO release in layer V was partially but significantly suppressed by non-NMDA (P < 0.002) or NMDA (P < 0.002) receptor antagonists. Simultaneous application of the antagonists of the two types blocked NO release almost completely. 7. These results clearly indicate the NO dependence of the induction of LTPV, and the greater NO release in the deeper layer of the rat auditory cortex.

We have shown that a number of anticonvulsant drugs can reduce glutamate release at synapses in the rat entorhinal cortex (EC) in vitro. We have also shown that presynaptic NMDA receptors (NMDAr) tonically facilitate glutamate release at these synapses. In the present study we determined whether, phenytoin, gabapentin and felbamate may reduce glutamate release by blocking the presynaptic NMDAr. Whole cell patch clamp recordings of spontaneous excitatory postsynaptic currents (sEPSCs) were used as a monitor of presynaptic glutamate release. Postsynaptic NMDAr were blocked with internal dialysis with an NMDAr channel blocker. The antagonist, 2-AP5, reduced the frequency of sEPSCs by blocking the presynaptic facilitatory NMDAr, but did not occlude a reduction in sEPSC frequency by gabapentin or phenytoin. Felbamate also reduced sEPSC frequency, but this effect was occluded by prior application of 2-AP5. Thus, whilst all three drugs can reduce glutamate release, only the action of felbamate seems to be due to interaction with presynaptic NMDAr.

Changes in gene expression are an important mechanism by which activity levels are regulated in the nervous system. It is not known, however, how network activity influences gene expression in interneurons; since they themselves provide negative feedback in the form of synaptic inhibition, there exists a potential conflict between their cellular homeostatic tendencies and those of the network. We present a means of examining this issue, utilizing simple in vitro models showing different patterns of intense network activity. We found that the degree of concurrent pyramidal activation changed the polarity of the induced gene transcription. When pyramidal cells were quiescent, interneuronal activation led to an upregulation of glutamate decarboxylase 1 ( GAD1) and parvalbumin ( Pvalb) gene transcriptions, mediated by activation of the Ras/extracellular signal-related kinase mitogen-activated protein kinase (Ras/ERK MAPK) pathway. In contrast, coactivation of pyramidal cells led to an ionotropic glutamate receptor N-methyl-d-aspartate 2B-dependent decrease in transcription. Our results demonstrate a hitherto unrecognized complexity in how activity-dependent gene expression changes are manifest in cortical networks. NEW & NOTEWORTHY We demonstrate a novel feedback mechanism in cortical networks, by which glutamatergic drive, mediated through the Ras/ERK MAPK pathway, regulates gene transcription in interneurons. Using a unique feature of certain in vitro epilepsy models, we show that without this glutamatergic feedback, intense activation of interneurons causes parvalbumin and glutamate decarboxylase 1 mRNA expression to increase. If, on the other hand, pyramidal cells are coactivated with interneurons, this leads to a downregulation of these genes.

Several previous studies have shown that postnatal blockade of thalamocortical activity with either tetrodotoxin (TTX) or the N-methyl-D-aspartate (NMDA) receptor antagonist D,L-2-amino-5-phosphonovalerate (APV) does not prevent the formation of vibrissa-related patterns in the primary somatosensory cortex of rats. One limitation of these studies is that this pattern forms very shortly after birth in rats, and there may be only a very limited time over which it may be influenced by activity blockade. In the present study, the effect of activity blockade was evaluated in a more altricial rodent, the hamster. The present study showed that a pattern of thalamocortical afferents corresponding to the vibrissae is not observed until the fourth postnatal day in hamsters. Nevertheless, application of TTX-impregnated implants to the cortices of newborn hamsters had no qualitative or quantitative effect upon vibrissa-related patterns in the primary somatosensory cortices of these animals. Moreover, TTX implants did not prevent the changes in patterns that followed cauterization of a row of vibrissa follicles.

High-frequency magnetic stimulation (HFMS) can elicit N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP) at Schaffer collateral-CA1 pyramidal cell synapses. Here, we investigated the priming effect of HFMS on the subsequent magnitude of electrically induced LTP in the CA1 region of rat hippocampal slices using field excitatory postsynaptic potential (fEPSP) recordings. In control slices, electrical high-frequency conditioning stimulation (CS) could reliably induce LTP. In contrast, the same CS protocol resulted in long-term depression when HFMS was delivered to the slice 30 min prior to the electrical stimulation. HFMS-priming was diminished when applied in the presence of the metabotropic glutamate receptor antagonists (RS)-α-methylserine-O-phosphate (MSOP) and (RS)-α-methyl-4-carboxyphenylglycine (MCPG). Moreover, when HFMS was delivered in the presence of the NMDA receptor-antagonist D-2-amino-5-phosphonovalerate (50 µM), CS-induced electrical LTP was again as high as under control conditions in slices without priming. These results demonstrate that HFMS significantly reduced the propensity of subsequent electrical LTP and show that both metabotropic glutamate and NMDA receptor activation were involved in this form of HFMS-induced metaplasticity.

The Ca2+ signal in supragranular layers of the rat auditory cortex (AC) was studied in slice preparations using rhod-2, a Ca2+ indicator. White matter stimulation elicited an increase in the Ca2+ signal, which was maximal in the image taken 34 ms after stimulation. This peak time was the same as that of the Ca2+ signal in pyramidal neurons injected with rhod-2. The intensity of the Ca2+ signal was proportional to the amplitude of the field potentials in supragranular layers. The Ca2+ signal was inhibited almost completely by 200 microM Ni2+ , but only slightly by 50 microM D-2-amino-5-phosphonovalerate (APV), an NMDA-receptor antagonist. Tetanic stimulation of the white matter or supragranular layers elicited long-term potentiation (LTP) of the Ca2+ signal in AC slices, but the potentiation was not clear in slices of the visual cortex (VC). The induction of LTP of the field potentials in AC slices was blocked by 50 microM APV or 50 microM Ni2+. These results indicate that Ca2+ influx through Ni2+ -sensitive Ca2+ channels in pyramidal neurons is potentiated by tetanic stimulation in parallel with LTP of neural activities and might be important for the induction of LTP in AC slices.

Metabotropic glutamate receptors have been shown to stimulate phosphatidylinositol metabolism, and subsequently liberate Ca2+ from intracellular stores, in a variety of tissue and cell types. We previously demonstrated that glutamate could stimulate phosphatidylinositol metabolism, generating inositol-1,4,5-trisphosphate (IP3), in isolated cochlear nucleus tissue from the chick. Using the calcium indicator dye fura-2 and ratiometric fluorescent imaging, this study examined the ability of glutamate and its analogs to liberate Ca2+ from intracellular stores of neurons of the avian cochlear nucleus, and qualitatively characterized the pharmacological profile of such an action. In normal, Ca(2+)-containing medium, glutamate, kainate (KA), alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), NMDA, quisqualate (QUIS), and (+/-)-aminocyclopentane-trans-dicarboxylate (ACPD) elicited increases in intracellular calcium concentrations ([Ca2+]i). In the absence of external Ca2+, glutamate, quisqualate, and ACPD evoked increases in [Ca2+]i. In normal medium, the ionotropic glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the NMDA receptor antagonist 2-amino-5-phosphonovalerate (APV) attenuated but did not abolish the glutamate-evoked response and had no effect on the ACPD-evoked response. The putative metabotropic glutamate receptor antagonist 2-amino-3-phosphonopropionate (AP3) was without effect on the glutamate- and ACPD-evoked increases in [Ca2+]i in Ca(2+)-free medium. We conclude that a metabotropic glutamate receptor (mGluR) is present on cochlear nucleus neurons and is able to stimulate the phosphatidylinositol metabolism--Ca2+ signal transduction cascade.

Corticosterone is known to accumulate in brain after various stressors including alcohol intoxication. Just as severe alcohol intoxication is typically required to impair memory formation only high concentrations of ethanol (60 mM) acutely inhibit long-term potentiation (LTP), a cellular memory mechanism, in naïve hippocampal slices. This LTP inhibition involves synthesis of neurosteroids, including allopregnanolone, and appears to involve a form of cellular stress. In the CA1 region of rat hippocampal slices, we examined whether a lower concentration of ethanol (20 mM) inhibits LTP in the presence of corticosterone, a stress-related modulator, and whether corticosterone stimulates local neurosteroid synthesis. Although low micromolar corticosterone alone did not inhibit LTP induction, we found that 20 mM ethanol inhibited LTP in the presence of corticosterone. At 20 mM, ethanol alone did not stimulate neurosteroid synthesis or inhibit LTP. LTP inhibition by corticosterone plus ethanol was blocked by finasteride, an inhibitor of 5α-reductase, suggesting a role for neurosteroid synthesis. We also found that corticosterone alone enhanced neurosteroid immunostaining in CA1 pyramidal neurons and that this immunostaining was further augmented by 20 mM ethanol. The enhanced neurosteroid staining was blocked by finasteride and the N-methyl-D-aspartate antagonist, 2-amino-5-phosphonovalerate (APV). These results indicate that corticosterone promotes neurosteroid synthesis in hippocampal pyramidal neurons and can participate in ethanol-mediated synaptic dysfunction even at moderate ethanol levels. These effects may contribute to the influence of stress on alcohol-induced cognitive impairment.

Considerable work demonstrates that Pavlovian fear conditioning depends on N-methyl-D-aspartate (NMDA) receptor-dependent plasticity within the amygdala. In addition, the bed nucleus of the stria terminalis (BNST) has also been implicated in fear conditioning, particularly in the expression of fear to poor predictors of threat. We recently found that the expression of backward (BW) fear conditioning, in which an auditory conditioned stimulus (CS) follows a footshock unconditioned stimulus (US), requires the BNST; the expression of forward (FW) fear conditioning was not disrupted by BNST inactivation. However, whether NMDA receptors within the BNST contribute to the acquisition of fear conditioning is unknown. Moreover, the central nucleus of the amygdala (CeA), which has extensive connections with the BNST, is critically involved in FW conditioning, however whether it participates in BW conditioning has not been explored. Here we test the specific hypothesis that the CeA and the BNST mediate the acquisition of FW and BW fear conditioning, respectively. Adult female and male rats were randomly assigned to receive bilateral infusions of the NMDA receptor antagonist, D,L-2-amino-5-phosphonovalerate (APV), into the CeA or BNST prior to FW or BW fear conditioning. We found that intra-CeA APV impaired the acquisition of both FW and BW conditioning, whereas intra-BNST APV produced selective deficits in BW conditioning. Moreover, APV in the BNST significantly reduced contextual freezing, whereas CeA NMDA receptor antagonism impeded early but not long-lasting contextual fear. Collectively, these data reveal that CeA and BNST NMDA receptors have unique roles in fear conditioning.

In the trigeminal ganglion (TG), satellite glial cells (SGCs) form a functional unit with neurons. It has been proposed that SGCs participate in regulating extracellular glutamate levels and that dysfunction of this SGC capacity can impact nociceptive transmission in craniofacial pain conditions. This study investigated whether SGCs release glutamate and whether elevation of TG glutamate concentration alters response properties of trigeminal afferent fibers. Immunohistochemistry was used to assess glutamate content and the expression of excitatory amino acid transporter (EAAT)1 and EAAT2 in TG sections. SGCs contained glutamate and expressed EAAT1 and EAAT2. Potassium chloride (10 mM) was used to evoke glutamate release from cultured rat SGCs treated with the EAAT1/2 inhibitor (3S)-3-[[3-[[4-(trifluoromethyl)ben zoyl]amino]phenyl]methoxy]-L-aspartic acid (TFB-TBOA) or control. Treatment with TFB-TBOA (1 and 10 μM) significantly reduced the glutamate concentration from 10.6 ± 1.1 to 5.8 ± 1.4 μM and 3.0 ± 0.8 μM, respectively (p<0.05). Electrophysiology experiments were conducted in anaesthetized rats to determine the effect of intraganglionic injections of glutamate on the response properties of ganglion neurons that innervated either the temporalis or masseter muscle. Intraganglionic injection of glutamate (500 mM, 3 μl) evoked afferent discharge and significantly reduced muscle afferent mechanical threshold. Glutamate-evoked discharge was attenuated bythe N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovalerate (APV) and increased by TFB-TBOA, whereas mechanical sensitization was only sensitive to APV. Antidromic invasion of muscle afferent fibers by electrical stimulation of the caudal brainstem (10 Hz) or local anesthesia of the brainstem with lidocaine did not alter glutamate-induced mechanical sensitization. These findings provide a novel mechanism whereby dysfunctional trigeminal SGCs could contribute to cranial muscle tenderness in craniofacial pain conditions such as migraine headache.

Synaptic activation of central neurons has been associated with rapid extracellular alkalinization. In this report, we directly activated CA1 pyramidal cells by antidromic invasion, or by field stimulation. Antidromic activation produced no pH change, despite a robust population spike in five of 11 slices. In six slices, antidromic stimulation at 10 Hz evoked a small alkalinization in stratum pyramidale (0.04 +/- 0.01 unit pH) which grew to 0.10-0.20 unit pH at 50-100 Hz, and was blocked in 0 Ca2+ media. Simultaneous pH recordings revealed no alkalinizations in stratum radiatum, despite robust alkaline shifts in stratum pyramidale. When synaptic transmission was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione, DL-2-amino-5-phosphonovalerate and picrotoxin, the Schaffer collateral-induced alkaline shift in stratum radiatum was abolished. With adequate stimulus strength and orientation, however, alkaline shifts in stratum radiatum could still be elicited, presumably by direct activation of the CA1 population. The non-synaptic alkaline shifts ranged from 0.10-0.20 unit pH, were amplified by benzolamide, and blocked by tetrodotoxin, 0 Ca2+ saline, and 300-400 microM Cd2+. Although directly activated alkaline shifts were never observed in 0 Ca2+ saline, large stimulus evoked responses could be elicited upon addition of 5-10 mM Ba2+. The Ba(2+)-dependent responses were also amplified by benzolamide and blocked by tetrodotoxin, Cd2+ or high Mg2+. These data demonstrate that stratum pyramidale can undergo an extracellular alkaline shift independent of stratum radiatum. The ionic dependence and pharmacologic sensitivity of the alkaline shifts suggest that voltage-gated Ca2+ channels are instrumental in triggering the alkalinizing mechanism. However, the ability of Ba2+ to support the alkaline shifts indicates that Ca2+ entry is not an absolute requirement. Implications for the mechanism of these pH changes are discussed.

During postnatal development, the dendrites of spinal motor neurons are refined in an activity-dependent manner that can be influenced by blocking activation of N-methyl-D-aspartate (NMDA) receptors. In late postnatal life, dendritic refinement ceases, and dendrite architecture is unaffected by NMDA antagonists; however the molecular substrate for limiting dendritic plasticity is not understood. During late postnatal development, expression of the NR3B NMDA receptor subunit, a putative dominant-negative subunit that reduces glutamate-induced ionic currents, is upregulated within motor neurons. To investigate whether increasing NR3B expression may contribute to the loss in late development of activity-dependent dendritic reorganization in the spinal cord, we over-expressed NR3B in cultured rat spinal motor neurons, and compared its effects on dendrite morphology with the effects of pharmacological blockade of NMDA receptors. We found that over-expression of the NR3B receptor subunit increased the length and complexity of dendritic arbor, and increased numbers of dendritic filopodia, suggesting that NR3B promotes the addition of branch segments in developing motor neurons. In contrast, blockade of NMDA receptor activity by the NMDA antagonist DL-2-amino-5-phosphonovalerate (AP5) had little effect on the overall length or complexity of dendritic arbor. Instead, treatment with AP5 resulted in significant reorganization of dendritic arbor in a manner that favored addition of dendritic segments of high branch orders, at the expense of those closer to the cell body. These results suggest that expression of the NR3B subunit may participate in activity-dependent reorganization of dendritic architecture, but via a mechanism that may be inconsistent with loss of NMDA receptor activity.

Using fluorometric and immunocytochemical techniques, we found that high glycine concentrations or blockade of glycine receptors increases neurite outgrowth in developing mouse spinal cord neurons. Glycine- and GABA(A)-activated currents were demonstrated during applications of glycine and GABA (50-100 microM) in 5 days in vitro (DIV) neurons. Long application (> or =10 min) of 100 microM glycine desensitized the membrane response by more than 95%. Application of glutamate in the absence of external Mg(2+), at several membrane potentials, did not produce any detectable membrane response in these cells. Immunocytochemical studies with NR1 and GluR1 antibodies showed a delayed appearance of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors respectively. Spontaneous synaptic activity was readily observed in 5 DIV neurons. The use of various receptor antagonists (strychnine, bicuculline, DL-2-amino-5-phosphonovalerate [APV], 6-cyano-7-nitroquinoxaline-2,3-dione [CNQX]) revealed that this activity was predominantly glycinergic, and to a smaller extent, GABAergic. In the presence of bicuculline, APV and CNQX, we detected abundant spontaneous depolarizing potentials which often reached the action potential threshold. Further evidence for functional synaptic activity was provided by the detection of co-localization of gephyrin and synaptophysin at 5 DIV using confocal microscopy. Fluorometric studies with Fluo-3, a Ca(2+) indicator, in 5 DIV cultures showed the presence of spontaneous fluctuations associated with tetrodotoxin-sensitive synaptic events. The number of neurons displaying these fluctuations was significantly increased (>100%) when the cells were bathed in a strychnine-containing solution. On the other hand, these synaptically mediated Ca(2+) events were blocked by the co-application of strychnine and bicuculline. This suggests that glycine and GABA(A) receptors provide a fundamental regulation of both neuronal excitability and intracellular Ca(2+) at this early time of development.The neurotrophic effects of agonists and antagonists for glycine, GABA(A) and glutamate receptors were examined in neurons cultured for 2 or 5 DIV. From all the agonists used, only high concentrations of glycine increased neurite outgrowth in 5 DIV neurons. We found that strychnine also increased neurite outgrowth, whereas tetrodotoxin (1 microM), nimodipine (4 microM) and bicuculline (20 microM) completely blocked it. On the other hand, APV (50 microM) and CNQX (20 microM) were unable to affect neurite outgrowth. These data suggest that spinal glycine receptors depress neurite outgrowth by shunting neuronal excitability. Outgrowth induction possibly results from the enhanced activity found after the inhibition of glycinergic activity. We postulate that this resets the intracellular calcium at a concentration that favors neurite outgrowth.

Cortical structures such as the hippocampus and cerebral cortex are considered to be particularly susceptible to seizure and epileptiform electrical activity and, as such, are the focus of intense investigation relative to hyperexcitability. To determine whether parallel glutamate-mediated hyperexcitability and seizure-like activity in the rat can be generated by neurons irrespective of their origin within the CNS, we maintained cells from the spinal cord,hippocampus, olfactory bulb, striatum, hypothalamus, and cortex in the long-term presence of glutamate receptor antagonists 2-amino-5-phosphonovalerate and 6-cyano-7-nitroquinoxaline-2-3-dione. After removal of chronic (three to 11 weeks) glutamate receptor block, whole-cell patch-clamp recordings from current-clamped neurons (n = 94) revealed an immediate increase in large excitatory postsynaptic potentials and a depolarization of 20-35 mV that was often sustained for recording periods lasting 5 min (54% of 66 neurons from all six areas). The intense activity was not seen in age-matched control neurons not subjected to chronic glutamate receptor block. Selective blockade of ionotropic glutamate receptors showed that the hyperexcitability was due to an enhanced response through both AMPA/kainate and N-methyl-D-aspartate receptors. Relief from chronic glutamate receptor block also increased inhibitory activity, as revealed by an increase in inhibitory postsynaptic currents while neurons were voltage-clamped at -25 mV. These inhibitory postsynaptic currents could be blocked with bicuculline, indicating that they were mediated by an enhanced GABA release. This enhanced GABA activity reduced, but did not eliminate, the glutamate-mediated hyperactivity, shown by an increase in both intracellular Ca2+ and excitatory electrical activity when bicuculline was added. When the glutamate receptor block was removed, cells (n > 1000) from all six regions showed exaggerated Ca2+ activity, characterized by abnormally high increases in intracellular Ca2+, rising from basal levels of 50-100 nM up to 150-1600 nM. Cd2+ eliminated the hyperexcitability by blocking Ca2+ channels, and reducing excitatory transmitter release and response. Fura-2 digital imaging revealed Ca2+ oscillations with periods ranging from 4 to 60 s. Ca2+ peaks in oscillations in oscillations were synchronized among most neurons recorded simultaneously. That synchronization was dependent on a mechanism involving voltage-dependent Na+ channels was demonstrated with experiments with tetrodotoxin that blocked Ca2+ rises and synchronous cellular behavior. Removal of the glutamate receptor antagonists resulted in the glutamate-mediated death of 44% of the cells after 23 days of chronic block and 82% cell death after 40 days of chronic block. Nimodipine substantially reduced cell death, indicating that one mechanism responsible for the enhanced cell death after relief from chronic glutamate receptor block was increased intracellular Ca2+ entry through L-type voltage-gated calcium channels. These data indicate that glutamate is released by neurons from all areas studied, including the spinal cord. Sufficient amounts of glutamate can be released from axon terminals from all areas to cause cell hippocampal and cortical neurons, but also by neurons from any of the brain regions tested after chronic deprivation of glutamate receptor stimulation during development. This hyperexcitability is mediated by glutamatergic mechanisms independent of the specific excitatory connections existing in vivo. The epileptiform activity of neurons from one region is indistinguishable from that of another in culture, underlining the importance of synaptic connections in vivo that define the responses characteristic of neurons from different brain regions.