Antagonist 2-amino-5-phosphonovalerate Research Articles

Nucleus Reuniens Is Required for Encoding and Retrieving Precise, Hippocampal-Dependent Contextual Fear Memories in Rats.

The nucleus reuniens (RE) is a ventral midline thalamic nucleus that interconnects the medial prefrontal cortex (mPFC) and hippocampus (HPC). Considerable data indicate that HPC-mPFC circuits are involved in contextual and spatial memory; however, it is not clear whether the RE mediates the acquisition or retrieval of these memories. To examine this question, we inactivated the RE with muscimol before either the acquisition or retrieval of pavlovian fear conditioning in rats; freezing served as the index of fear. We found that RE inactivation before conditioning impaired the acquisition of contextual freezing, whereas inactivation of the RE before retrieval testing increased the generalization of freezing to a novel context; inactivation of the RE did not affect either the acquisition or expression of auditory fear conditioning. Interestingly, contextual conditioning impairments were absent when retrieval testing was also conducted after RE inactivation. Contextual memories acquired under RE inactivation were hippocampal independent, insofar as contextual freezing in rats conditioned under RE inactivation was insensitive to intrahippocampal infusions of the NMDA receptor antagonist aminophosphonovalerate. Together, these data reveal that the RE supports hippocampal-dependent encoding of precise contextual memories that allow discrimination of dangerous contexts from safe contexts. When the RE is inactive, however, alternate neural systems acquire an impoverished contextual memory that is expressed only when the RE is off-line.SIGNIFICANCE STATEMENT The midline thalamic nucleus reuniens (RE) coordinates communication between the hippocampus and medial prefrontal cortex, brain areas that are critical for contextual and spatial memory. Here we show that temporary pharmacological inactivation of RE impairs the acquisition and precision of contextual fear memories after pavlovian fear conditioning in rats. However, inactivating the RE before retrieval testing restored contextual memory in rats conditioned after RE inactivation. Critically, we show that imprecise contextual memories acquired under RE inactivation are learned independently of the hippocampus. These data reveal that the RE is required for hippocampal-dependent encoding of precise contextual memories to support the discrimination of safe and dangerous contexts.

Corticosterone enhances the potency of ethanol against hippocampal long-term potentiation via local neurosteroid synthesis.

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.

Glutamate dysregulation in the trigeminal ganglion: A novel mechanism for peripheral sensitization of the craniofacial region

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 (10mM) 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 10μM) significantly reduced the glutamate concentration from 10.6±1.1 to 5.8±1.4μM and 3.0±0.8μM, 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 (500mM, 3μl) 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 (10Hz) 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.

Spermine modulates neuronal excitability and NMDA receptors in juvenile gerbil auditory thalamus

Medial geniculate body (MGB) neurons process synaptic inputs from auditory cortex. Corticothalamic stimulation evokes glutamatergic excitatory postsynaptic potentials (EPSPs) that vary markedly in amplitude and duration during development. The EPSP decay phase is prolonged during second postnatal week but then shortens, significantly, until adulthood. The EPSP prolongation depends on spermine interactions with a polyamine-sensitive site on receptors for N-methyl- D-aspartate (NMDA). We examined effects of spermine application on EPSPs, firing modes, and membrane properties in gerbil MGB neurons during the P14 period of highest polyamine sensitivity. Spermine slowed EPSP decay and promoted firing on EPSPs, without changing passive membrane properties. Spermine increased membrane rectification on depolarization, which is mediated by tetrodotoxin (TTX)-sensitive, persistent Na + conductance. As a result, spermine lowered threshold and increased tonic firing evoked with current injection by up to ∼150%. These effects were concentration-dependent (ED 50=100 μM), reversible, and eliminated by NMDA receptor antagonist, 2-amino-5-phosphonovalerate (APV). In contrast, spermine increased d V/d t of the low threshold Ca 2+ spike (LTS) and burst firing, evoked from hyperpolarized potentials. LTS enhancement was greater at −55 mV than at hyperpolarized potentials and did not result from persistent Na + conductance or glutamate receptor mechanisms. In summary, spermine increased excitability by modulating NMDA receptors in juvenile gerbil neurons.

Differential effects of PACAP-38 on synaptic responses in rat hippocampal CA1 region.

Pituitary adenylate cyclase-activating polypeptide (PACAP-38) is a member of the vasointestinal polypeptide (VIP)/secretin/glucagon family of neuropeptides for which neuroregulatory functions have been postulated. PACAP-38 receptors are expressed in different brain regions, including hippocampus. In this study, we examined the dose-dependent effects of PACAP-38 on the excitatory postsynaptic field potential (fEPSP) evoked at the Schaffer collateral-CA1 synapse in rat hippocampal slices. Bath application of low dose (0.05 nM) of PACAP-38 induced long-lasting facilitation of the fEPSP. This enhancement was blocked by the cholinergic receptor antagonist atropine and partially by the NMDA receptor antagonist 2-amino-5-phosphonovalerate (APV) and therefore, shares a common mechanism with LTP. In contrast, a high dose (1 microM) of PACAP-38 induced a persistent depression of the fEPSP that was not blocked by antagonists of cholinergic receptors (i.e., atropine and mecamylamine), adenosine receptors (i.e., DCPCX), or glutamatergic NMDA receptors (APV). Intermediate doses (0.1-0.5 microM) of PACAP-38 produced an initial decrease of the fEPSP followed by an enhancement. This decrease was not blocked by atropine whereas the facilitation was. These results show that PACAP-38 modulates CA1 synaptic transmission in a dose-dependent manner and that the peptide interacts with cholinergic and glutamatergic systems.

A brain slice model for in vitro analyses of astrocytic gap junction and connexin43 regulation: actions of ischemia, glutamate and elevated potassium

Brain slices prepared from adult rats and maintained for up to 3 h in vitro were used to investigate the effects of pharmacological treatments on the phosphorylation state, immunolabelling characteristics and ultrastructural localization of astrocytic gap junctions and connexin43 (Cx43). Slices deprived of glucose/oxygen to mimic ischemia or those exposed to 1 mM glutamate for 1 h exhibited Cx43 dephosphorylation, epitope masking and gap junction internalization as revealed by Western blotting and Cx43 immunolocalization with various antibodies. Treatment with 15 mM K+ caused Cx43 dephosphorylation without junction internalization. The effects of glutamate and K+ were completely blocked by the N-methyl-D-aspartate (NMDA) glutamate receptor antagonist 2-amino-5-phosphonovalerate (APV), which acts largely on neuronal NMDA receptors, suggesting neuronal mediation of glial gap junction responses to these treatments. Astrocytes contained a dephosphorylated form of Cx43 with a typical migration profile at 41 kDa as well as novel, apparently dephosphorylated or partially phosphorylated, forms migrating at 43 kDa. These results indicate that slices prepared from adult brain can serve as a convenient model to investigate the molecular basis and receptor-mediated mechanisms underlying astrocytic Cx43 responses that have been observed in vivo following cerebral ischemia or neural activation. These processes can be related in part to neuronal regulation of astrocytic gap junctional coupling state, which is also amenable to analysis in brain slices.

Quantitative ultrastructure of physiologically identified premotoneuron terminals in the trigeminal motor nucleus in the cat

Little is known about the ultrastructure of synaptic boutons contacting trigeminal motoneurons. To address this issue, physiologically identified premotor neurons (n = 5) in the rostrodorsomedial part of the oral nucleus (Vo.r) were labeled by intracellular injections of horseradish peroxidase (HRP) in cats. The ultrastructure of 182 serially sectioned axon terminals from the five neurons was both qualitatively and quantitatively analyzed. In addition, the effects of the glycine antagonist strychnine, GABAA antagonist bicuculline, NMDA antagonist 2-amino-5-phosphonovalerate (APV), and non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on Vo.r-induced postsynaptic potentials in trigeminal motoneurons (n = 11) were examined to evaluate potential signaling substances of the premotor neurons. Labeled boutons made synaptic contacts with either jaw-closing or -opening motoneurons. All the boutons contained pleomorphic vesicles, and most formed a single symmetric synapse either on the somata or on primary dendrites. Morphometric analyses indicated that bouton volume, bouton surface area, apposed surface area, total active zone area, and mitochondrial volume were not different between boutons on jaw-closing and -opening motoneurons. Vesicle number and density, however, were higher for boutons on jaw-closing motoneurons. The five morphological parameters were positively correlated with bouton volume. Vesicle density was the exception, which tending to be negatively correlated. Intravenous infusion of strychnine or bicuculline suppressed Vo.r-induced inhibitory postsynaptic potentials (IPSPs) in jaw-closing motoneurons. Abolition of Vo.r-induced excitatory postsynaptic potentials in jaw-opening motoneurons with APV and CNQX unmasked IPSPs. The present results suggest that premotor neurons in the Vo.r are inhibitory and that positive correlations between the ultrastructural parameters associated with synaptic release and bouton size are applicable to the interneurons, as they are in primary afferents. J. Comp. Neurol. 426:13–30, 2000. © 2000 Wiley-Liss, Inc.

Quantitative ultrastructure of physiologically identified premotoneuron terminals in the trigeminal motor nucleus in the cat

Little is known about the ultrastructure of synaptic boutons contacting trigeminal motoneurons. To address this issue, physiologically identified premotor neurons (n = 5) in the rostrodorsomedial part of the oral nucleus (Vo.r) were labeled by intracellular injections of horseradish peroxidase (HRP) in cats. The ultrastructure of 182 serially sectioned axon terminals from the five neurons was both qualitatively and quantitatively analyzed. In addition, the effects of the glycine antagonist strychnine, GABA(A) antagonist bicuculline, NMDA antagonist 2-amino-5-phosphonovalerate (APV), and non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on Vo.r-induced postsynaptic potentials in trigeminal motoneurons (n = 11) were examined to evaluate potential signaling substances of the premotor neurons. Labeled boutons made synaptic contacts with either jaw-closing or -opening motoneurons. All the boutons contained pleomorphic vesicles, and most formed a single symmetric synapse either on the somata or on primary dendrites. Morphometric analyses indicated that bouton volume, bouton surface area, apposed surface area, total active zone area, and mitochondrial volume were not different between boutons on jaw-closing and -opening motoneurons. Vesicle number and density, however, were higher for boutons on jaw-closing motoneurons. The five morphological parameters were positively correlated with bouton volume. Vesicle density was the exception, which tending to be negatively correlated. Intravenous infusion of strychnine or bicuculline suppressed Vo.r-induced inhibitory postsynaptic potentials (IPSPs) in jaw-closing motoneurons. Abolition of Vo. r-induced excitatory postsynaptic potentials in jaw-opening motoneurons with APV and CNQX unmasked IPSPs. The present results suggest that premotor neurons in the Vo.r are inhibitory and that positive correlations between the ultrastructural parameters associated with synaptic release and bouton size are applicable to the interneurons, as they are in primary afferents.

Effect of glutamate receptor antagonists on excitatory postsynaptic potentials in striatum

Glutamate, as the main transmitter of corticostriatal pathway, has a crucial role in the regulation of the activity of striatal cells as well as in pathogenesis of some diseases characterized by striatal malfunction caused by overexcitation of neurons. In the present study, the role of ionotropic excitatory amino acid receptors was investigated in the striatal synaptic transmission. Using conventional intracellular electrophysiological methods in brain slices, we have investigated the effects of the N-methyl- D-aspartate (NMDA) antagonist (±) 2-amino-5-phosphono-valerate (APV) and the α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) antagonist (±) 1-(4-aminophenyl)-3-methyl-carbamoyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 53655) on the excitatory postsynaptic potentials (EPSPs) evoked by electrical stimulation of corpus callosum. The AMPA antagonist significantly decreased electrically evoked responses and a weak inhibition was also observed after APV application. The results were compared to similar data obtained in a cortical slice study.

Poor correlation between postsynaptic [Ca] i increases and long-term potentiation induced by 2-deoxyglucose inCA1 hippocampal neurons

2-Deoxyglucose (2-DG; 10 mM) replacing glucose for ∼15 min regularly causes a very sustained enhancement of synaptic transmission in the CA1 hippocampal zone (2-DG-induced long-term potentiation, 2-DG-LTP). This LTP is prevented by dantrolene (10μM) or by the NMDA antagonist aminophosphonovalerate (APV; 50–100 μM); it is Ca-dependent [1, 2], and is not depressed by adenosine antagonists (DPCPX and 8-SPT), which prevent the initial block of EPSP caused by 2-DG, but is not observed if glucose is replaced by sucrose instead of 2-DG. To clarify the role of [Ca]i, in 2-DG-LTP, we monitored fluo-3 fluorescence inCA1 neurons, in submerged slices kept at 30°C. 2-DG applied as above consistently led to a rapid, but reversible rise in fluorescece. Isomodar sucrose (for 15–40 min) elicited smaller, but significant increases in fluorescence. Dantrolene and APV greatly reduced 2-DG's effect, but Ca-free medium did not abolish it. Paradoxically, it was fully suppressed by 8-SPT, but not at all by DPCPX. The discrepancies between the effects of sucrose and 8-SPT on the fluorescence changes and on 2-DG-LTP suggest that the observed increases in the postsynaptic [Ca] i are not essential for 2-DG-LTP. In further experiments, intracellular recordings showed that 2-DG applications consistently hyperpolarizeCA1 neurons, probably owing to adenosine-mediated activation of K+ conductance (which could be prevented by DPCPX or 8-SPT, but not by a KATP antagonist, glyburide). The 2-DG-LTP of EPSP was not suppressed by EGTA leakage from electrodes, in keeping with the above evidence that the [Ca] i changes needed for the induction of 2-DG-LTP are not those observed postsynaptically. In this respect, as well as in the absence of postsynaptic depolarization, 2-DG-LTP offers a marked contrast to more conventional forms of LTP.

Implication of protein kinase C in mechanisms of potassium- induced long-term potentiation in rat hippocampal slices

The involvement of Ca 2+/phospholipid-dependent ( α, β, γ PKCs) and Ca 2+-independent PKC ( ϵ, and ζ isoforms) in mechanisms of long-term potentiation was investigated in CA1 hippocampal slices, using a brief high potassium pulse (50 mM, 40 s) to induce long-term potentiation (K +/LTP). The K + pulse induced first, in 15 s a translocation of PKC activity to the membrane. This was rapidly followed, from 1 to 60 min after the pulse, by a selective activation of PKC in the cytosol. This activation, which could be blocked by the NMDA ( N-methyl- d-aspartate) receptor antagonist 2-amino-5- phosphonovalerate (APV), was associated with a significant increase in immunoreactivity for γ PKC in the cytosol, and also to a less degree for β PKC. In contrast, application of the phorbol ester PMA (phorbol 12- mirystate 13 acetate) to other slices induced a rapid and persistent translocation to the membrane of α, β, ϵ and ζ PKCs. A major role for the activation of cytosolic γ PKC in the maintenance of LTP is discussed.

Effects of NMDA antagonists on seizure thresholds induced by intrahippocampal microdialysis of picrotoxin in freely moving rats

The role of N-methyl- d-aspartate (NMDA) receptors on individual variations in animal seizure threshold induced by picrotoxin microperfusion in the hippocampus of freely moving rats was analyzed by testing the effects of NMDA receptor antagonists 2-amino 5-phosphonovalerate (AP-5) and dizocilpine (MK-801) on individual seizure thresholds. We found a correlation between the increased threshold levels induced by AP-5 and control seizure levels of each individual animal. However, no correlation was found with the increased threshold level induced by MK-801. These data suggest that the individual variations in seizure threshold in different animals reflect dynamic processes of NMDA mediated activity and emphasizes the value of whole-animal models to analyze NMDA receptor functions.

A field potential analysis on the effects of lamotrigine, GP 47779, and felbamate in neocortical slices.

We studied the action of the new antiepileptic drugs lamotrigine (LTG), GP 47779 (the active metabolite of oxcarbazepine), and felbamate (FBM) on stimulus-evoked field potentials recorded from rat prefrontal and frontal cortical slices. In the presence of physiologic concentrations of extracellular magnesium (1.2 mM) the field potential amplitude was not affected by the N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, 2-amino-5-phosphonovalerate (APV), while it was blocked by the non-NMDA glutamate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). When magnesium was removed from the bathing medium, there was a significant NMDA-mediated component of the field potential. LTG and GP 47779 decreased, in a dose-dependent manner, the field potential amplitude under both experimental conditions. FBM caused a dose-related decrease of the field potential amplitude only in the absence of external magnesium, suggesting a selective interaction with an NMDA-mediated component of this potential. These findings indicate that the reduction of cortical excitatory transmission might represent a common target for new antiepileptic drugs.

PH‐dependent Facilitation of Synaptic Transmission by Histamine in the CA1 Region of Mouse Hippocampus

The action of histamine on excitatory synaptic transmission in the CA1 region of the mouse hippocampus was investigated. In a medium at pH 7.4 population spikes were increased for approximately 60 min after a brief (5 min) perfusion with histamine (5 microM) but excitatory postsynaptic potentials (EPSPs) were unaffected, as previously reported in the rat. At pH 7.0, however, a late component of extra- and intracellularly registered EPSPs was enhanced in two phases: a shorter (by 30%) and a longer lasting one (> 1 h by 10%). The NMDA antagonist amino-phosphonovalerate (60 microM) blocked this late component and prevented the EPSP broadening by histamine. In some cells histamine induced burst-firing and prolonged EPSPs. The actions on EPSPs were not mediated by any of the known histamine receptors as they were not mimicked by histamine H1, H2 and H3 agonists or blocked by H1, H2 or H3 antagonists. We conclude that histamine enhances a late (NMDA) component of hippocampal EPSPs when protonization is increased by a slight shift of the pH in the acidic direction. Such shifts occur during intense nervous discharges, e.g. in epileptic tissue or following tetanic stimulation, and in hypoxic conditions.

Glutamate enhances the adenylyl cyclase-cAMP system-induced beta-endorphin secretion and POMC mRNA expression in rat hypothalamic neurons in culture: NMDA receptor-mediated modulation

l-Glutamate, a major excitatory amino acid of the central nervous system, plays important roles as neurotransmitter and neuromodulator in the brain. Increasing evidence suggests that glutamate may also involve in the regulation of the neuroendocrine system at the hypothalamus. Employing long term monolayer hypothalamic cell cultures prepared from neonatal rats, we reported here that whereas glutamate significantly enhanced forskolin-, or N 6,2′-O-dibutyrrladenosine-3′5′-cyclic monophosphate [(Bu) 2cAMP]-stimulated immunoreactive (ir)-β EP release form cultures treated daily for 4 consecutive days, the excitatory amino acid alone produced little effect. This potentiation of glutamate was time-related and dose-dependent with an E max value of the amino acid being approximately 50 μM; at this concentration glutamate augmented ir-β EP secretion about 1.8 times ( P<0.05) that induced by 2 μM forskolin alone. Similar effects were also observed for POMC and mRNA levels in cultures subjected to 6 h of the above treatment regime. This potentiating effect of glutamate appears to be mediated specifically through NMDA receptor as it can be mimicked by NMDA but not by kainic acid or quisqualic acid, and blocked by the NMDA receptor antagonist 2-amino-5-phosphonovalerate (APV), but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA glutamate receptor antagonist. Interestingly, glutamate was found not to enhance high doses of forskolin (10 μM) or (Bu) 2 cAMP (100 μM) stimulated β EP release and POMC mRNA levels in hypothalamic cell cultures. These novel findings suggests that (i) hypothalamic β EP neurons are subjected to direct glutamatergic modulation, and (ii) the modulating effect of glutamate is mediated, at least in part, through the adenylyl cyclases-cAMP system. Our novel findings have provided a direct evidence supporting the assertion that cortical glutamatergical system may directly involve in the functional modulation of hypothalamic β EP producing neurons.

Amygdala N-methyl- d-aspartate receptors participate in the induction of long-term potentiation in the dentate gyrus in vivo

We investigated the effects of injection of N-methyl- d-aspartate (NMDA) receptor antagonists, 2-amino-5-phosphonovalerate (APV) and 7-chlorokynurenate (7-Cl-Kyn), into the basolateral amygdala (BLA) on long-term potentiation (LTP) in the medial perforant path-dentate gyrus granule cell synapses of anesthetized rats. Injection of APV or 7-Cl-Kyn into the ipsilateral BLA did not affect the baseline synaptic responses, but significantly attenuated the dentate gyrus LTP induced by tetanic stimulation. Injection of APV into the contralateral BLA did not affect the induction of LTP. When APV was injected after tetanic stimulation, it did not affect the maintenance phase of LTP. These results suggest that NMDA receptors in the ipsilateral BLA partly participate in the induction of LTP in the dentate gyrus in vivo.

Activation of a metabotropic glutamate receptor increases intracellular calcium concentrations in neurons of the avian cochlear nucleus

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.

Evaluation of the hypothesis that hippocampal interictal spikes are caused by long-term potentiation.

Hippocampal spontaneous interictal spikes (SISs) occur in the EEG after repeated afterdischarges (ADs) induced by high-frequency (200 Hz) stimulation trains. Because SISs resemble population excitatory postsynaptic potentials (EPSPs), and SISs persist for several days like some types of hippocampal long-term potentiation (LTP), LTP has been suggested as a mechanism for SISs. We specifically examined the hypothesis that SISs are caused by basal-dendritic LTP in CA1. Rats were chronically implanted with bilateral electrodes in the hippocampal CA1 region. In the first experiment, 10-18 patterned primed burst (PB) stimulations were delivered hourly for 2-3 days to activate the commissural basal-dendritic EPSP in CA1. Robust LTP of the basal-dendritic CA1 synapse was detected, typically saturating at 100% enhancement after five stimulations. However, few SISs were detected if ADs were not elicited. In the second experiment, repeated commissurally evoked ADs induced a high rate of SISs, together with LTP of the basal-dendritic and apical-dendritic EPSP in CA1, but the SIS rate was not necessarily related to the level of LTP. In the third experiment, an intraventricular dose (20 micrograms) of an N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (APV) was used to block the basal-dendritic LTP in CA1. The increase in SISs induced by a single AD was not blocked, however, suggesting that NMDA receptors were not critical in generation of SISs. In the fourth experiment, PBs (that induced LTP but no ADs) were able to increase the rate of SISs marginally when SISs were already present. In all, the experiments suggest that LTP at the basal dendrites of CA1 is not critical in generation of hippocampal SISs, although an increase in LTP may increase the rate of SISs marginally when SISs are already present.

Regulation of NMDA receptor mRNA during visual map formation and after receptor blockade.

The topographic refinement of the rat retinocollicular projection is dependent on normal NMDA receptor function. Here we examined the expression of NMDA and non-NMDA glutamate receptor mRNA in the rat superior colliculus (SC) during this postnatal refinement period. The temporal expression pattern of mRNA coding for the NMDA receptor subunit NR1 in the superficial SC followed the time course of collicular synaptogenesis. A pronounced increase of NR1 mRNA levels occurred during the late stages of retinocollicular map refinement. In cortex, the time course of the expression of NR1 mRNA in cortex was found to be similar to that observed in SC, with low levels during the first postnatal week, a maximum at P19, and a decrease thereafter. In SC, but not in cortex, there was a change in the ratio of the two NR1 transcripts during the second postnatal week that parallels a previously demonstrated developmental change in the mean open time of NMDA channels in collicular neurons. In contrast, the mRNA expression pattern of the non-NMDA receptor subunit GluR2 in the developing SC was not closely correlated with synaptic changes. Chronic treatment of the SC with the NMDA receptor antagonist 2-amino-5-phosphonovalerate (APV) for 12 or 19 days, which disrupts retinocollicular map formation, appears to block the developmental rise in NR1 mRNA levels. These findings support a specific role for the NMDA receptor subtype of glutamate receptors in the control of synaptogenesis and developmental plasticity in the SC.

Glutamate-stimulated phosphatidylinositol metabolism in the avian cochlear nucleus

This study examined the ability of the excitatory amino acid glutamate and its analogs to stimulate phosphatidylinositol metabolism in isolated cochlear nucleus tissue from young chicks. In the presence of lithium chloride, glutamate and (±)-1-aminocyclopentyl- trans-1,3-dicarboxylate (ACPD) stimulated the formation of inositol phosphates to levels significantly above unstimulated control levels. Unexpectedly, quisqualate did not stimulate inositol phosphates formation. The N- methyl- d-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (APV), the ionotropic kainate/quisqualate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the putative metabotropic glutamate receptor antagonist 2-amino-3-phosphonopropionate (AP3) had no effect on the glutamate stimulated formation of inositol phosphates. We conclude that a metabotropic glutamate receptor is present on cochlear nucleus neurons of posthatch chicks and is able to stimulate formation of inositol phosphates.