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d -Serine plays an important role in modulating N-methyl- d -aspartate receptor (NMDAR) neurotransmission in the mammalian brain by binding to the receptor’s glycine modulatory site (GMS). The cytosolic enzyme serine racemase (SR) converts L-serine to d -serine, while the peroxisomal enzyme d -amino acid oxidase (DAAO) catalyzes the breakdown of d -serine. Although it is important to understand how the activities of SR and DAAO regulate d -serine levels, very little is known about the mechanisms that regulate the expression of SR and DAAO. In this study, we investigated whether the different centrally active drugs affect the expression of SR and DAAO in adult mouse brain. We found that the NMDAR antagonist, MK801, and cocaine, psychotropic drugs that both augment glutamate release, reduce the expression of SR and DAAO. This regulation is brain region selective, and in the case of cocaine, is reversed in part byα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX). However, d -serine and antipsychotics do not regulate SR and DAAO protein levels. In a genetic model of SR disruption, we found that DAAO expression was unaltered in SR conditional knockout mice, in which tissue d -serine content remains fairly stable despite marked reduction in SR expression. This study reveals a new mechanism by which AMPAR activity could regulate NMDAR function via d -serine availability.

Made available in DSpace on 2020-12-12T01:33:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-11-15 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Insular cortex is a brain structure involved in the modulation of autonomic activity and cardiovascular function. The nitric oxide/cyclic guanosine-3′,5′-monophosphate pathway is a prominent signaling mechanism in the central nervous system, controlling behavioral and physiological responses. Nevertheless, despite evidence regarding the presence of nitric oxide-synthesizing neurons in the insular cortex, its role in the control of autonomic and cardiovascular function has never been reported. Thus, the present study aimed to investigate the involvement of nitric oxide/cyclic guanosine-3′,5′-monophosphate pathway mediated by neuronal nitric oxide synthase (nNOS) activation within the insular cortex in the modulation of baroreflex responses in unanesthetized rats. For this, we evaluated the effect of bilateral microinjection of either the nitric oxide scavenger carboxy-PTIO, the selective neuronal nitric oxide synthase inhibitor Nω-Propyl-L-arginine or the soluble guanylate cyclase inhibitor ODQ into the insular cortex on the bradycardia evoked by blood pressure increases in response to intravenous infusion of phenylephrine, and the tachycardia caused by blood pressure decreases evoked by intravenous infusion of sodium nitroprusside. Bilateral microinjection of either NPLA or carboxy-PTIO into the insular cortex increased the reflex bradycardic response, whereas the reflex tachycardia was decreased by these treatments. Bilateral microinjection of the soluble guanylate cyclase inhibitor into the insular cortex did not affect any parameter of baroreflex function evaluated. Overall, our findings provide evidence that insular cortex nitrergic signaling, acting via neuronal nitric oxide synthase, plays a prominent role in control of baroreflex function. However, control of reflex responses seems to be independent of soluble guanylate cyclase activation. Department of Pharmacology School of Medicine of Ribeirão Preto University of São Paulo Laboratory of Pharmacology School of Pharmaceutical Sciences São Paulo State University (UNESP) Department of Health Sciences Faculty of Medicine – Federal University of Lavras Laboratory of Pharmacology School of Pharmaceutical Sciences São Paulo State University (UNESP) CNPq: 141992/2016-6 FAPESP: 2015/05922-9 CNPq: 456405/2014-3 FAPEMIG: APQ-01316-16

The spinal trigeminal subnucleus caudalis (Vc) receives preferentially nociceptive afferent signals from the orofacial area. Nociceptive stimuli to the orofacial area induce cyclooxygenase both peripherally and centrally, which can synthesize a major prostanoid prostaglandin E2 (PGE2) that implicates in diverse physiological functions. To clarify the roles of centrally-synthesized PGE2 in nociception, effects of exogenous PGE2 on synaptic transmission in the Vc neurons were investigated in the rat brainstem slice. Spontaneously occurring excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs) were recorded, respectively, under pharmacological blockade of inhibitory and excitatory transmission by whole-cell patch-clamp mode. Perfusion of PGE2 (1-5 μM) increased the frequency of sIPSCs in a concentration-dependent manner but had no significant effect on the amplitude. Similarly to the effects on sIPSCs, PGE2 increased the sEPSC frequency without any effect on the amplitude. These facilitatory effects of PGE2 on spontaneous synaptic transmissions were blocked by an EP1 antagonist SC19220 but not by an EP4 antagonist AH23848. Electrical stimulation of the trigeminal tract evoked short latency EPSCs (eEPSCs) in the Vc neurons. PGE2 (5 μM) was ineffective on the eEPSCs. The present study demonstrated that PGE2 facilitated spontaneous synaptic transmissions in the Vc neurons through activating the presynaptic EP1 receptors but had no effect on the trigeminal tract-mediated excitatory transmission. These results suggest that centrally-synthesized PGE2 modifies the synaptic transmission in the Vc region, thereby contributing to the processing of nociceptive signals originated from the orofacial area.

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

Cofilin is the major actin-depolymerizing factor in the CNS for the regulation of actin dynamics. Neurodegenerative stimuli can induce the formation of cofilin rod, a pathological structure composed of cofilin and actin. The formation of cofilin rod was found to disrupt synapse function and cause neurite loss. The aim of the present study is to study the whole process of cofilin rod formation pattern in cultured hippocampal neurons under excitotoxic stimulation and to explore its underlying pharmacological mechanism. By using live cell imaging of neurons overexpressing EGFP-tagged wild type cofilin, we found a two-phase pattern of rod formation induced by glutamate stimulation. The early phase of rod formation occurred shortly after stimulation (∼0.5h) but quickly dissolved within 2h. The second phase happened within a much longer time window, 8h after stimulation. Immunostaining of endogenous cofilin in neurons also confirmed this glutamate stimulation induced two-phase rod formation pattern. The first phase was co-related with intracellular calcium concentration and pH increase while the second phase was not. These two phases of cofilin rod formation induced by glutamate stimulation was antagonized by both non-NMDA and NMDA receptor antagonist DNQX and AP5, respectively. Our results for the first time demonstrate the dynamic cofilin rod formation pattern under stress stimulation in detail by time lapse imaging. These findings reveal a novel time course of excitotoxicity induced neuronal damage and indicate a potential target of neuropathy treatment of neurodegenerative diseases.

Receptor subunit composition is believed to play a major role in the synaptic trafficking of AMPA receptors (AMPARs), and thus in activity-dependent synaptic plasticity. To isolate a physiological role of GluA1-containing AMPARs in area CA3 of the hippocampus, pair recordings were performed in organotypic hippocampal slices taken from genetically modified mice lacking the GluA1 subunit. We report here that long-term potentiation (LTP) is impaired not only at active but also at silent synapses when the GluA1 subunit is absent. The GluA1 knockout mice also exhibited reduced AMPAR-mediated evoked currents between pairs of CA3 pyramidal neurons under baseline conditions suggesting a significant role for GluA1-containing AMPARs in regulating basal synaptic transmission. In two independent measures, however, long-term depression (LTD) was unaffected in tissue from these mice. These data provide a further demonstration of the fundamental role that GluA1-containing AMPARs play in activity-dependent increases in synaptic strength but do not support a GluA1-dependent mechanism for reductions in synaptic strength., National Institute of Mental Health (U.S.) (Grant MH080310)

Previous work had demonstrated that there was elevated regional cerebral O 2 consumption in the brains of a tuberous sclerosis model (Eker rat). We tested the hypothesis that the increased cerebral O 2 consumption was related to an increased contribution of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors to the control of cerebral metabolism. Young (4 weeks) male control Long Evans ( N = 14) and Eker ( N = 14) rats (70–100 g) were divided into control and NBQX (50 mg/kg, AMPA antagonist) treated animals. Cerebral regional blood flow ( 14 C-iodoantipyrine) and O 2 consumption (cryomicrospectrophotometry) were determined in isoflurane anesthetized rats. We found significantly increased basal O 2 consumption (+ 85%) in the cortex, hippocampus, cerebellum and pons of the Eker rats. Regional cerebral blood flow was also elevated in Eker rats at baseline (+ 53%) as was cerebral O 2 extraction (+ 22%). NBQX significantly lowered O 2 consumption in all regions of the Long Evans rats (− 41%). Cerebral O 2 consumption was lowered in the cortex to a lesser extent (− 24%) and there were no changes in other regions of the Eker rats after NBQX. Cerebral blood flow was reduced in the Long Evans, but not the Eker rats, after NBQX. In conclusion, Eker rats had significantly elevated cerebral O 2 consumption, O 2 extraction and blood flow, but this was not related to AMPA receptor activation. In fact, the importance of AMPA receptors in the control of basal cerebral O 2 consumption was reduced.

Neonatal (postnatal days 8-21) exposure of rats to the selective serotonin reuptake inhibitor (SSRI), citalopram, results in persistent changes in behavior including decreased sexual activity in adult animals. We hypothesized that this effect was a consequence of abnormal stimulation of serotonergic receptors 5- HT1A or/and 5-HT1B as a result of increased synaptic availability of serotonin during a critical period of development. We examined whether neonatal exposure to a 5-HT1A (8OH-DPAT) and/or a 5-HT1B (CGS 12066B) receptor agonist can mimic the effect of neonatal exposure to citalopram on adult sexual behavior. Results showed that neonatal treatment with 5-HT1B receptor agonist robustly impaired sexual behavior similar to the effect of citalopram whereas exposure to 5-HT1A receptor agonist only moderately influenced male sexual activity in adult animals. These data support the hypothesis that stimulation of serotonin autoreceptors during development contributes to the adult sexual deficit in rats neonatally exposed to citalopram.

Excitatory amino acids play an important role in generation of epileptic seizures. To study the participation of different types of their receptors in cortical epileptic afterdischarges, a noncompetitive NMDA receptor antagonist dizocilpine and a competitive AMPA receptor antagonist NBQX were used. Adult rats with implanted epidural stimulation and registration electrodes were pretreated either with NBQX (30 or 60 mg/kg i.p.) or with dizocilpine (0.1 or 0.5 mg/kg i.p.) and low-frequency stimulation of sensorimotor cortical area was repeatedly applied with stepwise increased current intensities. Lower dose of NBQX unexpectedly decreased thresholds for elicitation of spike-and-wave afterdischarges (ADs), clonic seizures accompanying this type of ADs and for transition into the second, limbic type of ADs. Lower dose of dizocilpine increased these three thresholds. Higher doses of either drug did not significantly change threshold intensities. Duration of ADs was also influenced by the two antagonists in opposite directions: higher dose of NBQX resulted in prolongation of ADs mainly due to an increased duration of the spike-and-wave part of ADs whereas dizocilpine shortened ADs in a dose-dependent manner affecting both types of ADs. In addition, NBQX did not influence interhemispheric responses meanwhile dizocilpine moderately suppressed these evoked potentials. According to our results, NMDA receptors are important for generation of cortical epileptic afterdischarges meanwhile the role of AMPA receptors is not clear and has to be analyzed.

γ-Aminobutyric acid (GABA) is inhibitory in adult, but excitatory in neonatal, neurons. The switch from excitatory to inhibitory action is due to a negative shift in the equilibrium potential for the GABAA receptor-mediated postsynaptic current (EGABA-PSC). Here, we report that, in neonatal rat hippocampal CA1 neurons, presynaptic theta-burst activation induces not only a shift in EGABA-PSC towards that in adult neurons, but also a recruitment of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-receptor-mediated postsynaptic currents.

In previous studies, we found that chronic intermittent ethanol (CIE) treatment–a model of ethanol consumption in which animals are exposed to and withdrawn from intoxicating levels of ethanol on a daily basis–produces neuroadaptive changes in hippocampal area CA1 excitatory synaptic transmission and plasticity. Synaptic responses mediated by N-methyl- d -aspartate (NMDA) receptors are known to be sensitive to ethanol and could play an important role in the neuroadaptive changes induced by CIE treatment. To address this issue, we compared electrophysiological recordings of pharmacologically isolated NMDA-receptor-mediated field excitatory postsynaptic potentials (fEPSPs) in the CA1 region of hippocampal slices prepared from control rats and rats exposed to 2 weeks of CIE treatment administered by vapor inhalation. We found that fEPSPs induced by NMDA receptor activation were unaltered in slices prepared shortly after cessation of CIE treatment (i.e., ≤1 day of withdrawal from CIE). However, following 7 days of withdrawal from CIE treatment, NMDA-receptor-mediated fEPSPs were augmented relative to age-matched controls. Western blot analysis of NMDA receptor subunit expression showed that, at 7 days of withdrawal, the level of protein for NR2A and NR2B subunits was elevated in the CA1 region of hippocampal slices from CIE-treated animals compared with slices from age-matched controls. These results are consistent with an involvement of NMDA-receptor-mediated synaptic responses in the neuroadaptive effects of CIE on hippocampal physiology and suggest that such changes may contribute to ethanol-induced changes in processes dependent on NMDA-receptor-mediated synaptic responses such as learning and memory, neural development, hyperexcitability and seizures, and neurotoxicity.

(1R,2R,3R,5R,6R)-2-amino-3-(3,4-dichlorobenzyloxy)-6-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (MGS0039), a selective group II metabotropic glutamate receptor (mGluR) antagonist, exhibits antidepressant-like activities in rodent models. In the present studies, to clarify the involvement of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor activation in exhibition of the antidepressant-like properties of MGS0039, we examined the effect of an AMPA receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), on the antidepressant-like effect of MGS0039 in the mouse tail suspension test. We also examined the effects of NBQX on increased serotonin release after treatment with MGS0039 in the rat medial prefrontal cortex (mPFC) using in vivo microdialysis evaluation. In the tail suspension test, MGS0039 (0.3-3 mg/kg, i.p.) treatment dose-dependently and significantly reduced immobility time. Pretreatment with NBQX (10 mg/kg, s.c.) significantly prevented the antidepressant-like effect of MGS0039 in the tail suspension test, while NBQX itself had no effect on immobility time. In the microdialysis evaluation, administration of MGS0039 (10 mg/kg, i.p.) significantly increased serotonin levels in mPFC in freely moving rats, while NBQX (1 mg/kg, i.p.) itself had no effect on serotonin release in this region. Pretreatment with NBQX significantly attenuated the increase in serotonin release by MGS0039. These findings suggest that stimulation of postsynaptic AMPA receptors plays a role in mediating the pharmacological effects of MGS0039.

This study aims to explore the effects of a selective 5-HT1B receptor agonist, CGS-12066A, on the neuronal excitability of the rat medial vestibular nuclear neurons. The spontaneous firing rate was decreased, and the membrane potential was hyperpolarized by CGS-12066A. The whole potassium currents were inhibited by CGS-12066A. After the calcium-dependent potassium, currents were blocked, however, CGS-12066A did not inhibit the potassium currents, suggesting that the 5-HT action site is calcium-dependent potassium currents.

It has been reported that the inhibitory postsynaptic potential (IPSP) is abolished before the excitatory postsynaptic potential (EPSP) when the extracellular concentration of Ca(2+) ([Ca(2+)](o)) is removed gradually in hippocampal slices. However, the low-Ca(2+) nonsynaptic epileptiform activity does not appear until the [Ca(2+)](o) is decreased to a level sufficient to depress the excitatory synaptic transmission. This suggests the hypothesis that the suppression of excitatory synaptic transmission itself could facilitate the generation of epileptiform activity. In the present study, we tested this hypothesis and developed a new model of nonsynaptic epileptiform activity by gradually raising the neuronal excitability and blocking the synaptic transmission with high K(+), zero Ca(2+) and calcium chelator ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) in the CA1 region of hippocampus in vivo. The changes of synaptic transmission and recurrent inhibitory activity during this process were evaluated by measuring the amplitude of the population spikes (PS) in response to paired-pulse orthodromic stimulation. The results show that the epileptiform activity appeared only when the excitatory synaptic transmission was depressed by further lowering [Ca(2+)](o) with EGTA. Similar epileptiform activity could be induced when EGTA was replaced by the excitatory postsynaptic amino acid antagonists D-(-)-2-amino-5-phosphonopentanoic acid (APV) plus 6,7-dinitroquinoxaline-2,3-dione (DNQX) or APV alone but not DNQX alone. The combination application of APV and cadmium enhanced the epileptiform activity. These results suggest that the suppression of excitatory synaptic transmission can facilitate the appearance of epileptiform activity in solution with high K(+) and low Ca(2+) in vivo. These data provide new information to be considered in the development of antiepileptic drugs. They also suggest a possible mechanism to explain the fact that low-frequency electrical stimulation can suppress epileptiform activity.

Kainic acid (KA) treatment induced neuronal death and apoptosis in murine cerebellar granule cells (CGNs) cultures from both wild-type and knockout p21(-/-) mice. There was not statistically significant difference in the percentage of neuronal apoptosis among strains. KA-induced neurotoxicity was prevented in the presence of NBQX (20 microM) and GYKI 52446 (20 microM), but not by z-VAD-fmk, suggesting that caspases are not involved in the apoptotic process. Data suggest that p21(WAF/Cip) was unable to modulate KA-induced apoptosis in murine CGNs.

The depolarizing afterpotential (DAP) following an action potential increases the excitability of a neuron. Mechanisms related to the DAP following an antidromic or current-induced spike were studied in CA1 pyramidal cells by whole-cell recordings in hippocampal slices in vitro. In DAP-holding voltage curves, the DAP at 10 ms after the spike peak (DAP10) was extrapolated to reverse at about −50 mV. Increase of extracellular K + concentration increased DAP and neuronal bursting. DAP10 reversal potential shifted positively with an increase in [K + ] o and with the blockade of K + conductance using pipettes filled with Cs + . Similarly, extracellular tetraethylammonium (TEA; 10 mM), 4-aminopyridine (3–10 mM) increased DAP and shifted the DAP10 reversal potential to a depolarizing direction. Decrease of [Ca 2+ ] o did not alter DAP significantly, suggesting a nonessential role of Ca 2+ in the DAP. Perfusion of tetrodotoxin (TTX; 0.1–1 μM) and replacement of extracellular Na + by choline + suppressed both spike height and DAP simultaneously. Replacement of extracellular Na + by Li + increased DAP and spike bursts, and caused a positive shift of the DAP10 reversal potential. It is suggested that Li + increased DAP by blocking an Na + -activated K + current. In summary, multiple K + conductances are normally active during the DAP following a single action potential.

Seizures have been shown to promote the proliferation of granule cell precursors in the adult brain, but the underlying mechanisms remain largely unknown. Using systemic bromodeoxyuridine (BrdU) to label dividing cells, we examined the effects of selective ionotropic glutamate receptor antagonists on granule cell precursor proliferation in adult rats after pentylenetrazol (PTZ)-induced generalized clonic seizures. We found that the NMDA receptor antagonist MK-801 significantly inhibited behavioral and EEG seizures and completely blocked seizure-induced increase in the number of BrdU-labeled cells in the dentate gyrus. Although the AMPA/KA receptor antagonist DNQX was not observed to affect seizures, it significantly suppressed the number of BrdU-labeled cells in the dentate gyrus. Double immunohistochemical staining showed that both the mature granule cells and the majority of BrdU-labeled, mitotically active cells expressed the NMDA receptor subunit NR1 and the AMPA/KA receptor subunit GluR2. Because accumulated evidence showed that mild seizures are sufficient to promote precursor cell proliferation, the present findings that MK-801 inhibited seizures and completely blocked seizure-induced increase in precursor cell proliferation suggest that the direct blockade action of MK-801 on NMDA receptors on the granule cell precursors may play an important role in blocking seizure-induced precursor cell proliferation. The suppression of seizure-induced proliferation of granule cell precursors by DNQX may be achieved by the direct action of DNQX on AMPA/KA receptors on the granule cell precursors. Thus, our findings indicate that seizures may promote cell proliferation in the adult rat dentate gyrus through glutamatergic mechanisms acting on both NMDA and AMPA/KA receptors.

Noradrenergic projections to the cortex modulate a variety of cortical activities and calcium channels are one likely target for such modulation. We used the whole-cell patch-clamp technique to study noradrenergic modulation of barium currents in acutely dissociated pyramidal neurons from rat sensorimotor cortex. Extracellular application of specific agonists and antagonists revealed that norepinephrine (NE) reduced Ca 2+ current. A major component of this modulation was due to activation of α 2 receptors. Activation of α 2 -adrenergic receptors resulted in a fast, voltage-dependent pathway involving G i /G o G-proteins. This pathway targeted N- and P-type calcium channels The α 2 modulation was partially reversed by repeated action potential waveforms (APWs). N- and P-type channels have been implicated in synaptic transmission and activation of afterhyperpolarizations in these cells. Our findings suggest that NE can regulate these cellular processes by mechanisms sensitive to spike activity.

Numerous data indicate that nonsynaptic release of glutamate occurs both in normal and pathophysiological conditions. When reaching receptors in the postsynaptic density (PSD), glutamate (Glu) could affect the synaptic transmission. We have tested this possibility in the hippocampal CA1 synapses of rats, either by applying exogenous Glu to the CA1 neurons or by disruption of Glu transporter activity. L-Glu (400 microM) was directly applied to the hippocampal slices acutely isolated from the rats. It produced a strong inhibition of both ortho- and antidromically elicited action potentials fired by CA1 neurons while the excitatory postsynaptic current (EPSC) measured in these neurons remained totally unaffected. The optical isomer D-Glu which is not transported by the systems of Glu uptake inhibited not only orthodromic and antidromic spikes, but also EPSC. Non-specific glutamate transporter inhibitor DL-threo-beta-hydroxyaspartic acid (THA, 400 microM) mimicked the effects of exogenous Glu and produced strong inhibition of both orthodromic and antidromic spikes, without any influence on the amplitude of EPSCs. Dihydrokainate (DHK, 300 microM), selective inhibitor of GLT-1 subtype of glutamate transporter, exerted a significant inhibitory action on the orthodromically evoked spikes and also on the EPSC. Our results indicate that extrasynaptic and PSD membranes of CA1 neurons form separate compartments differing in the mechanisms and efficiency of external Glu processing: the protection of PSD markedly prevails.

We have previously shown that hypothalamic injections of glutamate, or agonists of its ionotropic receptors (iGluRs), elicit intense feeding responses in satiated rats [Brain Res. 613 (1993) 88, Brain Res. 630 (1993) 41]. While attempting to clarify the role of the AMPA and kainate (KA) receptor subtypes in glutamatergic feeding systems, we discovered that lateral hypothalamic (LH) injection of high doses of the competitive AMPA/KA receptor antagonist, NBQX (10 and 30 nmol), elicited a pronounced feeding response. We questioned whether this effect was due to inactivation of AMPA or possibly KA receptors. To determine whether other AMPA/KA antagonists can also elicit feeding, we tested whether injection of CNQX, another AMPA/KA receptor antagonist, also stimulates eating and whether these feeding stimulatory effects were due to antagonists' actions in the LH or in other hypothalamic sites. Here we report that NBQX and CNQX elicit feeding in a dose dependent manner and are most effective when injected into the perifornical hypothalamus (PFH), or into the paraventricular nucleus (PVN) and, to a lesser extent, into the LH of satiated rats. In contrast, AMPA was most effective in stimulating feeding when injected into the LH, confirming previous reports. These data suggest that either activation or inactivation of AMPA/KA receptors in distinct but overlapping hypothalamic sites may be sufficient to induce feeding behavior, indicating a broadened role for glutamate in hypothalamic feeding mechanisms.

The present experiments were conducted to investigate the role of the alpha(1A)-, alpha(1B)-, beta(1)-, beta(2)-adrenoceptors, and the effects of losartan and CGP42112A (selective ligands of the AT(1) and AT(2) angiotensin receptors, respectively) on the water and sodium intake elicited by paraventricular nucleus (PVN) injection of adrenaline. Male Holtzman rats with a stainless steel cannula implanted into the PVN were used. The ingestion of water and sodium was determined in separate groups submitted to water deprivation or sodium depletion with the diuretic furosemide (20 mg/rat). 5-Methylurapidil (an alpha(1A)-adrenergic antagonist) and ICI-118,551 (a beta(2)-adrenergic antagonist) injected into the PVN produced a dose-dependent increase, whereas cyclazosin (an alpha(1B)-adrenergic antagonist) and atenolol (a beta(1)-adrenergic antagonist) do not affect the inhibitory effect of water intake induced by adrenaline. On the other hand, the PVN administration of adrenaline increased the sodium intake in a dose-dependent manner. Previous injection of the alpha(1A) and beta(1) antagonists decreased, whereas injection of the alpha(1B) and beta(2) antagonists increased the salt intake induced by adrenaline. In rats with several doses of adrenaline into PVN, the previous administration of losartan increased in a dose-dependent manner the inhibitory effect of adrenaline and decreased the salt intake induced by adrenaline, while PVN CGP42112A was without effect. These results indicate that both appetites are mediated primarily by brain AT(1) receptors. However, the doses of losartan were more effective when combined with the doses of CGP42112A than given alone p0.05, suggesting that the water and salt intake effects of PVN adrenaline may involve activation of multiple angiotensin II (ANG II) receptors subtypes.

The present study was done to determine if estrogen interacts with excitatory and/or inhibitory amino acid neurotransmitters to alter neuronal excitability within the parabrachial nucleus (PBN) and modulate autonomic tone. First, the role of estrogen in modulating autonomic tone was investigated in male rats anesthetized with Inactin (100 mg/kg). Animals were instrumented to record blood pressure, heart rate, vagal parasympathetic and renal sympathetic nerve activities as well as baroreflex sensitivity. Direct, bilateral injection of 17beta-estradiol (0.5 microM; 200 nl/side) into the PBN resulted in a significant decrease in blood pressure (17+/-4 mmHg), sympathetic tone (20+/-5%) and heart rate (22+/-5 beats/min) while increasing parasympathetic tone (34+/-4%) 30 min post-injection. These estrogen-induced effects were completely blocked by the co-injection of estrogen with the estrogen receptor antagonist, ICI 182,780 (20 microM; 200 nl/side). Co-injection of the NMDA receptor antagonist, (+/-)-3-(2-carboxypiperazine-4-yl) propyl-1-phosphonic acid (CPP; 10 microM; 200 nl/side), with estradiol resulted in complete blockade of the estrogen-induced decrease in heart rate and increase in parasympathetic tone only. Co-injection of estradiol with the GABA(A) receptor antagonist, (+)-bicuculline (0.1 microM; 200 nl/side), resulted in complete blockade of the estrogen-induced decrease in blood pressure and sympathetic nerve activity only. These results suggest that estrogen acts on estrogen receptors on neurons in the PBN to modulate GABA(A)-receptor mediated inhibitory neurotransmission to alter sympathetic tone and blood pressure and on neurons in a separate, parallel pathway to modulate NMDA-receptor mediated neurotransmission to alter parasympathetic tone and heart rate.

Although stroke is a major cause of death and disability in the elderly, the inhibitory effects of neuroprotectants in acute stroke have been investigated using experimental cerebral ischemic models of young animals. Recent clinical trials have found that few neuroprotectants are effective. These observations indicate that effects in the clinical setting do not always reflect data from young animals. Thus, we compared the effects of the NMDA receptor antagonist MK-801 and of the AMPA receptor antagonist NBQX [2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinixaline] on ischemic cerebral damage in the photothrombosis model of aged and young rats. MK-801 administered immediately after MCA occlusion significantly (P

YM872, an AMPA receptor antagonist, was administered together with t-PA to investigate the effects of coadministration on neuroprotection in a rat embolic stroke model, when administered 2 h after embolism. T-PA or YM872 alone decreased infarct volume and improved the neurological deficit score. Coadministration of YM872 and t-PA resulted in a further decrease in infarct volume and improvement of the neurological score as compared with single administration of t-PA. These data demonstrate that coadministration of YM872 and t-PA produces more potent neuroprotective effects than when t-PA is administered alone.

We determined the effect of nilvadipine, a dihydropyridine-type calcium channel blocker, in preventing glutamate neurotoxicity in purified retinal ganglion cells (RGCs). RGCs were purified from dissociated rat retinal cells (postnatal days 6-8), using a modified two-step panning method, and cultured in serum-free medium containing neurotrophic factors and forskolin. RGC survival after exposure to glutamate (25 microM) with nilvadipine or other calcium channel blockers was measured by calcein-acetoxymethyl ester staining after 3 days in culture. Changes in the level of intracellular Ca(2+) ([Ca(2+)](i)) were measured with fura-2 fluorescence. Induction of apoptosis was evaluated using the TDT-dUTP terminal nick-end labeling technique. The neurotoxic effects of low doses of glutamate were blocked by a specific alpha-amino-3-dihydro-5-methylisoxazole-4-propionate-kainate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (20 microM). Simultaneous application of nilvadipine (1-100 nM) with glutamate protected against glutamate neurotoxicity in a dose-dependent manner. Calcium-imaging experiments showed that the glutamate-evoked [Ca(2+)](i) increase was significantly blocked by nilvadipine (P

Recent studies suggest that nuclear factor NF-kappaB may be involved in excitotoxin-induced cell apopotosis. To analyze the variation of NF-kappaB, levels of NF-kappaB were measured after the rats were subjected to 30 min of four-vessel occlusion and sacrificed in selected reperfusion time points. Induction of NF-kappaB consisting mainly of p65 and p50 subunits was detected by Western blot with anti p65, p50 antibodies, respectively. DNA binding activity of NF-kappaB was performed by electrophoretic mobility-shift analysis. Our studies indicate that ischemia-induced NF-kappaB nuclear translocation is time-dependent. Inductions or binding activity of NF-kappaB in nucleus increased about 10-fold from 6 to 12 h as compared with that of the control group, then gradually declined in the following 24, 72 h. To further analyze the regulation by ionotropic glutamate receptor and L-type voltage-gated Ca(2+) channel (L-VGCC) in vivo, N-methyl-D-aspartate (NMDA) receptor antagonist ketamine, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3 (1H,4H)-dione and L-VGCC antagonist nifedipine were given 20 min prior to 30 min of ischemia. The NF-kappaB nuclear translocation was completely blocked by these three antagonists in a dose-dependent manner after ischemia/reperfusion 6 h. Increased phosphorylation of the NF-kappaB regulatory unit IkappaB-alpha was detected by Western blot. Decrement of IkappaB-alpha was found after 3 h reperfusion in the cytoplasm following global ischemia, which was also blocked by such three antagonists. These results illustrate that glutamate-gated ionotropic NMDA or non-NMDA receptors and voltage-gated Ca(2+) channels are important routes to mediate NF-kappaB activation during brain ischemic injury. Active NF-kappaB may attend the excitotoxin-induced cell death in turn. Our studies also suggest that IkappaB-alpha is an important regulatory unit that controls the activation of NF-kappaB after its phosphorylation and degradation and resynthesis in rat hippocampus following global ischemia.

Responses of cultured rat cortical and spinal cord neurones to kainate (KA) have been related to the expression of KA receptor subunits revealed by single-cell reverse transcription polymerase chain reaction (RT-PCR). Semi-rapid application of KA evoked non-desensitizing responses with EC(50) values of 82 microM for cortical and 67 microM for spinal cord neurones. In the presence of concanavalin A, GYKI 53655 (100 microM) reduced responses of both types of neurone to KA by about 80% to leave a KA receptor-mediated response with an EC(50) of 4 microM on spinal cord neurones and 27 microM (P

This study characterizes the receptor subtypes and investigates some of the mechanisms by which glutamate, injected intraplantarly (i.pl.) into the mouse paw, produces nociception and paw oedema. I.pl. injection of glutamate induced a rapid-onset, dose-related pain response associated with oedema formation, with mean ED(50) values of 2.6 (1.6-4.3) and 0.5 (0.4-0.7) micromol/kg, respectively. Pretreatment with Chicago sky blue 6B (100 microg/kg), an inhibitor of glutamate uptake, caused a significant (about sixfold) reduction of the mean ED(50) value for glutamate-induced nociception, but not paw oedema. NMDA receptor antagonist MK 801, given by systemic (i.p.), intracerebroventricular (i.c.v.), i.pl. or intrathecal (i.t.) routes, produced graded inhibition of glutamate-induced nociception. Non-NMDA receptor antagonists NBQX or GAMS, metabotropic antagonist E4CPG, and also the antagonist that acts at the NMDA receptor-associated glycine binding site felbamate, significantly inhibited the nociception induced by glutamate. L(omega)-N-nitro-arginine (given i.p., i.t., i.pl. or i.c.v.) prevented the nociception and paw oedema caused by glutamate, an effect that was reversed by L-arginine but not by D-arginine. S-nitroso-N-acetyl-D,L-penicillamine (SNAP), given i.pl., greatly potentiated glutamate-induced nociception and oedema formation. Finally, the i.pl. injection of glutamate was accompanied by a graded increase in the nitrite levels of the hindpaw exudate. It is concluded that the nociception caused by i.pl. injection of glutamate probably involves the activation of NMDA and non-NMDA receptors by a mechanism which largely depends on the activation of L-arginine-nitric oxide pathway. Glutamate-induced paw oedema seems to be primarily mediated by non-NMDA ionotropic glutamate receptors and release of nitric oxide.

Glial cells limit local K(+)-accumulation by K(+)-uptake through different mechanisms, sensitive to Ba(2+), ouabaine, furosemide, or DIDS. Since the relative contribution of these mechanisms has not yet been determined, we studied the effects of bath-applied barium (2 mM), ouabaine (9 microM), furosemide (2 mM), and DIDS (1 mM) on ionophoretically-induced rises in [K(+)](o) in the pyramidal layer of area CA1 from normal rat slices, in the presence of glutamate receptor (Glu-R) antagonists. We also investigated the effect of barium on ionophoretically-induced tetrapropylammonium (TPA(+))-signals in order to test for barium-induced changes of the extracellular space. Finally, we repeated the barium experiment on slices from human non-sclerotic and sclerotic hippocampal specimens to assess a reduced glial capability for barium-sensitive K(+)-uptake in sclerotic tissue from epilepsy patients. In normal rat slices barium augmented ionophoretically-induced rises in [K(+)](o) by approximately 120%, also in the presence of tetrodotoxin (TTX) (by approximately 150%), but did not significantly affect the TPA(+)-signal. Ouabaine also augmented the K(+)-signal, but only by 27%. Furosemide and DIDS had negligible effects. In slices from sclerotic human hippocampus an augmentation of the K(+)-signal by barium was absent. Thus barium augments ionophoretically-induced K(+)-signals to a similar extent as previously shown for stimulus-induced signals. We suggest that glial barium-sensitive K(+)-buffer mechanisms reduce fast local rises of [K(+)](o) by at least 50%. This capability of glial cells is extremely reduced in area CA1 of slices from human sclerotic hippocampal specimens.

Brimonidine, a selective alpha(2)-adrenoceptor agonist, has recently been shown to be neuroprotective as it significantly improves survival of retinal ganglion cells (RGCs) after calibrated optic nerve injury in rats. In the present study, we examined the effect of brimonidine (alpha(2)-adrenoceptor agonist) on RGC survival after increased intraocular pressure (IOP) in adult rats. RGCs were prelabeled by bilateral tectal injection of 5% Fluoro-Gold (FG). Two days later, unilaterally IOP was increased 2.2-2.5 times (28-30.5 mmHg) that of the normal pressure (12.5-14.5 mmHg) by cauterization of three episcleral veins. The elevated IOP was maintained throughout the duration of the experiment. Rats were treated intraperitoneally with brimonidine (1 mg/kg) or phosphate-buffered saline (PBS) once per week beginning either before (group A) or after (group B) increasing the IOP. Another group of rats was left as the control with elevated IOP but without any brimonidine/PBS treatment. Rats were euthanized at 3, 4 and 5 weeks after IOP elevation. Identifiable RGCs were counted and compared between control and experimental groups. Brimonidine significantly protected RGCs from elevated IOP-induced cell death. In control rats with three-vein cauterization, there was 5-6% cell death per week. Almost all RGCs were protected following brimonidine treatment for 3 weeks both in groups A and B. At 4 weeks, there was 4.5% cell death in group A and 6.5% in group B. At 5 weeks, cell death was 5.9% in group A and 6.2% in group B. The difference in cell death in groups A and B was insignificant. No significant differences were observed between PBS-treated and control groups. No significant changes in elevated IOP was found after brimonidine or PBS treatment when compared with the nontreated control group. Although pressure remained elevated throughout the length of the experiment, 3 weeks later the amount of cell death gradually increased in brimonidine-treated animals.

Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded with the whole-cell patch-clamp technique from 41 pyramidal cells in the layers V-VI of the prelimbic (PL) cortex. The sEPSCs occurred randomly and the averaged frequency in 41 cells was 1.81+/-0.27 Hz. The amplitude distribution was skewed toward larger events and could be adequately fitted by a sum of two or three Gaussian distributions, but they could not be fitted by a sum of Gaussian distributions with equidistant separation in all cells studied (n=24). In eight of 24 cells, after the transformation of the amplitudes into logarithms, the skewed histogram became bell-shaped and could be adequately fitted by a single Gaussian distribution, whereas in the other 16 cells, after the transformation the histograms were still skewed. However, for those latter cells, when the logarithms were transformed into difference, the distribution of the differences in 15 of 16 cells became bell-shaped and could be adequately fitted by a single Gaussian distribution. The pie distribution of different rise times within one cell in 1 ms bin showed that there were four different patterns of the rise time distribution. The amplitude distribution of the sEPSCs was unchanged in 10 of 22 cells after TTX, but in the other 12 cells, it was changed significantly. However, for these cells although TTX had a marked effect, it could not change the skewed distribution into a single Gaussian distribution in case of both original and transformed data.

The developing cortical neurons have been well documented to be extremely vulnerable to the toxic effect of methylmercury (MeHg). In the present study, a possible involvement of N-methyl-D-aspartate (NMDA) receptors in MeHg neurotoxicity was examined because the sensitivity of cortical neurons to NMDA neurotoxicity has a similar developmental profile. Rats on postnatal day 2 (P2), P16, and P60 were orally administered MeHg (10 mg/kg) for 7 consecutive days. The most severe neuronal damage was observed in the occipital cortex of P16 rats. When MK-801 (0.1 mg/kg), a non-competitive antagonist of NMDA, was administered intraperitoneally with MeHg, MeHg-induced neurodegeneration was markedly ameliorated. Furthermore, there was a marked accumulation of nitrotyrosine, a reaction product of peroxynitrite and L-tyrosine, after chronic treatment of MeHg in the occipital cortex of P16 rats. The accumulation of nitrotyrosine was also significantly suppressed by MK-801. In the present electrophysiological study, the amplitude of synaptic responses mediated by NMDA receptors recorded in cortical neurons of P16 rats was significantly larger than those from P2 and P60 rats. These observations strongly suggest that a generation of peroxynitrite through activation of NMDA receptors is a major causal factor for MeHg neurotoxicity in the developing cortical neurons. Furthermore, enhanced sensitivity of NMDA receptors may make the cortical neurons of P16 rats most susceptible to MeHg neurotoxicity.

The potential toxic effects of the metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) and its interactions with the N-methyl-D-aspartate (NMDA) receptor were studied in hippocampal brain slice cultures, using densitometric measurements of the cellular uptake of propidium iodide (PI) to quantify neuronal degeneration. Cultures exposed to ACPD, showed a concentration (2-5 mM) and time (1-4 days) dependent increase in PI uptake in CA1, CA3 and dentate subfields after 24 h and 48 h of exposure, with CA1 pyramidal cells being most sensitive. The neurodegeneration induced by 2 mM ACPD was completely abolished by addition of 10 microM of the NMDA receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), while 20 microM of the 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainic acid receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) had no effect. Co-exposing cultures to a subtoxic dose of 300 microM ACPD together with 10 microM NMDA, which at this dose is known to induce a fairly selective degeneration of CA1 pyramidal cells, significantly increased the PI uptake in both CA1 and CA3, compared to cultures exposed to 10 microM NMDA only. Adding the 300 microM ACPD as pretreatment for 30 min followed by a 30 min wash in normal medium before the ACPD/NMDA co-exposure, eliminated the potentiation of NMDA toxicity. The potentiation was also blocked by addition of 10 or 100 microM 2-methyl-6-(phenylethynyl)pyridine (MPEP) (mGluR5 antagonist) during the co-exposure, while a corresponding addition of 10 or 100 microM 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) (mGluR1 antagonist) had no effect. We conclude that, stimulation of metabotropic glutamate receptors with ACPD at concentrations of 2 mM or higher induces a distinct subfield-related and time and concentration dependent pattern of hippocampal degeneration, and that ACPD at subtoxic concentrations modulates NMDA-induced excitotoxicity through the mGluR5 receptor in a time dependent way.

Extracellular signal-regulated kinases (ERK1/ERK2) have been shown transiently activated and involved in excitotoxicity. We searched for upstream molecules responsible for the regulation of glutamate-induced ERK1/ERK2 activation and ERK1/ERK2-mediated apototic-like death in cultured rat cortical neurons. ERK1/ERK2 activation (monitored by anti-active ERK1/ERK2 antibody) was almost completely prevented by blockage of NMDA receptor (NMDA-R) or elimination of extracellular Ca2+, but not any other glutamate receptor or L-type voltage-gated Ca2+ channel. It was prevented largely by inhibition of protein kinase C (PKC), protein-tyrosine kinases (PTK), respectively, but mildly by that of CaM kinase II. Combined inhibition of CaM kinase II (but not PTK) and PKC had an additive effect. Reversion of ERK1/ERK2 activation was largely prevented by inhibition of protein phosphatase (PP) 1 or protein tyrosine phosphatase (PTP). Combined inhibition of PP 1 and PTP had no additive effect. Glutamate-induced apoptotic-like death (determined by DAPI staining) was largely prevented by inhibition of NMDA-R, PKC, CaM kinase II, PTK and MEK1/MEK2 (ERK1/ERK2 kinase), respectively. Combined inhibition of CaM kinase II (but not PKC or PTK) and MEK1/MEK2 had an additive effect. Glutamate-induced apoptotic-like death was promoted by inhibition of PP1 and PTP, respectively. The above results suggested that in glutamate-induced cortical neurotoxicity ERK1/ERK2 activation be mainly mediated by NMDA-R. Subsequently, a pathway dependent on both PKC and PTK was mainly involved, which was also mainly responsible for ERK1/ERK2-mediated apoptotic-like death, and a CaM kinase II-dependent pathway was relatively mildly involved. Reversion of ERK1/ERK2 activation was mainly mediated by a pathway dependent on both PP1 and PTP, which might be involved in the restrain of glutamate-induced neurotoxicity.

Previous work showed that the dorsal periaqueductal gray is involved in the inhibition of fear-potentiated startle. The present study investigated the effects of blockade and stimulation of Kainate/AMPA and GABA(A) receptors within the dorsal periaqueductal gray on expression and conditioned inhibition of fear-potentiated startle. Blockade of the Kainate/AMPA receptors enhanced whereas stimulation of the Kainate/AMPA receptors decreased expression of fear-potentiated startle. These effects do not reflect conditioned inhibition since this modulation was not changed by injections of Kainate/AMPA receptor agonists or antagonists into the dorsal periaqueductal gray. Stimulation and blockade of GABA(A) receptors within the dorsal periaqueductal gray neither affected expression of fear-potentiated startle nor its conditioned inhibition. The present results together with findings from the literature indicate that glutamate in the dorsal periaqueductal gray is a critical substrate for the expression and modulation of fear-related behaviours.

The effects of exo- and endogenous cGMP on the resting activity (RA) of afferent crista fibers were studied in isolated preparations of the statocysts of the cuttlefish Sepia officinalis and the squid Sepioteuthis lessoniana. Bath application of the membrane-permeable cGMP analogs 8-bromo-cGMP (B-cGMP) and N2,2′-o-dibutyryl 3′,5′-cyclic guanosine monophosphate (dB-cGMP), and of the selective inhibitor of cGMP-phosphodiesterase zaprinast (ZAP), caused an inhibition of RA. The inhibitory effects of B-cGMP and dB-cGMP remained when the preparation was pre-treated with: (i) the guanylate cyclase inhibitors 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) or cystamine (CYS); (ii) the adenylate cyclase inhibitors nicotinic acid (NIC-A), 2′,3′dideoxyadenosine (DDA), or MDL-12330A (MDL); (iii) the guanylate cyclase inhibitor methylene blue (M-BLU) and the adenylate cyclase inhibitor MDL combined; or (iv) the nitric oxide (NO) synthase inhibitors NG-nitric- l -arginine methyl ester HCl ( l -NAME) or NG-nitro- l -arginine ( l -NOARG). These data indicate that cGMP, as an intracellular messenger, has a tonic inhibitory effect on the RA of afferent crista fibers in cephalopod statocysts.

Quantitative autoradiography was used to characterise the binding of selective radiolabelled antagonists for the N-methyl-D-aspartate (NMDA) receptor and the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor in the dorsal, intermediate and ventral subregions of the grey matter of the upper thoracic spinal cord in male and female lean and obese-diabetic (ob/ob) mice. The density of binding sites for both receptor subtypes was greater in diabetic mice, in all three subregions of the grey matter, than the corresponding subregions in the lean mice. The affinity of the binding site for the NMDA antagonist was significantly higher in obese mice than lean mice, consistent with the presence of two subpopulations of NMDA receptors with different ligand binding affinities in obese mice. The increase in expression of the glutamate receptor subtypes, and altered ligand affinity for the NMDA receptor subtype in the obese mice may be causally involved in the peripheral neuropathies which can accompany diabetes mellitus.

Striatal function is heavily influenced by glutamatergic and dopaminergic afferent input. To ultimately better understand how the N-methyl-D-aspartate (NMDA) antagonist, phencyclidine (PCP), alters striatal function, we sought to determine how NMDA receptor function is influenced by activation of other glutamatergic receptors and by dopaminergic receptors. To this end, we used NMDA-stimulated efflux of [14C]GABA and [3H]acetylcholine (ACh) from striatal slices to assess the influence of these receptors on NMDA function. NMDA-stimulated [14C]GABA release was more sensitive to NMDA and glycine antagonists than was [3H]ACh release, suggesting that different NMDA receptors regulate the release of these neurotransmitters. Furthermore, NMDA-stimulated [3H]ACh release was inhibited by a D2 receptor mechanism whereas NMDA-stimulated [14C]GABA release was enhanced by D1 receptor activation. NMDA and (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) interact additively to evoke [3H]ACh release, and synergistically to evoke [14C]GABA release. An additive effect of NMDA and kainate (KA) was found on [14C]GABA release, but NMDA and KA acted in a less than additive manner in evoking [3H]ACh release. KA-stimulated [3H]ACh release was largely blocked by NMDA antagonists, suggesting mediation through activation of NMDA receptors, probably secondary to KA-induced glutamate release. A selective group II metabotropic receptor agonist inhibited NMDA-stimulated [14C]GABA and [3H]ACh release. On the other hand, NMDA-stimulated [14C]GABA release was potentiated by activation of group I metabotropic receptors. Thus, in addition to the differential modulation by D1- and D2-like receptors, the release of striatal neurotransmitters by NMDA receptor activation depends on the extent to which the other glutamate receptors, both ionotropic and metabotropic, are activated.

The effects of bath application of the nitric oxide (NO) precursor l -arginine ( l -ARG) on the resting activity (RA) of afferent crista fibers were studied in isolated statocysts of the cuttlefish Sepia officinalis under various experimental conditions. l -ARG (threshold 10 −7 M) had three different effects: inhibition, excitation, and excitation followed by an inhibition; only the inhibitory effect of l -ARG was dose-dependent. d -Arginine ( d -ARG) had no effect. When the preparation was pre-treated with NO synthase inhibitors ( N G -Nitric- l -arginine methyl ester HCl ( l -NAME), N G -Nitro- l -arginine ( l -NOARG)), both the inhibitory and the excitatory effects of l -ARG significantly decreased at higher concentrations (10 −5 to −4 M), or were completely blocked at lower concentrations (10 −7 to −6 M), of l -ARG. When the preparation was pre-treated with guanylate cyclase inhibitors (1 H -[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), methylene blue (M-BLU), cystamine (CYS)), l -ARG had only excitatory effects, whereas its effects were only inhibitory when the preparation was pre-treated with adenylate cyclase inhibitors 2′,3′-dideoxyadenosine (DDA), MDL-12330A (MDL), nicotinic acid (NIC-A)). l -ARG had no effects when the pre-treatment was with a guanylate cyclase inhibitor and an adenylate cyclase inhibitor combined; in that situation, the RA of the afferent fibers remained. These data indicate that in cephalopod statocysts, a cGMP and a cAMP signal transduction pathway (presumably via the generation of NO) are responsible for the effects of l -ARG on the RA of crista afferent fibers. They also indicate that the l -ARG–cGMP pathway is the dominant pathway and is inhibitory, and that both pathways have only modulatory effects on, but are not essential for, the generation of the RA.

To study the roles of peripheral excitatory amino acids receptor subtypes N-methyl-D-aspartate (NMDA) and non-NMDA receptors in persistent nociception, extracellular single unit recording technique was used to assess the effects of a single dose NMDA and non-NMDA receptor antagonists, AP(5) (5-aminophosphonovaleric acid) and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) or DNQX (6,7-dinitroquinoxaline-2,3-dione), on s.c. bee venom-induced increase in firing of wide-dynamic-range (WDR) neurons in the spinal dorsal horn of the urethane-chloralose anesthetized cats. Subcutaneous bee venom injection into the cutaneous receptive field resulted in a single phase of increased firing of WDR neurons over the background activity for more than 1 h. Local pre-administration of AP(5) (200 microg/100 microl) or CNQX (8.3 microg/100 microl) into the bee venom injection site produced 94% (1.01+/-0.96 spikes/s, n=5) or 76% (2.97+/-0.58 spikes/s, n=4) suppression of the increased neuronal firing when compared with local saline (16.32+/-4.55 spikes/s, n=10) or dimethyl sulfoxide (DMSO) (12.37+/-6.36 spikes/s, n=4) pre-treated group, respectively. Local post-administration of the same dose of AP(5) produced a similar result to the pre-treatment group with a 67% inhibition of the mean firing rate, however, the same treatment with CNQX and even a higher dose of DNQX (100 microg/100 microl) did not produce any inhibition of the neuronal firing induced by s.c. bee venom injection (DNQX vs. DMSO: 23.91+/-0. 25 vs. 22.14+/-0.04 spikes/s, P=0.0298, n=5). In the control experiments, local pre-administration of the same dose of AP(5) or CNQX into a region on the contralateral hindpaw symmetrical to the bee venom injection site produced no significant influence on the increased firing of the WDR neurons [contralateral AP(5) vs. saline: 14.17+/-6.27 spikes/s (n=5) vs. 16.32+/-4.55 spikes/s (n=10), P0.05; contralateral CNQX vs. DMSO: 12.85+/-6.38 spikes/s (n=4) vs. 12. 37+/-6.36 spikes/s (n=4), P0.05], implicating that the suppressive action of local AP(5) or CNQX was not the result of systemic effects. The present results suggest that activation of the peripheral NMDA receptors is involved in both induction and maintenance, while activation of non-NMDA receptors is only involved in induction, but not in the maintenance of persistent firing of the dorsal horn WDR neurons induced by s.c. bee venom injection.

The effect of glutamate (GLUT) and its ionotropic receptor agonists on K+-evoked GABA release from the neurointermediate lobe (NIL) was investigated in diestrus, ovariectomized, ovariectomized–estrogenized female rats and intact male rats. GLUT and N-methyl- d -aspartate (NMDA) increased K+-evoked GABA release from the NIL in all the experimental groups. This stimulatory effect of NMDA was blocked by specific NMDA receptor antagonists but not by non-NMDA receptor antagonists. However, kainate did not modify evoked GABA release from the NIL in any of these groups. Neither GLUT nor NMDA modified nitric oxide synthase activity. These results indicate that GLUT, acting through NMDA receptors, stimulates evoked GABA release from the NIL of female and male rats. This effect is not influenced by gonadal status and does not appear to be mediated by nitric oxide production.

Two patterns of transforming growth factor-beta1 (TGF-beta1) expression were identified in brains of normotensive rats following permanent occlusion of the middle cerebral artery (MCAO). First, a relative increase of TGF-beta1 mRNA by 37% was found at 12 h after MCAO in the ipsilateral cingulate cortex as compared to the homotopic contralateral region. The cingulate cortex is located distant from the ischemic territory. Treatment with the glutamate receptor antagonists MK-801 and NBQX did not reduce this expression (34% and 26% increase, respectively). Therefore, peri-infarct depolarization waves were probably not responsible for induction. Secondly, an increase of TGF-beta1 mRNA by 116% was found at 7 days after MCAO within infarcted tissue. This expression was not reduced by the glutamate receptor antagonists MK-801 (increase 140%) and NBQX (increase 137%), either. TGF-beta1 mRNA expression in the cingulate cortex at 12 h after MCAO is possibly mediated by neurons and astroglia and may support cell survival. Expression in the infarcted tissue at 7 days after MCAO is most likely related to the invasion of monocytes and may be involved in the downregulation of inflammatory events, in neoangiogenesis, and in formation of a glial scar around the infarct.

Postmortem alterations in the neuronal cytoskeleton resemble some aspects of the cytoskeletal disruption associated with neurodegenerative disorders, and are also similar to those observed following ischemia and produced by excitotoxins in vivo and in vitro. This suggests the involvement of excitotoxic mechanisms during the postmortem interval. The purpose of this study was to determine if extracellular levels of glutamate are elevated postmortem. Extracellular levels of GABA and taurine were also monitored using in vivo microdialysis. These three amino acids were analyzed using high-performance liquid chromatography. When postmortem rat brain temperature cooled rapidly to near room temperature, dialysate concentrations of glutamate were not increased in the hippocampal CA1 region during a 2-h postmortem interval, although increased extracellular levels of GABA and taurine were observed. In contrast, maintenance of brain temperature at 37 degrees C resulted in a 12-to-40 fold elevation in extracellular glutamate levels 20-120 min postmortem. In addition, the elevation in dialysate taurine concentration was greater than that observed in rats in which postmortem brain temperature was not maintained. Excitatory amino acid antagonists, NBQX (2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline) and MK-801 (dizocilpine, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cylohepten-5, 10-imine hydrogen maleate blocked the additional elevation in taurine associated with maintaining brain at 37 degrees C, but had less robust effects against glutamate and GABA release. The results indicate that extracellular concentrations of glutamate, taurine and GABA increase in postmortem rat brain when physiologic temperatures are maintained, but that these increases are blunted when brain temperature decreases. After death, the human brain cools much more slowly than does the rat brain. Therefore, extracellular glutamate levels are likely to increase in the postmortem human brain and may contribute to excitotoxic neuronal damage occurring in the interval between death and autopsy.

This study tested the hypothesis that selective antagonism of excitatory amino acid (EAA) receptors within the ventral respiratory group (VRG) would induce changes in both respiratory rhythm and pattern. In the paralyzed, decerebrate, vagotomized and ventilated cat, baseline values for respiratory (Ttot), inspiratory (Ti), and expiratory (Te) durations and peak integrated phrenic nerve (integralPN) amplitude were established. Microinjection of the non-NMDA (N-methyl-d-aspartate) receptor antagonist NBQX (2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline) into rostral/inspiratory-modulated (iVRG) or caudal/expiratory-modulated VRG elicited an immediate apnea. When PN activity resumed, Ttot was significantly decreased, and integralPN amplitude was attenuated. NMDA receptor antagonism with microinjections of AP5 (2-amino-5-phosphonopentanoic acid) into iVRG decreased Te for more than 30 min. NMDA receptor antagonism in inspiratory/expiratory-modulated VRG (level of obex, transitional VRG) yielded either apnea or a significant reduction in Ttot, Ti and integralPN amplitude. Our data suggest that endogenous EAA receptor-mediated neurotransmission throughout the VRG is active in the determination of both respiratory timing and pattern. Our data further suggest that tVRG serves a unique function within the respiratory network.

Responses of cerebral blood vessels to nitric oxide (NO) are mediated by soluble guanylate cyclase (sGC)-dependent and potentially by sGC-independent mechanisms. One sGC-independent mechanism by which NO may produce vasodilatation is inhibition of formation of a vasoconstrictor metabolite produced through the cytochrome P450 pathway. In these experiments, we examined the hypothesis that dilatation of cerebral microvessels in response to NO is dependent on activation of sGC. Diameters of cerebral arterioles (baseline diameter=94+/-5 micrometers, mean+/-S.E.) were measured using a closed cranial window in anesthetized rabbits. Under control conditions, YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole], an NO-independent activator of sGC, produced vasodilation that was blocked by ODQ (1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one)(10 microM), an inhibitor of sGC. These findings indicate that sGC is functionally important in cerebral arterioles. In addition, acetylcholine (which stimulates endogenous production of NO by endothelium) produced dilatation of cerebral arterioles that was inhibited by ODQ. For example, 1 microM acetylcholine dilated cerebral arterioles by 34+/-7 and 5+/-1% in the absence and presence of ODQ (10 microM), respectively. Increases in arteriolar diameter in response to sodium nitroprusside (1 microM, an NO donor) were inhibited by approximately 80% by ODQ, but were not affected by 17-ODYA (10 microM) or clotrimazole (10 microM), inhibitors of the cytochrome P450 pathway. Thus, dilatation of the cerebral microcirculation in response to exogenously applied and endogenously produced NO is dependent, in large part, on activation of sGC.

An in vitro model of ischemia was developed and characterized using the acute rat hippocampal slice preparation. Neuroprotective concentrations of several competitive and noncompetitive glutamate subtype-selective antagonists (CGS-19755, MK-801, YM90K and GYKI-52466) were initially determined in anoxia-enhanced agonist-induced excitotoxicity experiments. Concentrations which proved to be effective in these studies were subsequently tested for their effectiveness against an ischemic episode. Ischemia was defined as a 30-min exposure to aglycemic media ending in 5 min of concurrent anoxia, a protocol which was arrived at by empirically determining the effect of various hypoglycemic and anoxic insults on the ability of hippocampal slices to retain their electrophysiological viability. Exposure to such an ischemic episode resulted in a loss of viability by most slices, an effect which was strongly dependent on extracellular calcium. AMPA antagonists applied alone produced no neuroprotective effect in the present model of in vitro ischemia, while NMDA antagonists applied alone had a modest neuroprotective effect. In contrast, the coapplication of 10 microM MK-801 and 300 microM GYKI-52466, noncompetitive NMDA and AMPA receptor antagonists, respectively, resulted in almost complete neuroprotection. This protection was comparable to that obtained by withholding extracellular calcium, indicating that the toxic effects of glutamate receptor overstimulation can be accounted for solely by calcium influx. The effect of this combination treatment on the survival rate of hippocampal slices was synergistic, that is greater than the sum of the effects of the individual compounds. The results indicate that neuroprotection against acute ischemic insults may require a combination therapy approach.

Due to loss of afferent innervation, synaptic reorganization occurs in organotypic hippocampal slice cultures. With extra- and intracellular recordings, we confirm that the excitatory loop from the dentate gyrus (DG) to CA3 and further to CA1 is preserved. However, hilar stimulation evoked antidromic population spikes in the DG which were followed by a population postsynaptic potential (PPSP); intracellularly, an antidromic spike with a broad shoulder or EPSP/IPSP sequences were induced. Synaptic responses were blocked by glutamate receptor antagonists. Stimulation of CA1 induced a PPSP in DG. Dextranamine stained pyramidal cells of CA1 were shown to project to DG. After removal of area CA3, DG's and mossy fibers' (MF) stimulation still elicited PPSPs and EPSP/IPSP sequences in area CA1 which disappeared when a cut was made through the hippocampal fissure. During bicuculline perfusion, hilar stimulation caused EPSPs in granule cells and spontaneous and evoked repetitive firing appeared even after its isolation from areas CA3 and CA1. Collateral excitatory synaptic coupling between granule cells was confirmed by paired recordings. Besides the preservation of the trisynaptic pathway in this preparation, new functional synaptic contacts appear, presumably due to MF collateral sprouting and formation of pathways between areas CA1 and DG.