Ionotropic Glutamate Receptor Antagonists Research Articles

A calcium-dependent glutamate release induced by metabotropic glutamate receptors I/II promotes GABA efflux from amacrine cells via a transporter-mediated process

Glutamate and GABA are, respectively, the major excitatory and inhibitory neurotransmitters in the retina, participating in the two pathways through which the retina processes light information. It has already been shown that glutamate induces GABA release from amacrine cells through a transporter-mediated mechanism, and that this process is mediated by ionotropic glutamate receptors. It is well established that glutamate can also activate metabotropic glutamate receptors, which are widely distributed in the retina, and can be detected in amacrine cell bodies and synaptic contacts. Thus, we decided to investigate the role of the activation of groups I and II metabotropic glutamate receptors in GABA release from amacrine cells in the chicken retina. Group I/II agonist trans-ACPD promoted a 40% decrease in the number of GABA-positive cells in relation to the control, effect that was prevented by antagonists of both groups. Also, the trans-ACPD effect was blocked by GAT-1 inhibitor or by antagonists of ionotropic glutamate receptors. Trans-ACPD induced release of GABA was abolished when the experiment was conducted in absence of calcium ions. Under the superfusing conditions used, trans-ACPD promoted an increase in endogenous glutamate release that was prevented when calcium was omitted from the bathing medium. The results suggest that mGluRI/II regulate the release of glutamate, likely from bipolar cells, that in turn activates GABA release from amacrine cells via a transporter mediated process.

Induction of theta-frequency oscillations in the rat medial septal diagonal band slice by metabotropic glutamate receptor agonists

The aim of this study was to examine the role of metabotropic glutamate receptors (mGluR) in the generation of oscillatory field activity at theta frequency (4–12 Hz) in the medial septal slice prepared from rat brain. Bath application of mGluR agonists and antagonists showed that activation of mGluR1-type receptors produces persistent theta frequency oscillations in a dose-responsive manner. This activity, induced by the group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG), was reduced by ionotropic glutamate receptor antagonists and abolished by further addition of a GABAA receptor antagonist. However, addition of a GABAA receptor antagonist on its own converted the DHPG-induced oscillations to intermittent episodes of accentuated theta frequency activity following a burst. In a proportion of slices, DHPG induced large amplitude field population spiking activity (100–300 μV) which is correlated linearly with the field theta oscillations and is sensitive to glutamate receptor antagonists, suggesting a role of this type of spikes in theta generation induced by DHPG. These data demonstrate that DHPG-sensitive neuronal networks within medial septum generate theta rhythmic activity and are differentially modulated by excitatory and inhibitory ionotropic neurotransmissions.

Modulatory effect of a new KYNA derivative on the trigeminal activation

Event Abstract Back to Event Modulatory effect of a new KYNA derivative on the trigeminal activation A. Fejes1*, E. Vámos2, Z. Bohár1, J. Tajti1, F. Fülöp3, L. Vécsei1 and Á. Párdutz1 1 University of Szeged, Department of Neurology, Hungary 2 University of Szeged, Department of Physiology, Anatomy and Neuroscience, Hungary 3 University of Szeged, Department of Pharmaceutical Chemistry, Hungary The exact pathomechanism of trigeminal nociception is not fully understood, but the role of glutamate seems crucial. One of the endogenous ionotropic glutamate receptor antagonists is the kynurenic acid (KYNA) formed from L-kynurenine during the tryptophan metabolism. KYNA may modulate nociception, but its therapeutic use is difficult because of its poor penetration properties across the blood brain barrier (BBB). One of the new KYNA derivatives (KYNAd) having better penetrance properties across the BBB could well use in treatment of trigeminal nociception. One of the animal models of somatic trigeminal nociception is the orofacial formalin test, which leads to activation of the trigeminal system. We examined the effect of pretreatment with the new KYNAd on the formalin-induced changes in the CGRP and TRPV1 immunoreactivity in the trigeminal ganglion (TG) and in the c-Fos immunoreactivity in the caudal trigeminal nucleus (TNC). Four hours after the injection of formalin in the right upper lip of rats, the percentage of CGRP-immunoreactive (IR) neurones in the TG was decreased, while that of TRPV1-IR neurones was increased, as was the number of c-Fos-IR neurones in the TNC. The pretreatment with the new KYNAd significantly modulated the formalin-induced changes in the TG and in the TNC. The new KYNAd can modulate the activation of neurones in the trigeminal system acting probably on the glutamate receptors, thus it can be new candidate in the treatment of trigeminal nociception. Keywords: Sensory and motor activity, Neuroscience Conference: 13th Conference of the Hungarian Neuroscience Society (MITT), Budapest, Hungary, 20 Jan - 22 Jan, 2011. Presentation Type: Abstract Topic: Sensory and motor activity Citation: Fejes A, Vámos E, Bohár Z, Tajti J, Fülöp F, Vécsei L and Párdutz Á (2011). Modulatory effect of a new KYNA derivative on the trigeminal activation. Front. Neurosci. Conference Abstract: 13th Conference of the Hungarian Neuroscience Society (MITT). doi: 10.3389/conf.fnins.2011.84.00118 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 03 Mar 2011; Published Online: 23 Mar 2011. * Correspondence: Dr. A. Fejes, University of Szeged, Department of Neurology, Szeged, Hungary, fejesannamaria@yahoo.co.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers A. Fejes E. Vámos Z. Bohár J. Tajti F. Fülöp L. Vécsei Á. Párdutz Google A. Fejes E. Vámos Z. Bohár J. Tajti F. Fülöp L. Vécsei Á. Párdutz Google Scholar A. Fejes E. Vámos Z. Bohár J. Tajti F. Fülöp L. Vécsei Á. Párdutz PubMed A. Fejes E. Vámos Z. Bohár J. Tajti F. Fülöp L. Vécsei Á. Párdutz Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Involvement of Peripheral Ionotropic Glutamate Receptors in Orofacial Thermal Hyperalgesia in Rats

BackgroundThe purpose of the present study was to elucidate the mechanisms that may underlie the sensitization of trigeminal spinal subnucleus caudalis (Vc) and upper cervical spinal cord (C1-C2) neurons to heat or cold stimulation of the orofacial region following glutamate (Glu) injection.ResultsGlu application to the tongue or whisker pad skin caused an enhancement of head-withdrawal reflex and extracellular signal-regulated kinase (ERK) phosphorylation in Vc-C2 neurons. Head-withdrawal reflex and ERK phosphorylation were also enhanced following cold stimulation of the tongue but not whisker pad skin in Glu-injected rats, and the head-withdrawal reflex and ERK phosphorylation were enhanced following heat stimulation of the tongue or whisker pad skin. The enhanced head-withdrawal reflex and ERK phosphorylation after heat stimulation of the tongue or whisker pad skin, and those following cold stimulation of the tongue but not whisker pad skin were suppressed following ionotropic glutamate receptor antagonists administration into the tongue or whisker pad skin. Furthermore, intrathecal administration of MEK1/2 inhibitor PD98059 caused significant suppression of enhanced head-withdrawal reflex in Glu-injected rats, heat head-withdrawal reflex in the rats with Glu injection into the tongue or whisker pad skin and cold head-withdrawal reflex in the rats with Glu injection into the tongue.ConclusionsThe present findings suggest that peripheral Glu receptor mechanisms may contribute to cold hyperalgesia in the tongue but not in the facial skin, and also contribute to heat hyperalgesia in the tongue and facial skin, and that the mitogen-activated protein kinase cascade in Vc-C2 neurons may be involved in these Glu-evoked hyperalgesic effects.

Microinjections of urocortin1 into the nucleus ambiguus of the rat elicit bradycardia.

Urocortins are members of the hypothalamic corticotropin-releasing factor (CRF) peptide family. Urocortin1 (UCN1) mRNA has been reported to be expressed in the brainstem neurons. The present investigation was carried out to test the hypothesis that microinjections of UCN1 into the nucleus ambiguus (nAmb) may elicit cardiac effects. Urethane-anesthetized, artificially ventilated, adult male Wistar rats, weighing between 300-350 g, were used. nAmb was identified by microinjections of l-glutamate (5 mM, 30 nl). Microinjections (30 nl) of different concentrations (0.062, 0.125, 0.25, and 0.5 mM) of UCN1 into the nAmb elicited bradycardic responses (26.5 ± 1, 30.1 ± 1.7, 46.9 ± 1.7, and 40.3 ± 2.6 beats/min, respectively). These heart rate responses were not accompanied by significant changes in mean arterial pressure. The bradycardic responses to maximally effective concentration of UCN1 (0.25 mM) were significantly (P < 0.05) attenuated by prior microinjections of a selective antagonist (NBI 27914, 1.5 mM) for CRF type 1 receptor (CRF1R). Prior microinjections of ionotropic glutamate receptor (iGLUR) antagonists [d-(-)-2-amino-7-phosphono-heptanoic acid and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo-(f)quinoxaline-7-sulfonamide disodium] also attenuated the bradycardia elicited by UCN1 microinjections into the nAmb. Microinjections of NBI 27914 (1.5 mM) into the nAmb did not alter baroreflex responses. Bilateral vagotomy abolished the bradycardic responses to microinjections of UCN1 into the nAmb. These results indicated that 1) microinjections of UCN1 into the nAmb elicited bradycardia, 2) the bradycardia was vagally mediated, 3) activation of CRF1Rs in the nAmb was responsible for the actions of UCN1, and 4) activation of iGLURs in the nAmb also participated in the bradycardia elicited by UCN1.

Prototypic Seizure Activity Driven by Mature Hippocampal Fast-Spiking Interneurons

A variety of epileptic seizure models have shown that activation of glutamatergic pyramidal cells is usually required for rhythm generation and/or synchronization in hippocampal seizure-like oscillations in vitro. However, it still remains unclear whether GABAergic interneurons may be able to drive the seizure-like oscillations without glutamatergic transmission. Here, we found that electrical stimulation in rat hippocampal CA1 slices induced a putative prototype of seizure-like oscillations ("prototypic afterdischarge," 1.8-3.8 Hz) in mature pyramidal cells and interneurons in the presence of ionotropic glutamate receptor antagonists. The prototypic afterdischarge was abolished by GABA(A) receptor antagonists or gap junction blockers, but not by a metabotropic glutamate receptor antagonist or a GABA(B) receptor antagonist. Gramicidin-perforated patch-clamp and voltage-clamp recordings revealed that pyramidal cells were depolarized and frequently excited directly through excitatory GABAergic transmissions in each cycle of the prototypic afterdischarge. Interneurons that were actively spiking during the prototypic afterdischarge were mostly fast-spiking (FS) interneurons located in the strata oriens and pyramidale. Morphologically, these interneurons that might be "potential seizure drivers" included basket, chandelier, and bistratified cells. Furthermore, they received direct excitatory GABAergic input during the prototypic afterdischarge. The O-LM cells and most of the interneurons in the strata radiatum and lacunosum moleculare were not essential for the generation of prototypic afterdischarge. The GABA-mediated prototypic afterdischarge was observed later than the third postnatal week in the rat hippocampus. Our results suggest that an FS interneuron network alone can drive the prototypic form of electrically induced seizure-like oscillations through their excitatory GABAergic transmissions and presumably through gap junction-mediated communications.

Tuberoinfundibular peptide of 39 residues modulates the mouse hypothalamic-pituitary-adrenal axis via paraventricular glutamatergic neurons.

Neurons in the subparafascicular area at the caudal border of the thalamus that contain the neuropeptide tuberoinfundibular peptide of 39 residues (TIP39) densely innervate several hypothalamic areas, including the paraventricular nucleus (PVN). These areas contain a matching distribution of TIP39's receptor, the parathyroid hormone receptor 2 (PTH2R). Frequent PTH2R coexpression with a vesicular glutamate transporter (VGlut2) suggests that TIP39 could presynaptically regulate glutamate release. By using immunohistochemistry we found CRH-ir neurons surrounded by PTH2R-ir fibers and TIP39-ir axonal projections in the PVN area of the mouse brain. Labeling hypothalamic neuroendocrine neurons by peripheral injection of fluorogold in PTH2R-lacZ knock-in mice showed that most PTH2Rs are on PVN and peri-PVN interneurons and not on neuroendocrine cells. Double fluorescent in situ hybridization revealed a high level of coexpression between PTH2R and VGlut2 mRNA by cells located in the PVN and nearby brain areas. Local TIP39 infusion (100 pmol) robustly increased pCREB-ir in the PVN and adjacent perinuclear zone. It also increased plasma corticosterone and decreased plasma prolactin. These effects of TIP39 on pCREB-ir, corticosterone, and prolactin were abolished by coinfusion of the ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and DL-2-amino-5-phosphonopentanoic acid (AP-5; 30 pmol each) and were absent in PTH2R knockout mice. Basal plasma corticosterone was slightly decreased in TIP39 knockout mice just before onset of their active phase. The present data indicate that the TIP39 ligand/PTH2 receptor system provides facilitatory regulation of the hypothalamic-pituitary-adrenal axis via hypothalamic glutamatergic neurons and that it may regulate other neuroendocrine systems by a similar mechanism.

The Protective Signaling of Metabotropic Glutamate Receptor 1 Is Mediated by Sustained, β-Arrestin-1-dependent ERK Phosphorylation

Metabotropic glutamate receptor 1 (mGlu1) is a G protein-coupled receptor that enhances the hydrolysis of membrane phosphoinositides. In addition to its role in synaptic transmission and plasticity, mGlu1 has been shown to be involved in neuroprotection and neurodegeneration. In this capacity, we have reported previously that in neuronal cells, mGlu1a exhibits the properties of a dependence receptor, inducing apoptosis in the absence of glutamate, while promoting neuronal survival in its presence (Pshenichkin, S., Dolińska, M., Klauzińska, M., Luchenko, V., Grajkowska, E., and Wroblewski, J. T. (2008) Neuropharmacology 55, 500-508). Here, using CHO cells expressing mGlu1a receptors, we show that the protective effect of glutamate does not rely on the classical mGlu1 signal transduction. Instead, mGlu1a protective signaling is mediated by a novel, G protein-independent, pathway which involves the activation of the MAPK pathway and a sustained phosphorylation of ERK, which is distinct from the G protein-mediated transient ERK phosphorylation. Moreover, the sustained phosphorylation of ERK and protective signaling through mGlu1a receptors require expression of beta-arrestin-1, suggesting a possible role for receptor internalization in this process. Our data reveal the existence of a novel, noncanonical signaling pathway associated with mGlu1a receptors, which mediates glutamate-induced protective signaling.

Retrograde release of endocannabinoids inhibits presynaptic GABA release to second-order baroreceptive neurons in NTS

In prior studies, we found that activation of cannabinoid-1 receptors in the nucleus tractus solitarii (NTS) prolonged baroreflex-induced sympathoinhibition in rats. In many regions of the central nervous system, activation of cannabinoid-1 receptors presynaptically inhibits γ-aminobutyric acid (GABA) release, disinhibiting postsynaptic neurons. To determine if cannabinoid-1 receptor-mediated presynaptic inhibition of GABA release occurs in the NTS, we recorded miniature inhibitory postsynaptic currents in anatomically identified second-order baroreceptive NTS neurons in the presence of ionotropic glutamate receptor antagonists and tetrodotoxin. The cannabinoid-1 receptor agonists, WIN 55212-2 (0.3–30 μM) and methanandamide (3 μM) decreased the frequency of miniature inhibitory postsynaptic currents in a concentration-dependent manner, an effect that was blocked by the cannabinoid-1 receptor antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM 251, 5 μM). Importantly, depolarization of second-order baroreceptive neurons decreased the frequency of miniature inhibitory postsynaptic currents; an effect which was blocked by the cannabinoid-1 receptor antagonist. The data indicate that depolarization of second-order baroreceptive NTS neurons induces endocannabinoid release from the neurons, leading to activation of presynaptic cannabinoid-1 receptors, inhibition of GABA release and subsequent enhanced baroreflex signaling in the NTS. The data suggest that endocannabinoid signaling in the NTS regulates short-term synaptic plasticity and provide a mechanism for endocannabinoid modulation of central baroreflex control.

Effects of ionotropic glutamate receptor antagonists on rat dural artery diameter in an intravital microscopy model

During migraine, trigeminal nerves may release calcitonin gene-related peptide (CGRP), inducing cranial vasodilatation and central nociception; hence, trigeminal inhibition or blockade of craniovascular CGRP receptors may prevent this vasodilatation and abort migraine headache. Several preclinical studies have shown that glutamate receptor antagonists affect the pathophysiology of migraine. This study investigated whether antagonists of NMDA (ketamine and MK801), AMPA (GYKI52466) and kainate (LY466195) glutamate receptors affected dural vasodilatation induced by alpha-CGRP, capsaicin and periarterial electrical stimulation in rats, using intravital microscopy. Male Sprague-Dawley rats were anaesthetized and the overlying bone was thinned to visualize the dural artery. Then, vasodilator responses to exogenous (i.v. alpha-CGRP) and endogenous (released by i.v. capsaicin and periarterial electrical stimulation) CGRP were elicited in the absence or presence of the above antagonists. alpha-CGRP, capsaicin and periarterial electrical stimulation increased dural artery diameter. Ketamine and MK801 inhibited the vasodilator responses to capsaicin and electrical stimulation, while only ketamine attenuated those to alpha-CGRP. In contrast, GYKI52466 only attenuated the vasodilatation to exogenous alpha-CGRP, while LY466195 did not affect the vasodilator responses to endogenous or exogenous CGRP. Although GYKI52466 has not been tested clinically, our data suggest that it would not inhibit migraine via vascular mechanisms. Similarly, the antimigraine efficacy of LY466195 seems unrelated to vascular CGRP-mediated pathways and/or receptors. In contrast, the cranial vascular effects of ketamine and MK801 may represent a therapeutic mechanism, although the same mechanism might contribute, peripherally, to cardiovascular side effects.

Insulin Activates a NMDA Pathway to the RVLM to Elevate Sympathetic Nerve Activity

Hyperinsulinemia elevates lumbar sympathetic nerve activity (SNA) via activation of ionotropic glutamatergic receptors in the rostral ventrolateral medulla (RVLM). Therefore, the present study determined the contribution of NMDA vs non‐NMDA receptors. Male Sprague‐Dawley rats (250–300 g) were anesthetized with α‐chloralose and infused with insulin (3.75 mU/kg/min) and 50% dextrose (.25–.75 ml/hr) for 120 min. At 90 min, hyperinsulinemia significantly increased lumbar SNA (40±6%) with no change in arterial blood pressure (ABP, 0 min: 117±5 vs 90 min: 119±6 mmHg) or plasma glucose levels (0 min: 72±5 vs 90 min: 68±5 mg/dl). RVLM microinjection of the ionotropic glutamate receptor antagonist KYN significantly reduced lumbar SNA (−28±10%) and ABP (−8±2 mmHg, p&lt;0.05). Similarly, RVLM microinjection of the NMDA receptor antagonist AP5 significantly attenuated lumbar SNA (−30±8%) and ABP (−5±3 mmHg, P&lt;0.05). The decrease in lumbar SNA and ABP after injection of KYN vs AP5 was not statistically different. In contrast, microinjection of the non‐NMDA receptor antagonist NBQX into the RVLM had no significant effect on lumbar SNA (6±3%) or ABP (5±4 mmHg). In addition, direct microinjection of insulin into the RVLM did not significantly increase lumbar SNA or ABP. These findings indicate that hyperinsulinemia increases lumbar SNA through a glutamatergic NMDA projection to the RVLM.Supported by NIH HL090826, AHA 0630202N &amp; 0815372D

Electrophysiological characteristics of identified kidney-related neurons in adult rat spinal cord slices

Whole-cell patch-clamp recordings were made from kidney-related neurons in the intermediolateral cell column (IML) in horizontal slices of thoracolumbar spinal cord from adult rats. Kidney-related neurons were identified in vitro subsequent to inoculation of the kidney with a fluorescent, retrograde, transynaptic pseudorabies viral label (i.e., PRV-152). Kidney-related neurons detected in the IML expressed choline acetyltransferase, characteristic of spinal preganglionic motor neurons. Their mean resting potential was −51±4mV and input resistance was 448±39MΩ. Both spontaneous inhibitory and excitatory post-synaptic currents (i.e., sIPSCs and sEPSCs) were observed in all neurons. The mean frequency for sEPSCs (3.1±1Hz) was approximately 2.5 times that for sIPSCs (1.4±0.3Hz). Application of the glycine and GABAA receptor-linked Cl− channel blocker, picrotoxin (100μM) blocked sIPSCs, while the ionotropic glutamate receptor antagonist, kynurenic acid (1mM) blocked all sEPSCs, indicating they were mediated by GABA/glycine and glutamate receptors, respectively. Thus, using PRV-152 labeling allowed whole-cell patch-clamp recording of neurons in the adult spinal cord, which were kidney-related. Excitatory glutamatergic input dominated synaptic responses in these cells, the membrane characteristics of which resembled those of immature IML neurons. Combined PRV-152 pre-labeling and whole-cell patch-clamp recordings may allow more effective analysis of synaptic plasticity seen in adult models of injury or chronic disease.

Selective Excitatory Actions of DNQX and CNQX in Rat Thalamic Neurons

The thalamic reticular nucleus (TRN) consists of GABA-containing neurons that form reciprocal synaptic connections with thalamic relay nuclei. Excitatory synaptic innervation of TRN neurons arises from glutamatergic afferents from thalamocortical relay neurons and deep layer corticothalamic neurons, and they produce excitation via both N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Quinoxaline derivatives [e.g., 6,7-dinitroquinoxaline-2,3-dione (DNQX), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)] have routinely been used as non-NMDA receptor antagonists over the last two decades. In this study, we examined whether quinoxaline derivatives alter the intrinsic properties of thalamic neurons in light of recent findings indicating that these compounds can alter neuronal excitability in hippocampal and cerebellar neurons via transmembrane AMPA receptor (AMPAR) regulatory proteins (TARPs). Whole cell recordings were obtained from TRN and ventrobasal (VB) thalamic relay neurons in vitro. DNQX and CNQX produced a consistent depolarization in all TRN neurons tested. The depolarization persisted in tetrodotoxin and low Ca²+/high Mg²+ conditions, suggesting a postsynaptic site of action. In contrast, DNQX and CNQX produced little or no change in VB thalamocortical relay neurons. The nonspecific ionotropic glutamate receptor antagonist, kynurenic acid, and the selective AMPAR antagonist, 4-(8-methyl-9H-1,3-dioxolo[4,5-h][2,3]benzodiazepin-5-yl)-benzenamine hydrochloride, blocked the DNQX-mediated depolarizations. Our results indicate that the DNQX- and CNQX-mediated depolarizations are mediated by AMPAR but not kainate receptors in TRN neurons. The AMPAR-positive allosteric modulator, trichloromethiazide, potentiated the DNQX-mediated depolarization in TRN neurons but did not unmask any excitatory actions of DNQX/CNQX in relay neurons. This selective action may not only reveal a differential TARP distribution among thalamic neurons but also may provide insight into distinct characteristics of AMPA receptors of thalamic neurons that could be exploited by future pharmacological development. Furthermore, these data suggest that quinoxaline derivatives could modulate synaptic transmission and alter neuronal excitability.

Distribution and role of Kv3.1b in neurons in the medial septum diagonal band complex

The medial septum diagonal band complex (MS/DB) projects via cholinergic and GABAergic pathways to the hippocampus and plays a key role in the hippocampal theta rhythm. In the MS/DB we have previously described a population of fast spiking GABAergic neurons that contain parvalbumin and mediate theta frequency activity in vitro. The Kv3.1 potassium channel is a delayed rectifier channel that plays a major role in fast spiking neurons in the CNS, and has previously been localized in the MS/DB. To determine which cell types in the MS/DB express the Kv3.1b ion channel subunit, transgenic mice in which the expression of GABAergic and glutamate markers are associated with the expression of green fluorescent protein (GFP; GAD67-GFP and VGluT2-GFP mice, respectively) were used for immunofluorescence and axonal tract tracing. Electrophysiological studies were also carried out on rat MS/DB slices to examine the role of the Kv3.1 channel in theta frequency oscillations. The results for the MS/DB were as follows: (1) cholinergic cells did not express GFP in either GAD67-GFP or VGluT2-GFP mice, and there was GAD67 immunoreactivity in GFP-positive neurons in GAD67-GFP mice and in a small proportion (6%) of GFP-positive neurons in VGluT2-GFP mice. (2) Kv3.1b immunofluorescence was associated with the somata of GABAergic neurons, especially those that contained parvalbumin, and with a minority of glutamatergic neurons, but not with cholinergic neurons, and with GABAergic axonal terminal-like processes around certain GABAergic neurons. (3) Both Kv3.1b-positive and -negative GABAergic neurons were septo-hippocampal, and there was a minor projection to hippocampus from VGluT2-GFP neurons. (4) Kainate-induced theta oscillations in the MS/DB slice were potentiated rather than inhibited by the Kv3.1 blocker 4-aminopyridine, and this agent on its own produced theta frequency oscillations in MS/DB slices that were reduced by ionotropic glutamate and GABA receptor antagonists and abolished by low extracellular calcium. These studies confirm the presence of heterogeneous populations of septo-hippocampal neurons in the MS/DB, and suggest that presence of Kv3.1 in the GABAergic neurons does not contribute to theta activity through fast spiking properties, but possibly by the regulation of transmitter release from axonal terminals.

Glutamatergic Receptor Activation in the Rostral Ventrolateral Medulla Mediates the Sympathoexcitatory Response to Hyperinsulinemia

Hyperinsulinemia increases sympathetic nerve activity (SNA) and has been linked to cardiovascular morbidity in obesity. The rostral ventrolateral medulla (RVLM) plays a key role in the regulation of SNA and arterial blood pressure (ABP). Many sympathoexcitatory responses are mediated by glutamatergic receptor activation within the RVLM, and both the central renin-angiotensin and melanocortin systems are implicated in the sympathoexcitatory response to hyperinsulinemia. Therefore, we hypothesized that one or more of these neurotransmitters in the RVLM mediate the sympathoexcitatory response to insulin. Hyperinsulinemic-euglycemic clamps were performed in alpha-chloralose anesthetized, male Sprague-Dawley rats by infusion of insulin (3.75 mU/kg per minute, IV) and 50% dextrose solution for 120 minutes. Physiological increases in plasma insulin elevated lumbar SNA, with no change in renal SNA, ABP, or blood glucose. Microinjection of the ionotropic glutamate receptor antagonist kynurenic acid into the RVLM significantly reduced lumbar SNA and ABP. Selective blockade of NMDA but not non-NMDA glutamate receptors resulted in similar reductions of lumbar SNA. In marked contrast, microinjection of the angiotensin II type 1 receptor antagonist losartan or the melanocortin 3/4 antagonist SHU9119 had no effect on lumbar SNA or ABP. Western blot analysis showed that insulin receptor expression is significantly lower in the RVLM than the hypothalamus, and direct microinjection of insulin into the RVLM did not significantly increase lumbar SNA. These findings suggest that hyperinsulinemia increases lumbar SNA by activation of a glutamatergic NMDA-dependent projection to the RVLM.

Nicotine induction of theta frequency oscillations in rodent hippocampus in vitro

The hippocampus is an area important for learning and memory and exhibits prominent and behaviourally relevant theta (4–12 Hz) and gamma (30–100 Hz) frequency oscillations in vivo. Hippocampal slices produce similar types of oscillatory activity in response to bath-application of neurotransmitter receptor agonists. The medial septum diagonal band area (MS/DB) provides both a cholinergic and GABAergic projection to the hippocampus, and although it plays a major role in the generation and maintenance of the hippocampal theta rhythm in vivo, there is evidence for intrinsic theta generation mechanisms in the hippocampus, especially in area CA3. The aim of this study was to examine the role of the nicotinic receptor (nAChR) in the induction of oscillatory field activity in the in vitro preparation of the rat hippocampus. Bath-application of a low concentration of nicotine (1 μM) to transversely-cut hippocampal slices produced persistent theta-frequency oscillations in area CA3 of the hippocampus. These oscillations were reduced by both GABA A receptor antagonists and ionotropic glutamate receptor antagonists, indicating the involvement of local GABAergic and glutamatergic neurons in the production of the rhythmic theta activity. The nicotine-induced theta activity was inhibited by non-selective nAChR antagonists and partially by an α7* nAChR antagonist. The induction of theta frequency oscillations in CA3 by nicotine was mimicked α7* nAChR agonists but not by non-α7* nAChR agonists. In conclusion, theta activity in the hippocampus may be promoted by tonic stimulation of α7* nAChRs, possibly via selective stimulation of theta-preferring interneurons in the hippocampus that express post-synaptic α7* nAChRs.

Regulation of striatal nitric oxide synthesis by local dopamine and glutamate interactions

Nitric oxide (NO) is a key neuromodulator of corticostriatal synaptic transmission. We have shown previously that dopamine (DA) D1/5 receptor stimulation facilitates neuronal NO synthase (nNOS) activity in the intact striatum. To study the impact of local manipulations of D1/5 and glutamatergic NMDA receptors on striatal nNOS activity, we combined the techniques of in vivo amperometry and reverse microdialysis. Striatal NO efflux was monitored proximal to the microdialysis probe in urethane-anesthetized rats during local infusion of vehicle or drug. NO efflux elicited by systemic administration of SKF-81297 was blocked following intrastriatal infusion of: (i) the D1/5 receptor antagonist SCH-23390, (ii) the nNOS inhibitor 7-nitroindazole, (iii) the non-specific ionotropic glutamate receptor antagonist kynurenic acid, and (iv) the selective NMDA receptor antagonist 3-phosphonopropyl-piperazine-2-carboxylic acid. Glycine co-perfusion did not affect SKF-81297-induced NO efflux. Furthermore, intrastriatal infusion of SKF-81297 potentiated NO efflux evoked during electrical stimulation of the motor cortex. The facilitatory effects of cortical stimulation and SKF-81297 were both blocked by intrastriatal infusion of SCH-23390, indicating that striatal D1/5 receptor activation is necessary for the activation of nNOS by corticostriatal afferents. These studies demonstrate for the first time that reciprocal DA-glutamate interactions play a critical role in stimulating striatal nNOS activity.

Cardiovascular Responses to Hypothalamic Arcuate Nucleus Stimulation in the Rat

Experiments were carried out in urethane-anesthetized, artificially ventilated, adult male Wistar rats. Microinjections (50 nL) of N-methyl-d-aspartic acid (1, 5, and 10 mmol/L), but not artificial cerebrospinal fluid, into the hypothalamic arcuate nucleus (ARCN) elicited increases in mean arterial pressure (5.7+/-0.5, 13.2+/-1.4, and 17.3+/-1.1 mm Hg, respectively) and heart rate (24.3+/-4.3, 49.3+/-5.2, and 75.2+/-8.0 bpm, respectively). ARCN stimulation was accomplished by microinjections of a maximally effective concentration of N-methyl-d-aspartic acid (10 mmol/L). The tachycardic responses to the ARCN stimulation were significantly attenuated after bilateral vagotomy. Intrathecal injections of ionotropic glutamate receptor (iGLUR) antagonists completely blocked pressor responses to the ARCN stimulation, whereas the tachycardic responses were significantly attenuated but not abolished. Intrathecal injections of iGLUR antagonists at T9 to T10, combined with bilateral vagotomy, completely blocked the tachycardic responses to ARCN stimulation. ARCN stimulation with N-methyl-d-aspartic acid elicited increased activities of the greater splanchnic nerve (91.7+/-14.8%) and the renal nerve (109.3+/-13%). Intrathecal injections of iGLURs at T9 to T10 blocked the increase in the greater splanchnic nerve activity in response to ARCN stimulation. These results indicate the following: (1) the chemical stimulation of the ARCN elicits increases in mean arterial pressure, greater splanchnic nerve and renal nerve activity, and heart rate; (2) the increases in mean arterial pressure and sympathetic nerve activity are mediated via the activation of spinal cord iGLURs; and (3) the increases in heart rate are mediated via the activation of spinal cord iGLURs and decreases in vagal input to the heart.

Potent homocysteine-induced ERK phosphorylation in cultured neurons depends on self-sensitization via system Xc −

Homocysteine is increased during pathological conditions, endangering vascular and cognitive functions, and elevated homocysteine during pregnancy may be correlated with an increased incidence of schizophrenia in the offspring. This study showed that millimolar homocysteine concentrations in saline medium cause phosphorylation of extracellular-signal regulated kinases 1 and 2 (ERK 1/2) in cerebellar granule neurons, inhibitable by metabotropic but not ionotropic glutamate receptor antagonists. These findings are analogous to observations by Zieminska et al. (2003), that similar concentrations cause neuronal death. However, these concentrations are much higher than those occurring clinically during hyperhomocysteinemia. It is therefore important that a ∼ 10-fold increase in potency occurred in the presence of the glutamate precursor glutamine, when ERK 1/2 phosphorylation became inhibitable by NMDA or non-NMDA antagonists and dependent upon epidermal growth factor (EGF) receptor transactivation. However, glutamate release to the medium was reduced, suggesting that reversal of the cystine/glutamate antiporter, system X c − could be involved in potentiation of the response by causing a localized release of initially accumulated homocysteine. In agreement with this hypothesis further enhancement of ERK 1/2 phosphorylation occurred in the additional presence of cystine. Pharmacological inhibition of system X c − prevented the effect of micromolar homocysteine concentrations, and U0126-mediated inhibition of ERK 1/2 phosphorylation enhanced homocysteine-induced death. In conclusion, homocysteine interacts with system X c − like quisqualate (Venkatraman et al. 1994), by “self-sensitization” with initial accumulation and subsequent release in exchange with cystine and/or glutamate, establishing high local homocysteine concentrations, which activate adjacent ionotropic glutamate receptors and cause neurotoxicity.

Involvement of NMDAR2A tyrosine phosphorylation in depression-related behaviour

Major depressive and bipolar disorders are serious illnesses that affect millions of people. Growing evidence implicates glutamate signalling in depression, though the molecular mechanism by which glutamate signalling regulates depression-related behaviour remains unknown. In this study, we provide evidence suggesting that tyrosine phosphorylation of the NMDA receptor, an ionotropic glutamate receptor, contributes to depression-related behaviour. The NR2A subunit of the NMDA receptor is tyrosine-phosphorylated, with Tyr 1325 as its one of the major phosphorylation site. We have generated mice expressing mutant NR2A with a Tyr-1325-Phe mutation to prevent the phosphorylation of this site in vivo. The homozygous knock-in mice show antidepressant-like behaviour in the tail suspension test and in the forced swim test. In the striatum of the knock-in mice, DARPP-32 phosphorylation at Thr 34, which is important for the regulation of depression-related behaviour, is increased. We also show that the Tyr 1325 phosphorylation site is required for Src-induced potentiation of the NMDA receptor channel in the striatum. These data argue that Tyr 1325 phosphorylation regulates NMDA receptor channel properties and the NMDA receptor-mediated downstream signalling to modulate depression-related behaviour.