Expression of the ionotropic glutamate receptors on neuronostatin neurons in the periventricular nucleus of the hypothalamus.

BACKGROUND: Neuronostatin, a newly identified peptide, is accepted as an anorexigenic peptide since it suppresses food intake when given intracerebroventricularly. Although the effect mechanisms of neuronostatin have been shown in different studies, there are no reports in the literature describing the mechanisms controlling neuronostatin neurons. In this study, we aimed to determine the presence of the ionotropic glutamate receptor subunits (iGluRs) in neuronostatin neurons in the periventricular nucleus of the hypothalamus. MATERIALS AND METHODS: The presence of glutamate receptors in neuronostatin neurons was investigated by dual immunohistochemistry. Immunohistochemistry was performed on 40 µm thick coronal brain sections with antibodies against AMPA (GluA1-4), kainate (GluK1/2/3, and GluK5), and NMDA (GluN1 and GluN2A) receptor subunits. RESULTS: The results showed that the neuronostatin neurons expressed most of the NMDA and non-NMDA receptor subunits. The neuronostatin neurons in the anterior hypothalamic periventricular nucleus were particularly immunopositive for GluA1, GluA4, GluK1/2/3, GluK5 and GluN1 antibodies. No expression was observed for GluA2, GluA3 and GluN2A antibodies. CONCLUSIONS: For the first time in the literature, our study demonstrated that the neuronostatin neurons express glutamate receptor subunits which may form homomeric or heteromeric functional receptor complexes. Taken together, these results suggest that multiple subunits of iGluRs are responsible for glutamate transmission on neuronostatin neurons in the anterior hypothalamic periventricular nucleus.

Thalamic circuits for independent control of prefrontal signal and noise.

Interactions between the mediodorsal thalamus and the prefrontal cortex are critical for cognition. Studies in humans indicate that these interactions may resolve uncertainty in decision-making1, but the precise mechanisms are unknown. Here we identify two distinct mediodorsal projections to the prefrontal cortex that have complementary mechanistic roles in decision-making under uncertainty. Specifically, we found that a dopamine receptor (D2)-expressing projection amplifies prefrontal signals when task inputs are sparse and a kainate receptor (GRIK4) expressing-projection suppresses prefrontal noise when task inputs are dense but conflicting. Collectively, our data suggest that there are distinct brain mechanisms for handling uncertainty due to low signals versus uncertainty due to high noise, and provide a mechanistic entry point for correcting decision-making abnormalities in disorders that have a prominent prefrontal component2-6.

Impaired Glutamatergic Neurotransmission in the Ventromedial Hypothalamus May Contribute to Defective Counterregulation in Recurrently Hypoglycemic Rats.

The objectives of this study were to understand the role of glutamatergic neurotransmission in the ventromedial hypothalamus (VMH) in response to hypoglycemia and to elucidate the effects of recurrent hypoglycemia (RH) on this neurotransmitter. We 1) measured changes in interstitial VMH glutamate levels by using microdialysis and biosensors, 2) identified the receptors that mediate glutamate's stimulatory effects on the counterregulatory responses, 3) quantified glutamate metabolic enzyme levels in the VMH, 4) examined astrocytic glutamate reuptake mechanisms, and 5) used 1H-[13C]-nuclear magnetic resonance (NMR) spectroscopy to evaluate the effects of RH on neuronal glutamate metabolism. We demonstrated that glutamate acts through kainic acid receptors in the VMH to augment counterregulatory responses. Biosensors showed that the normal transient rise in glutamate levels in response to hypoglycemia is absent in RH animals. More importantly, RH reduced extracellular glutamate concentrations partly as a result of decreased glutaminase expression. Decreased glutamate was also associated with reduced astrocytic glutamate transport in the VMH. NMR analysis revealed a decrease in [4-13C]glutamate but unaltered [4-13C]glutamine concentrations in the VMH of RH animals. The data suggest that glutamate release is important for proper activation of the counterregulatory response to hypoglycemia and that impairment of glutamate metabolic and resynthetic pathways with RH may contribute to counterregulatory failure.

AMPA/kainate glutamate receptors contribute to inflammation, degeneration and pain related behaviour in inflammatory stages of arthritis.

OBJECTIVES: Synovial fluid glutamate concentrations increase in arthritis. Activation of kainate (KA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors (GluRs) increase interleukin-6 (IL-6) release and cause arthritic pain, respectively. We hypothesised that AMPA and KA GluRs are expressed in human arthritis, and that intra-articular NBQX (AMPA/KA GluR antagonist) prevents pain and pathology in antigen-induced arthritis (AIA). METHODS: GluR immunohistochemistry was related to synovial inflammation and degradation in osteoarthritis (OA) and rheumatoid arthritis (RA). A single intra-articular NBQX injection was given at induction, and knee swelling and gait of AIA and AIA+NBQX rats compared over 21 days, before imaging, RT-qPCR, histology and immunohistochemistry of joints. Effects of NBQX on human primary osteoblast (HOB) activity were determined. RESULTS: AMPAR2 and KA1 immunolocalised to remodelling bone, cartilage and synovial cells in human OA and RA, and rat AIA. All arthritic tissues showed degradation and synovial inflammation. NBQX reduced GluR abundance, knee swelling (p<0.001, days 1-21), gait abnormalities (days 1-2), end-stage joint destruction (p<0.001), synovial inflammation (p<0.001), and messenger RNA expression of meniscal IL-6 (p<0.05) and whole joint cathepsin K (p<0.01). X-ray and MRI revealed fewer cartilage and bone erosions, and less inflammation after NBQX treatment. NBQX reduced HOB number and prevented mineralisation. CONCLUSIONS: AMPA/KA GluRs are expressed in human OA and RA, and in AIA, where a single intra-articular injection of NBQX reduced swelling by 33%, and inflammation and degeneration scores by 34% and 27%, respectively, exceeding the efficacy of approved drugs in the same model. AMPA/KA GluR antagonists represent a potential treatment for arthritis.

Radial symmetry in a chimeric glutamate receptor pore.

Ionotropic glutamate receptors comprise two conformationally different A/C and B/D subunit pairs. Closed channels exhibit fourfold radial symmetry in the transmembrane domain (TMD) but transition to twofold dimer-of-dimers symmetry for extracellular ligand binding and N-terminal domains. Here, to evaluate symmetry in open pores we analysed interaction between the Q/R editing site near the pore loop apex and the transmembrane M3 helix of kainate receptor subunit GluK2. Chimeric subunits that combined the GluK2 TMD with extracellular segments from NMDA receptors, which are obligate heteromers, yielded channels made up of A/C and B/D subunit pairs with distinct substitutions along M3 and/or Q/R site editing status, in an otherwise identical homotetrameric TMD. Our results indicate that Q/R site interaction with M3 occurs within individual subunits and is essentially the same for both A/C and B/D subunit conformations, suggesting that fourfold pore symmetry persists in the open state.

Nitric oxide signaling exerts bidirectional effects on plasticity inductions in amygdala.

It has been well known that long-term potentiation (LTP) of synaptic transmission in the lateral nucleus of the amygdala (LA) constitutes an essential cellular mechanism contributing to encoding of conditioned fear. Nitric oxide (NO), produced by activation of the postsynaptic N-methyl-D-aspartate receptors (NMDAR) in thalamic input to the LA, has been thought to promote LTP, contributing to the establishment of conditioned fear. However, it is not known whether and how NO, released from cortical input to the LA, plays the role on the plasticity induction and fear memory. Here we report that the diffusion of NO, released in response to activation of presynaptic NMDAR on cortical afferent fibers in the LA, could suppress heterosynaptically a form of presynaptic kainate receptor (KAR) dependent LTP (pre-LTP) in thalamic input, which was induced by low-frequency presynaptic stimuli without postsynaptic depolarization. We also confirmed that NO, produced by activation of postsynaptic NMDAR in thalamic input, can promote postsynaptic NMDAR-dependent LTP (post-LTP), which was induced by pairing protocol. These LTPs were occluded following fear conditioning, indicating that they could contribute to encoding of conditioned fear memory. However, their time courses are different; Post-LTP was more rapidly formed than pre-LTP in the course of fear conditioning. NO, produced by activation of presynaptic NMDAR in cortical input and postsynaptic NMDAR in thalamic input, may control conditioned fear by suppressing pre-LTP and promoting post-LTP, respectively, in thalamic input to the LA.

Kainate receptors in health and disease.

Our understanding of the molecular properties of kainate receptors and their involvement in synaptic physiology has progressed significantly over the last 30 years. A plethora of studies indicate that kainate receptors are important mediators of the pre- and postsynaptic actions of glutamate, although the mechanisms underlying such effects are still often a topic for discussion. Three clear fields related to their behavior have emerged: there are a number of interacting proteins that pace the properties of kainate receptors; their activity is unconventional since they can also signal through G proteins, behaving like metabotropic receptors; they seem to be linked to some devastating brain diseases. Despite the significant progress in their importance in brain function, kainate receptors remain somewhat puzzling. Here we examine discoveries linking these receptors to physiology and their probable implications in disease, in particular mood disorders, and propose some ideas to obtain a deeper understanding of these intriguing proteins.

[Effects of ionotropic glutamate receptor channel blockers on the development of audiogenic seizures in Krushinski-Molodkina rats].

The action of noncompetitive blockers of glutamate receptors has been investigated on Krushinski-Molodkina rats genetically-prone to audiogenic seizures. The selective blockers of NMDA receptor channels, memantine and IEM-1921, and their dicationic homologues, IEM-1925 and IEM-1754, capable of blocking in varying degrees both NMDA and Ca-permeable AMPA receptor channels, were studied. The drugs were injected intramuscularly to rats with the different time intervals (30 min, 1, 2 or 3 hours) before sound signal. The effects of the drugs on latent period of initial locomotor activity provoked by audio stimulation (8 kHz sine-wave tone, 90 dB volume), the appearance of clonic convulsions of different intensities, and, finally, tonic convulsions with limb and tail extension were evaluated. Within 30 min after injection IEM-1921 at a dose of 5 mg/kg, 33% of rats manifested a complete absence of convulsive reactions to sound, and in 59% of rats audiogenic seizures occured only in the form of motor excitation without a generalized clonic-tonic convulsions. Memantine at a dose of 5 mg/kg did not cause a complete blockade of seizures, but after 1 h of injection in 50% of the rats and after 2 h in 70% of rats a weakening of the audiogenic seizures to the level of motor excitation only was observed. After 3 hrs after administration of blockers its anticonvulsive action weakened significantly (p < 0.01). Dicationic blockers that block both NMDA and AMPA/kainate receptors, IEM-1925 (in doses of 0.001-20.0 mg/kg) and IEM-1754 (0.025-50.0 mg/kg), did not affect audiogenic clonic-tonic convulsive reactions. The involvement of activation of NMDA and calcium permeable AMPA/kainate receptors in the pathogenesis of audiogenic seizures is discussed.

Cations but not anions regulate the responsiveness of kainate receptors.

Kainate-selective ionotropic glutamate receptors are unique among ligand-gated ion channels in their obligate requirement of external anions and cations for activation. Although it is established that the degree of kainate receptor (KAR) activation is shaped by the chemical nature of the agonist molecule, the possible complementary role of external ions has yet to be examined. Here we show that external cations but not anions regulate the responsiveness to a range of full and partial agonists acting on rat GluK2 receptors. This observation is unexpected as previous work has assumed anions and cations affect KARs in an identical manner through functionally coupled binding sites. However, our data demonstrate that anion- and cation-binding pockets behave discretely. We suggest cations uniquely regulate a pregating or flipping step that impacts the closed-cleft stability of the agonist-binding domain (ABD). This model departs from a previous proposal that KAR agonist efficacy is governed by the degree of closure elicited in the ABD by ligand binding. Our findings are, however, in line with recent studies on Cys-loop ligand-gated ion channels suggesting that the "flipping" mechanism has been conserved by structurally diverse ligand-gated ion channel families as a common means of regulating neurotransmitter behavior.

Ionotropic glutamate receptor mRNA expression in the human thalamus: absence of change in schizophrenia.

Abnormalities in glutamate neurotransmission are thought to be among the major contributing factors to the pathophysiology of schizophrenia. Although schizophrenia has been regarded mostly as a disorder of higher cortical function, the cortex and thalamus work as a functional unit. Existing data regarding alterations of glutamate receptor subunit expression in the thalamus in schizophrenia remain equivocal. This postmortem study examined mRNA expression of ionotropic glutamate receptor (iGluR) subunits and PSD95 in 5 precisely defined and dissected thalamic subdivisions (medial and lateral sectors of the mediodorsal nucleus; and the ventral lateral posterior, ventral posterior, and centromedian nuclei) of persons with schizophrenia and matched controls using quantitative PCR with normalization to multiple endogenous controls. Among 15 genes examined (NR1 and NR2A-D subunits of the NMDA receptor; GluR1-4 subunits of the AMPA receptor; GluR5-7 and KA1-2 subunits of the kainate receptor; PSD95), all but two (GluR4 and KA1) were expressed at quantifiable levels. Differences in iGluR gene expression were seen between different thalamic nuclei but not between diagnostic groups. The relative abundance of transcripts was: NR1>>NR2A>NR2B>NR2D>NR2C for NMDA, GluR2>GluR1>GluR3 for AMPA, and KA2>GluR5>GluR7>GluR6 for kainate receptors. The expression of PSD95 correlated with the expression of NR1, NR2A, NR2B, NR2D and GluR6 in all nuclei. These results provide detailed and quantitative information on iGluR subunit expression in multiple nuclei of the human thalamus but suggest that alterations in their expression are not a prominent feature of schizophrenia.

Imbalance of a serotonergic system in frontotemporal dementia: implication for pharmacotherapy.

RATIONALE: Information is sparse on neurotransmitter deficiencies in frontotemporal dementia (FTD), in particular with reference to distinct histological subgroups and Alzheimer's disease (AD). OBJECTIVES: To evaluate in FTD with the major histologies, and compare with AD and controls, neurotransmission indices, as these may help in developing treatment. MATERIALS AND METHODS: Post-mortem grey matter from Brodmann Area 21, 9 and 7 of 51 brains was assayed for ten neurochemical parameters indexing neurotransmission. Repeated measures analyses of variance were carried out for each parameter comparing groups (FTD vs AD vs control) at each anatomical site. RESULTS: In FTD only the indices of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid, serotonin (5-HT)(1A) and 5-HT(2A) receptors were significantly reduced from control values. Of the ten parameters only 5-HT(1A) receptors showed significant group x site interaction. This reflected disproportionate reduction in frontal and temporal compared to parietal cortex. In FTD three other receptors (muscarinic, M(1), N-methyl-D: -aspartate, NMDA, and kainate), choline acetyltransferase (ChAT) activity, 5-HT and 5-hydroxyindoleacetic acid content and 5-HT reuptake site values were not significantly reduced from control values. Only 5-HT, 5-HT reuptake site and ChAT values were significantly higher in FTD than AD. NMDA receptor and ChAT values were significantly reduced from control only in AD. CONCLUSIONS: Neurochemical results in FTD indicate degeneration and loss of pyramidal neurones in frontotemporal neocortex, yet 5-HT afferents and 5-HT concentration, which are inhibitory on pyramidal neurones, were relatively preserved. This could lead to an excess of extraneural 5-HT causing underactivity of surviving pyramidal neurones. Pharmacotherapy with a 5-HT(1A) receptor antagonist may be indicated.

BMAA selectively injures motor neurons via AMPA/kainate receptor activation.

The toxin beta-methylamino-l-alanine (BMAA) has been proposed to contribute to amyotrophic lateral sclerosis-Parkinsonism Dementia Complex of Guam (ALS/PDC) based on its ability to induce a similar disease phenotype in primates and its presence in cycad seeds, which constituted a dietary item in afflicted populations. Concerns about the apparent low potency of this toxin in relation to estimated levels of human ingestion led to a slowing of BMAA research. However, recent reports identifying potential new routes of exposure compel a re-examination of the BMAA/cycad hypothesis. BMAA was found to induce selective motor neuron (MN) loss in dissociated mixed spinal cord cultures at concentrations ( approximately 30 muM) significantly lower than those previously found to induce widespread neuronal degeneration. The glutamate receptor antagonist NBQX prevented BMAA-induced death, implicating excitotoxic activation of AMPA/kainate receptors. Using microfluorimetric techniques, we further found that BMAA induced preferential [Ca(2+)](i) rises and selective reactive oxygen species (ROS) generation in MNs with minimal effect on other spinal neurons. Cycad seed extracts also triggered preferential AMPA/kainate-receptor-dependent MN injury, consistent with the idea that BMAA is a crucial toxic component in this plant. Present findings support the hypothesis that BMAA may contribute to the selective MN loss in ALS/PDC.

Different composition of glutamate receptors in corticothalamic and lemniscal synaptic responses and their roles in the firing responses of ventrobasal thalamic neurons in juvenile mice.

Thalamic ventrobasal (VB) relay neurons receive information via two major types of glutamatergic synapses, that is, from the medial lemniscus (lemniscal synapses) and primary somatosensory cortex (corticothalamic synapses). These two synapses influence and coordinate firing responses of VB neurons, but their precise operational mechanisms are not yet well understood. In this study, we compared the composition of glutamate receptors and synaptic properties of corticothalamic and lemniscal synapses. We found that the relative contribution of NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) to non-NMDA receptor-mediated EPSCs was significantly greater in corticothalamic synapses than in lemniscal synapses. Furthermore, NMDA receptor 2B-containing NMDA receptor- and kainate receptor-mediated currents were observed only in corticothalamic synapses, but not in lemniscal synapses. EPSCs in corticothalamic synapses displayed the postsynaptic summation in a frequency-dependent manner, in which the summation of the NMDA receptor-mediated component was largely involved. The summation of kainate receptor-mediated currents also partially contributed to the postsynaptic summation in corticothalamic synapses. In contrast, the contribution of NMDA receptor-mediated currents to the postsynaptic summation of lemniscal EPSCs was relatively minor. Furthermore, our results indicated that the prominent NMDA receptor-mediated component in corticothalamic synapses was the key determinant for the late-persistent firing of VB neurons in response to corticothalamic stimuli. In lemniscal synapses, in contrast, the onset-transient firing in response to lemniscal stimuli was regulated mainly by AMPA receptors.

Histone modifications in status epilepticus induced by kainate.

Animal models of epilepsy have allowed the determination of the basic molecular and cellular mechanisms of epileptogenesis. Generalized limbic seizures and subsequent status epilepticus can be induced by either pilocarpine, the muscarinic acetylcholine receptor agonist or kainate, the glutamate receptor agonist. There has been increasing interest that chromatin remodeling might play a critical role in gene regulation even in non-dividing cells such as neurons. One form of chromatin remodeling is histone amino-terminal modification that can generate synergistic or antagonistic affinities for the interactions of transcriptional factors, in turn causing changes in gene activity. Two widely studied histone modification processes are histone acetylation and phosphorylation. While histone hyperacetylation indicates an increase in gene activity, its hypoacetylation marks gene repression. Both states are controlled by a dynamic interplay of histone acetyltransferase (HAT) and histone deacetylase (HDAC). We have found the upregulation of acetylation and phosphorylation of histones, coupled with status epilepticus after kainate administration. c-fos and c-jun mRNA have been sequentially induced in response to kainate, in different hippocampal subpopulations starting from the dentate gyrus and spreading to the cornus ammonis regions well correlated with the spatio-temporal distribution of histone H4 hyperacetylation. Both histone modifications are associated with the c-fos gene promoter after kainate stimulation, while only histone acetylation with the c-jun gene. Pretreatment with curcumin, which has a HAT inhibitory activity specific for CBP/p300, attenuates histone modifications, IEGs expression and also the severity of status epilepticus after kainate treatment. Histone modifications may have a crucial role in the development of epilepsy induced by kainate.

Localization of kainate receptor subunit GluR5-immunoreactive cells in the rat hypothalamus.

Glutamate is the major excitatory neurotransmitter in the hypothalamus, which exerts its effects by activating ion channel-forming (ionotropic) or G-protein-coupled (metabotropic) receptors. Kainate-preferring glutamate receptor subunits (GluR5, GluR6, GluR7, KA1, and KA2) form one of the three ionotropic receptor families. In the present study, we analyzed the distribution of GluR5 subunit protein in the rat hypothalamus with immunohistochemistry. GluR5 immunoreactivity was observed in perikarya and processes of many hypothalamic cells some of which, based upon their morphological differentiation by size and structure, appeared to be neurons and others glial cells. Analyses revealed that higher number of glial cells were GluR5 positive when compared to the moderate number of GluR5-labeled neurons in the anteroventral periventricular nucleus. Numerous GluR5-expressing neurons and similar number of glia were detected in the suprachiasmatic nucleus. In the arcuate nucleus more glial cells were identified with GluR5 immunoreactivity than the number of labeled neurons. Scattered GluR5-positive cells were present in the periventricular nucleus. Specific immunostaining was not seen in the ventromedial nucleus or dorsomedial nucleus. In conclusion, it is suggested that the GluR5 subunits participate in the glutamatergic regulation of several neuroendocrine systems, such as the tubero-infundibular systems as well as in the control of circadian output through neuron-to-neuron and/or neuron-to-glia interactions.

Are unconventional NMDA receptors involved in slowly adapting type I mechanoreceptor responses?

Specific immunohistochemical staining for NMDA receptor NR2A/B subunits was found in the outer root sheath layer of rat sinus hair (whisker) follicle. Co-localization with CK 20 confirmed that Merkel cells were stained. The NR2A/B staining seen on Merkel cells was pericellular. In addition it appeared that NF70-positive staining was in close proximity to, but did not colocalise with NR2A/B immunoreactivity, indicating that NR2A/B was only expressed by Merkel cells and not their adjacent nerve terminals. Merkel cells and the nerve terminals have previously been associated with electrophysiological recordings from slowly adapting type I (St I) mechanoreceptor unit activity. Pharmacological experiments with isolated sinus hairs using a wide range of ionotropic glutamate receptor antagonists found that only certain NMDA receptor blockers depressed St I unit responses to mechanical stimuli. AMPA/kainate receptor antagonists (CNQX and NBQX, 100 microM) had no effect, nor did classical competitive NMDA receptor antagonists, D-AP5 (600 microM) and R-CPP (100 microM), nor the NMDA glycine site antagonist 5,7-dichlorokynurenic acid (100 microM). The only effective NMDA receptor blockers were those selective for the polyamine site: ifenprodil (IC50 20 microM) and Ro 25-6981 (IC50 approximately 50 microM), and the associated ion channel: MK 801, ketamine and (+/-)-1-(1,2-diphenylethyl)piperidine (IC50 < 100 microM). The two enantiomers of MK 801 were equipotent. All effects were long lasting, consistent with their non-/uncompetitive actions. The most potent drug tested, ifenprodil, at an effective dose of 30 microM, had a mean recovery time of 74 min. A three-fold increase in drug concentration was required to depress St II units (associated with non-synaptic lanceolate endings). Changes in Zn2+ did not affect St I unit responses. These data suggest that unconventional NMDA receptors are involved in St I unit responses, but question the notion of a glutamatergic synapse between the Merkel cell and nerve terminal.

NMDA and AMPA/KA receptors are involved in the c-Fos expression following mustard oil activation of C-fibres.

To examine whether mustard oil application to the skin activated c-Fos via glutamate receptors, a competitive NMDA receptor antagonist, 3-(2-carboxpiperazin-4-yl)propyl-1-phospionic acid (CPP), a selective AMPA/KA receptor antagonist, 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX), or both, were used intrathecally 10 min prior to noxious stimulation. Application of mustard oil to left hind foot of the vehicle-injected animals produced c-Fos expression mainly in superficial laminae (laminae I-II) of the dorsal horn on the side ipsilateral to the stimulation. CPP significantly reduced the number of c-Fos-positive nuclei in superficial laminae. But significant reduction of c-Fos expression by CNQX was seen in deeper laminae (laminae III-X). Administration of both CPP and CNQX extensively reduced the number of c-Fos-positive cells in both superficial and deeper laminae. However, they did not greatly change the number of c-Fos-positive cells in lamina I. This experiment revealed that NMDA and AMPA/KA receptors contribute to the mustard oil-induced c-Fos expression in the spinal cord. These data also suggest that other neurotransmitter receptors might be involved in the activation produced by algesic chemical activation of C-fibre primary afferents.

Dual roles in feeding for AMPA/kainate receptors: receptor activation or inactivation within distinct hypothalamic regions elicits feeding behavior.

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 roles of different types of glutamate receptors involved in the mediation of nucleus submedius (Sm) glutamate-evoked antinociception in the rat.

Based on our previous findings that glutamate microinjected into the thalamic nucleus submedius (Sm) inhibits dose-dependently the rat tail-flick (TF) reflex, this study investigated which glutamate receptor subtype is involved in mediating this effect. The effects of an NMDA (N-methyl-D-aspartate), non-NMDA or metabotropic glutamate receptor (mGluR) antagonist microinjected into Sm on the TF reflex were examined in untreated or in Sm glutamate treated (microinjection into the Sm) rats. The TF latencies were measured in each of these groups of rats every 5 min. Injection of DNQX [6,7-dinitroquinoxaline-2,3(1H,4H)-dione], a non-NMDA receptor antagonist, or (+/-)-MCPG [(+/-)-alpha-methyl-4-carboxyphenylglycine], a mGluR antagonist, into the Sm blocked the inhibitory effects induced by a subsequent microinjection of glutamate into the same Sm site. The TF latency increased only by 6.6+/-1.6 or 9.0+/-1.1%, respectively, of the baseline value, which was markedly less than that (51.3+/-8.4 or 50.7+/-5.3%) obtained from injection of glutamate only (P<0.001, n=8). However, pre-microinjection of MK-801 [(+)-5-methyl-10,11-dibenzo[a,d]cyclohepten-5,10-imine], an NMDA receptor antagonist, into the Sm had no effect on the Sm glutamate-evoked inhibition of the TF reflex. The TF latency change (40.0+/-11.1%) was not significantly different (P>0.05, n=8) compared with that obtained from glutamate injection alone. These observations suggest that non-NMDA and metabotropic glutamate receptors, but not NMDA receptors, are involved in mediating Sm glutamate-evoked antinociception.

The expression of dominant-negative subunits selectively suppresses neuronal AMPA and kainate receptors.

Glutamate-gated ion channels are widely expressed in neurons where they serve a host of cellular functions. An appealing, but yet unexplored, way to delineate the functions of particular glutamate receptor subtypes is to direct the expression of dominant-negative and gain-of-function mutant subunits. We tested the ability of two dominant-negative subunits, an alpha-amino-3-hydroxy-5-methyl-isoxazolproprionic acid receptor subunit and a kainate receptor subunit, to silence recombinant and neuronal glutamate receptors. Co-expression studies in non-neuronal cells indicated that the inclusion of a single mutant subunit was sufficient to silence the receptor. When expressed in cerebellar granule cells, the dominant-negative subunits silenced native channels in a subtype-specific fashion. Immunocytochemical staining of control and transfected neurons, as well as studies with a gain-of-function glutamate receptor-1 mutant, indicated that the mutant subunits were expressed at levels roughly equal to the total abundance of related native subunits, and both dominant-negatives suppressed native channel expression 60-65% when tested 24 h post-transfection. If co-assembly of the mutant subunits with related native subunits is combinatorial, this level of suppression gives receptor half-lives of approximately 20 h.