non-N-methyl-D-aspartate Receptor Research Articles

Inhibitory actions of borneol on the substantia gelatinosa neurons of the trigeminal subnucleus caudalis in mice.

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) is the first relay site for the orofacial nociceptive inputs via the thin myelinated Aδ and unmyelinated C primary afferent fibers. Borneol, one of the valuable time-honored herbal ingredients in traditional Chinese medicine, is a popular treatment for anxiety, anesthesia, and antinociception. However, to date, little is known as to how borneol acts on the SG neurons of the Vc. To close this gap, the whole-cell patch-clamp technique was applied to elucidate the antinociceptive mechanism responding for the actions of borneol on the SG neurons of the Vc in mice. In the voltage-clamp mode, holding at –60 mV, the borneol-induced non-desensitizing inward currents were not affected by tetrodotoxin, a voltage-gated Na+ channel blocker, 6-cyano-7-nitro-quinoxaline-2,3-dione, a non-N-methyl-d-aspartate (NMDA) glutamate receptor antagonist and DL-2-amino-5-phosphonopentanoic acid, an NMDA receptor antagonist. However, borneol-induced inward currents were partially decreased in the presence of picrotoxin, a γ-aminobutyric acid (GABA)A receptor antagonist, or strychnine, a glycine receptor antagonist, and was almost suppressed in the presence of picrotoxin and strychnine. Though borneol did not show any effect on the glycine-induced inward currents, borneol enhanced GABA-mediated responses. Beside, borneol enhanced the GABA-induced hyperpolarization under the current-clamp mode. Altogether, we suggest that borneol contributes in part toward mediating the inhibitory GABA and glycine transmission on the SG neurons of the Vc and may serve as an herbal therapeutic for orofacial pain ailments.

Action of citral on the substantia gelatinosa neurons of the trigeminal subnucleus caudalis in juvenile mice.

The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) is admitted as a pivotal site of integrating and regulating orofacial nociceptive inputs. Although citral (3,7-dimethyl-2,6-octadienal) is involved in antinociception, the action mechanism of citral on the SG neurons of the Vc has not been fully clarified yet. In this study, we examined the direct membrane effects of citral and how citral mediates responses on the SG neurons of the Vc in juvenile mice using a whole-cell patch-clamp technique. Under high chloride pipette solution, citral showed repeatable inward currents that persisted in the presence of tetrodotoxin, a voltage-gated Na+ channel blocker, and 6-cyano-7-nitro-quinoxaline-2,3-dione, a non-N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, D-2-amino-5-phosphonopentanoic acid, an NMDA receptor antagonist. However, the citral-induced inward currents were partially blocked by picrotoxin, a gamma-aminobutyric acid (GABAA)-receptor antagonist, or by strychnine, a glycine receptor antagonist. Further, the citral-induced responses were almost blocked by picrotoxin with strychnine. We also found that citral exhibited additive effect with GABA-induced inward currents and glycine-induced inward currents were potentiated by citral. In addition, citral suppressed the firing activities by positive current injection on the SG neurons of the Vc. Taken together, these results demonstrate that citral has glycine- and/or GABA-mimetic actions and suggest that citral might be a potential target for orofacial pain modulation by the activation of inhibitory neurotransmission in the SG area of the Vc.

Functional NMDA receptors are expressed by both AII and A17 amacrine cells in the rod pathway of the mammalian retina.

At many glutamatergic synapses, non-N-methyl-d-aspartate (NMDA) and NMDA receptors are coexpressed postsynaptically. In the mammalian retina, glutamatergic rod bipolar cells are presynaptic to two rod amacrine cells (AII and A17) that constitute dyad postsynaptic partners opposite each presynaptic active zone. Whereas there is strong evidence for expression of non-NMDA receptors by both AII and A17 amacrines, the expression of NMDA receptors by the pre- and postsynaptic neurons in this microcircuit has not been resolved. In this study, using patch-clamp recording from visually identified cells in rat retinal slices, we investigated the expression and functional properties of NMDA receptors in these cells with a combination of pharmacological and biophysical methods. Pressure application of NMDA did not evoke a response in rod bipolar cells, but for both AII and A17 amacrines, NMDA evoked responses that were blocked by a competitive antagonist (CPP) applied extracellularly and an open channel blocker (MK-801) applied intracellularly. NMDA-evoked responses also displayed strong Mg(2+)-dependent voltage block and were independent of gap junction coupling. With low-frequency application (60-s intervals), NMDA-evoked responses remained stable for up to 50 min, but with higher-frequency stimulation (10- to 20-s intervals), NMDA responses were strongly and reversibly suppressed. We observed strong potentiation when NMDA was applied in nominally Ca(2+)-free extracellular solution, potentially reflecting Ca(2+)-dependent NMDA receptor inactivation. These results indicate that expression of functional (i.e., conductance-increasing) NMDA receptors is common to both AII and A17 amacrine cells and suggest that these receptors could play an important role for synaptic signaling, integration, or plasticity in the rod pathway.

Ionotropic Glutamate Receptors in Hypothalamic Paraventricular and Supraoptic Nuclei Mediate Vasopressin and Oxytocin Release in Unanesthetized Rats

We report changes in plasma arginine vasopressin (AVP) and oxytocin (OT) concentrations evoked by the microinjection of l-glutamate (l-glu) into the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) of unanesthetized rats, as well as which local mechanisms are involved in their mediation. l-Glu microinjection (10 nmol/100 nl) into the SON increased the circulating levels of both AVP and OT. The AVP increases were blocked by local pretreatment with the selective non-N-methyl-d-aspartate (NMDA) receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) (2 nmol/100 nl), but it was not affected by pretreatment with the NMDA-receptor antagonist LY235959 (2 nmol/100 nl). The OT response to l-glu microinjection into the SON was blocked by local pretreatment with either NBQX or LY235959. Furthermore, the administration of either the non-NMDA receptor agonist (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) (5 nmol/100 nl) or NMDA receptor agonist NMDA (5 nmol/100 nl) into the SON had no effect on OT baseline plasma levels, but when both agonists were microinjected together these levels were increased. l-Glu microinjection into the PVN did not change circulating levels of either AVP or OT. However, after local pretreatment with LY235959, the l-glu microinjection increased plasma levels of the hormones. The l-glu microinjection into the PVN after the local treatment with NBQX did not affect the circulating AVP and OT levels. Therefore, results suggest the AVP release from the SON is mediated by activation of non-NMDA glutamate receptors, whereas the OT release from this nucleus is mediated by an interaction of NMDA and non-NMDA receptors. The present study also suggests an inhibitory role for NMDA receptors in the PVN on the release of AVP and OT.

Korean Red Ginseng Extract Activates Non-NMDA Glutamate and GABAAReceptors on the Substantia Gelatinosa Neurons of the Trigeminal Subnucleus Caudalis in Mice

Korean red ginseng (KRG) is a valuable and important traditional medicine in East Asian countries and is currently used extensively for botanical products in the world. KRG has both stimulatory and inhibitory effects on the central nervous system (CNS) suggesting its complicated action mechanisms. The substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis (Vc) are involved in orofacial nociceptive processing. Some studies reported that KRG has antinociceptive effects, but there are few reports of the functional studies of KRG on the SG neurons of the Vc. In this study, a whole cell patch clamp study was performed to examine the action mechanism of a KRG extract on the SG neurons of the Vc from juvenile mice. KRG induced short-lived and repeatable inward currents on all the SG neurons tested in the high chloride pipette solution. The KRG-induced inward currents were concentration dependent and were maintained in the presence of tetrodotoxin, a voltage gated Na channel blocker. The KRG-induced inward currents were suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione, a non-N-methyl-D-aspartate (NMDA) glutamate receptor antagonist and/or picrotoxin, a gamma-aminobutyric acid (GABA)A receptor antagonist. However, the inward currents were not suppressed by d,l-2-amino-5-phosphonopentanoic acid, an NMDA receptor antagonist. These results show that KRG has excitatory effects on the SG neurons of the Vc via the activation of non-NMDA glutamate receptor as well as an inhibitory effect by activation of the GABAA receptor, indicating the KRG has both stimulatory and inhibitory effects on the CNS. In addition, KRG may be a potential target for modulating orofacial pain processing.

Identification of a neurosteroid binding site contained within the GluR2‐S1S2 domain

Glutamate receptors play a major role in neural cell plasticity, growth, and maturation. The degree to which ionotropic glutamate receptors (iGluR) conduct current is dependent on binding of extracellular ligands, of which glutamate is the native agonist. Although the glutamate binding site of the GluR2 class of amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) iGluR has been structurally characterized, the allosteric sites attributed to neurosteroid binding have yet to be localized. Here, using intrinsic tryptophan fluorescence spectroscopy, we show that the extracellular glutamate binding core of the GluR2 class of AMPA receptors also binds to two neurosteroids, pregnenolone sulfate (PS) and 3alpha-hydroxy-5beta-pregnan-20-one sulfate, both of which negatively modulate its activity. Interest in these sulfated neurosteroids stems from their differential modulation of other members of the iGluR family and their potential use as endogeneous agents for stroke therapy. In particular, whereas PS inhibits AMPA and other non-N-methyl-D-aspartate (NMDA) family members, it activates the NMDA receptor. In addition to providing evidence for binding of these neurosteroids to the glutamate binding core of the GluR2 class of AMPA receptors, our data suggests that both neurosteroids bind in a similar manner, consistent with their modulation of activity of this class of iGluR. Interestingly, the conformational change induced upon binding of these neurosteroids is distinct from that induced upon glutamate binding.

Neural pathways between sacrocaudal afferents and lumbar pattern generators in neonatal rats.

Projections of sacrocaudal afferents (SCA) onto lumbar pattern generators were studied in isolated spinal cords of neonatal rats. A locomotor-like pattern could be produced by SCA stimulation in the majority of the preparations. The SCA-induced lumbar rhythm was abolished after blocking synaptic transmission in the sacrococcygeal (SC) cord by bathing its segments in a low-calcium, high-magnesium artificial cerebrospinal fluid and restored when the synaptic block was alleviated by local application of calcium onto specific SC segments prior to SCA stimulation. Thus the SCA evoked lumbar rhythm involves synaptic activation of relay neurons in the SC cord. Functional activation of these relays depends on non-N-methyl-D-aspartate (NMDA) receptors because the lumbar rhythm was abolished when the non-NMDA receptor antagonist CNQX was added to the SC cord. By contrast, pharmacological block of the rhythmicity in the SC cord by specific antagonists of NMDA receptors and alpha1 and alpha2 adrenoceptors did not impair the SCA-induced lumbar rhythm. Midsagittal splitting experiments of parts of the SC and lumbar cord revealed that crossed and uncrossed ascending/propriospinal pathways are coactivated by SCA stimulation. We suggest that these pathways ascend onto the thoracolumbar cord through the lateral, ventrolateral, and ventral funiculi, because a complete block of the lumbar rhythm could only be obtained with a bilateral interruption of all of these funiculi. The relevance of our findings to the neural control of the rhythmogenic networks in the spinal cord is discussed.

NBQX treatment improves mitochondrial function and reduces oxidative events after spinal cord injury.

The purpose of this study was to examine the effects of inhibiting ionotropic glutamate receptor subtypes on measures of oxidative stress events at acute times following traumatic spinal cord injury (SCI). Rats received a moderate contusion injury and 15 min later were treated with one of two doses of 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzol[f]quinoxaline-7-sulfonamide disodium (NBQX), MK-801, or the appropriate vehicle. At 4 h following injury, spinal cords were removed and a crude synaptosomal preparation obtained to examine mitochondrial function using the MTT assay, as well as measures of reactive oxygen species (ROS), lipid peroxidation, and glutamate and glucose uptake. We report here that intraspinal treatment with either 15 or 30 nmol of NBQX improves mitochondrial function and reduces the levels of ROS and lipid peroxidation products. In contrast, MK-801, given intravenously at doses of 1.0 or 5.0 mg/kg, was without effect on these same measures. Neither drug treatment had an effect on glutamate or glucose uptake, both of which are reduced at acute times following SCI. Previous studies have documented that drugs acting on non-N-methyl-D-aspartate (NMDA) receptors exhibit greater efficacy compared to NMDA receptor antagonists on recovery of function and tissue sparing following traumatic spinal cord injury. The results of this study provide a potential mechanism by which blockade of the non-NMDA ionotropic receptors exhibit positive effects following traumatic SCI.

Modulation of excitatory synaptic transmission by GABA(C) receptor-mediated feedback in the mouse inner retina.

In many vertebrate CNS synapses, the neurotransmitter glutamate activates postsynaptic non-N-methyl-D-aspartate (NMDA) and NMDA receptors. Since their biophysical properties are quite different, the time course of excitatory postsynaptic currents (EPSCs) depends largely on the relative contribution of their activation. To investigate whether the activation of the two receptor subtypes is affected by the synaptic interaction in the inner plexiform layer (IPL) of the mouse retina, we analyzed the properties of the light-evoked responses of ON-cone bipolar cells and ON-transient amacrine cells in a retinal slice preparation. ON-transient amacrine cells were whole cell voltage-clamped, and the glutamatergic synaptic input from bipolar cells was isolated by a cocktail of pharmacological agents (bicuculline, strychnine, curare, and atropine). Direct puff application of NMDA revealed the presence of functional NMDA receptors. However, the light-evoked EPSC was not significantly affected by D(-)-2-amino-5-phosphonopentanoic acid (D-AP5), but suppressed by 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) or 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466). These results indicate that the light-evoked EPSC is mediated mainly by AMPA receptors under this condition. Since bipolar cells have GABA(C) receptors at their terminals, it has been suggested that bipolar cells receive feedback inhibition from amacrine cells. Application of (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), a specific blocker of GABA(C) receptors, suppressed both the GABA-induced current and the light-evoked feedback inhibition observed in ON-cone bipolar cells and enhanced the light-evoked EPSC of ON-transient amacrine cells. In the presence of TPMPA, the light-evoked EPSC of amacrine cells was composed of AMPA and NMDA receptor-mediated components. Our results suggest that photoresponses of ON-transient amacrine cells in the mouse retina are modified by the activation of presynaptic GABA(C) receptors, which may control the extent of glutamate spillover.

Non-N-methyl-D-aspartate (NMDA) receptor antagonist 1,2,3, 4-tetrahydro-6-nitro-2,3-dioxo-benzo(f)quinoxaline-7-sulphonamide (NBQX) decreases functional disorders in cytotoxic brain oedema.

N-methyl-D-aspartate (NMDA) and non-NMDA receptors were found to be involved in development of functional disorders caused by hexachlorophene. In order to specify the role of glutamate receptors we studied the protective effects of the selective antagonist of the kainate/(+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor/channel 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo-benzo[f]quinoxaline-7-sulphonamide disodium (NBQX) and of the non-competitive NMDA receptor antagonist ifenprodil tartrate on coordinative motor behaviour of adult male Wistar rats as assessed in a simple 'ladder-test'. Neurotoxic injury of the cerebrum after hexachlorophene administration and putative amelioration after treatment with test substances was demonstrated histologically. Hexachlorophene-induced motor disturbance remitted spontaneously when stopping the noxis, but remittance occurred significantly earlier when NBQX [0.45 and 0.6 mg/kg intraperitoneal (i.p.)] was applied as well. Ifenprodil (0.15 to 1.2 mg/kg) did not improve the motor function. Vacuolation of white matter of the whole cerebrum was observed after 3 weeks of treatment with hexachlorophene. These morphological alterations caused by hexachlorophene treatment [central nervous system (CNS) vacuolation] spontaneously revert only after 5-6 weeks. The 5-day duration with test substances was too short for remission of vacuolation which thus may not apply to the situation after treatment with glutamate antagonists, despite improvement of motor function. The results suggest that kainate/AMPA receptor channels are at least partially involved in the mechanism of brain damage induced by hexachlorophene, however, the polyamine binding site of the NMDA receptor evidently is not involved.

Selective glutamate receptor antagonists can induce or prevent axonal sprouting in rat hippocampal slice cultures.

After the transection of the Schaffer collateral pathway in hippocampal slice cultures, reactive sprouting is induced in the CA3 area, and eventually synaptic transmission between areas CA1 and CA3 is restored. Using this model, we have studied the role of ionotropic glutamate receptors in the initiation of axonal sprouting and the regeneration of functional synapses. We show that neither reactive sprouting nor functional recovery of synaptic transmission occur in the presence of the non-N-methyl-D-aspartate (NMDA) receptor antagonist 6-nitro-7-sulfamoylbenzoquinoxaline-2,3-dione (CNQX). In contrast, the NMDA receptor antagonists methyl-10, 11-dihydro-5-H-dibenzocyclohepten-5,10-imine (MK-801) or 3-(RS)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP) did not interfere with these processes. Moreover, we observed that the application of NMDA receptor antagonists induced massive axonal sprouting and an increase in the frequency of miniature excitatory postsynaptic currents in unlesioned cultures. Our results thus indicate that NMDA and non-NMDA receptors exert a differential effect on reactive sprouting and the recovery of synaptic transmission after injury in the hippocampus. Activation of non-NMDA receptors appears necessary for these processes to occur, whereas activation of NMDA receptors suppresses growth-associated protein -43 expression and axonal outgrowth.

M�ller cell swelling, glutamate uptake, and excitotoxic neurodegeneration in the isolated rat retina

We characterized morphological effects of the endogenous excitotoxin, glutamate in ex vivo retinal segments prepared from 30-day-old rats. Initial changes induced by glutamate consisted of reversible, sodium-dependent Müller cell swelling. This glial swelling was mimicked by glutamate transport substrates but not by ionotropic glutamate receptor agonists. Only very high concentrations of exogenous glutamate (3,000 microM) produced excitotoxic neuronal damage. The neuronal damage was accompanied by severe glial swelling and was blocked by an antagonist of non-N-methyl-D-aspartate (NMDA) receptors but not by an NMDA receptor antagonist. Because glutamate uptake can be influenced by changes in cellular energy levels, we studied the effects of oxidative and glycolytic energy depletion on glutamate-mediated Müller cell swelling. Oxygen deprivation produced little morphological change and did not alter either glutamate-mediated Müller cell swelling or glutamate-induced excitotoxicity. In contrast, inhibition of glycolysis by iodoacetate produced severe neuronal damage without Müller cell swelling. In the presence of iodoacetate, exogenous glutamate failed to cause glial swelling. The neuronal damage produced by iodoacetate was inhibited by pyruvate, a substrate that sustains oxidative energy pathways. In the presence of iodoacetate plus pyruvate, glutamate failed to cause Müller cell swelling but became neurotoxic at low concentrations through activation of non-NMDA receptors. These results indicate that glycolytic energy metabolism plays a critical role in sustaining ionic balances required for Müller cell glutamate uptake and glial uptake helps to prevent glutamate-mediated excitotoxicity.

Retinal input induces three firing patterns in neurons of the superficial superior colliculus of neonatal rats.

By using an in vitro isolated brain stem preparation, we recorded extracellular responses to electrical stimulation of the optic tract (OT) from 71 neurons in the superficial superior colliculus (SC) of neonatal rats (P1-13). At postnatal day 1 (P1), all tested neurons (n = 10) already received excitatory input from the retina. Sixty-nine (97%) superficial SC neurons of neonatal rats showed three response patterns to OT stimulation, which depended on stimulus intensity. A weak stimulus evoked only one spike that was caused by activation of non-N-methyl-D-aspartate (NMDA) glutamate receptors. A moderate stimulus elicited a short train (<250 ms) of spikes, which was induced by activation of both NMDA and non-NMDA receptors. A strong stimulus gave rise to a long train (>300 ms) of spikes, which was associated with additional activation of L-type high-threshold calcium channels. The long train firing pattern could also be induced either by temporal summation of retinal inputs or by blocking gamma-aminobutyric acid-A receptors. Because retinal ganglion cells show synchronous bursting activity before eye opening at P14, the retinotectal inputs appear to be sufficient to activate L-type calcium channels in the absence of pattern vision. Therefore activation of L-type calcium channels is likely to be an important source for calcium influx into SC neurons in neonatal rats.

NMDA and non-NMDA receptors mediate responses in the primary gustatory nucleus in goldfish.

Primary gustatory afferents from the oropharynx of the goldfish, Carassius auratus, terminate in the vagal lobe, a laminated structure in the dorsal medulla comparable to the gustatory portion of the nucleus of the solitary tract in mammals. We utilized an in vitro brain slice preparation to test the role of different ionotropic glutamate receptor subtypes in synaptic transmission of gustatory information by recording changes in field potentials after application of various glutamate receptor antagonists. Electrical stimulation of the vagus nerve (NX) evokes two short-latency postsynaptic field potentials from sensory layers of the vagal lobe. 6,7-Dinitroquinoxaline-2,3-dione and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione, two non-N-methyl-D-aspartate (NMDA) ionotropic receptor antagonists, blocked these short-latency potentials. Slower potentials that were revealed under Mg2+ -free conditions, were abolished by the NMDA receptor antagonist, D(-)-2-amino-5-phosphonovaleric acid (APV). Repetitive stimulation produced short-term facilitation, which was attenuated by application of APV. These results indicate that the synaptic responses in the vagal lobe produced by stimulation of the gustatory roots of the NX involve both NMDA and non-NMDA receptors. An NMDA receptor-mediated facilitation may serve to amplify incoming bursts of primary afferent activity.

Dendritic Na+ channels amplify EPSPs in hippocampal CA1 pyramidal cells.

1. Whole cell recordings were performed on the somata of CA1 pyramidal neurons in the rat hippocampal slice preparation Remote synaptic events were evoked by electrical stimulation of Schaffer collateral/commissural fibers in outer stratum radiatum. To isolate non-N-methyl-D-aspartate (NMDA)-mediated excitatory postsynaptic potentials (EPSPs), bath solutions contained the NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (D-APV; 30 microM), the gamma-aminobutyric acid-A (GABAA) receptor antagonist, bicuculline (10 microM), and the GABAB receptor antagonists, CGP 35348 (30 microM) or, in some experiments, saclofen (100 microM). 2. Local application of tetrodotoxin (TTX; 0.5-10 microM) into the proximal region of the apical dendrite reduced the peak amplitude of somatically recorded EPSPs by 28% on average. In contrast to dendritic TTX application, injection of TTX into the axosomatic region of the recorded neuron reduced EPSP amplitude by only 12% on average. 3. Spill-over of dendritically applied TTX into stratum pyramidale or into outer stratum radiatum was ruled out experimentally: somatic action potentials and field EPSPs recorded near the stimulation site in outer stratum radiatum remained unaffected by local TTX application. 4. Variations of somatic membrane potential revealed a strong voltage dependence of EPSP reduction after dendritic TTX application with the effect increasing substantially with membrane depolarization. Together with the field recordings from stratum radiatum, this finding argues strongly against a predominantly presynaptic site of TTX action. 5. We therefore ascribe the EPSP decrease after local TTX application to the proximal dendrite to suppression of dendritic Na+ channels, which we assume to give rise to a noninactivating (persistent) Na+ current (INaP) in the subthreshold voltage range. Our data suggest that presumed dendritic INaP produces considerable elevation of remote excitatory signals, thereby compensating for much of their electrotonic attenuation. 6. The experimental findings were related to computer simulations performed on a reduced compartmental model of the CA1 neuron. Because the experimental evidence available so far yields only indirect clues on the strength and distribution of INaP, we allowed considerable variations in these parameters. We also varied both size and location of synaptic input. 7. The major conclusions drawn from these simulations are the following: somatic INaP alone produces little EPSP enhancement; INaP density at the axon hillock/initial segment has to be at least twice the density at the soma to produce substantial EPSP amplification; depending on the density and distribution of dendritic INaP, < or = 80% of a remote synaptic potential arrives at the soma (compared with only 52% in a passive dendrite); synaptic potentials receive progressively more elevation by dendritic INaP the stronger they are; even if restricted to the proximal segment of the apical dendrite, INaP also affects dendritic processing at more distal segments; and spatial distribution rather than local density appears to be the most important parameter determining the role of dendritic INaP in synaptic integration.

Extracellular free potassium and calcium during synchronous activity induced by 4-aminopyridine in the juvenile rat hippocampus.

1. Field potential recordings and measurements of the extracellular concentration of free K+ ([K+]o) and Ca2+ ([Ca2+]o) were made during application of 4-aminopyridine (4-AP, 50 microM) in hippocampal slices that were obtained from 11- to 32-day-old rats. 2. Spontaneous field potentials recorded under this experimental condition in the CA3 stratum radiatum of slices from rats < 23 days old consisted of interictal (duration, 0.2-1.4 s; intervals of occurrence, 0.9-3.4 s) and ictal epileptiform discharges (duration, 5-46 s; intervals of occurrence, 22-259 s) and negative-going potentials that often preceded the onset of ictal discharge. Ictal activity became rare in slices from rats > 25 days old. 3. The negative-going potential (which also corresponded to the ictal discharge onset) was associated with [K+]o increases to 9.4 +/- 3.6 mM (mean +/- S.D.) from 3.25 mM baseline (n = 11 slices). [K+]o remained elevated at 5-6 mM throughout the ictal event. Decreases in [Ca2+]o (from 1.8 mM baseline to 1.3 +/- 0.1 mM, n = 7) were observed during the ictal discharge. 4. Interictal and ictal discharges were abolished by the non-N-methyl-D-aspartate (NMDA) receptor antagonist 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX, 10 microM). CNQX and the NMDA receptor antagonist 3-((+/-)-2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (CPP) did not influence negative-going potentials or the associated [K+]o increases (peak values were 8.7 +/- 3.2 mM, n = 8), that were blocked, however, by bicuculline methiodide (BMI, 10 microM). 5. The mu-opioid receptor agonist (D-Ala2,N-Me-Phe4,Gly5-ol)-enkephalin (DAGO, 10 microM) which inhibits GABA release from interneurons, prevented the occurrence of both GABA-mediated synchronous potentials and subsequent ictal discharges (n = 6) as well as the [K+]o elevations. DAGO effects were antagonized by naloxone (10 microM; n = 4). 6. The GABA-mediated [K+]o elevations changed as a function of age. In hippocampal slices obtained from 11- to 17-day-old rats, peak values of 10.6 +/- 2.0 mM (n = 10) and half-width durations of 8.7 +/- 1.3 s (n = 7) were observed. In slices obtained from 25- to 32-day-old animals these parameters were 5.2 +/- 0.5 mM (n = 13) and 4.6 +/- 1.1 s (n = 4), respectively. 7. This study shows that, in the juvenile rat hippocampus, 4-AP induces a glutamatergic independent synchronous potential that is due to GABA released from inhibitory terminals and is associated with an increase in [K+]o. This [K+]o elevation undergoes age-dependent changes, and is instrumental in synchronizing neurons thus initiating prolonged epileptiform discharges.

Glutamate receptor subtypes mediate excitatory synaptic responses of rat lateral parabrachial neurons.

We examined the role of specific excitatory amino acid (EAA) receptors in synaptic transmission within the lateral parabrachial nucleus (LPBN) using whole cell patch-recording techniques in a slice preparation. Two types of excitatory postsynaptic responses were observed. The first involved the contribution of non-N-methyl-D-aspartate (NMDA) receptors, which mediated a fast component, and NMDA receptors, which governed the late component of the excitatory postsynaptic current (EPSC). The second EPSC response was mediated solely by non-NMDA receptors. Both EPSC responses reversed near 0 mV. The fast component of the EPSC was attenuated by the non-NMDA antagonists [6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 6,7-dinitroquinoxaline-2,3-dione (DNQX), and 6-nitro-7-sulfamobenzoquinoxaline-2-3,-dione (NBQX)]. The late component was reduced by D,L-2-amino-5-phosphonovaleric acid (APV) and augmented in Mg(2+)-free external medium. EPSCs mediated solely by non-NMDA receptors were completely blocked by CNQX and NBQX but not affected by APV or Mg(2+)-free external medium. EPSCs were also markedly attenuated by the metabotropic-receptor agonist, trans-(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD). We concluded that intra-LPBN stimulation causes the synaptic release of glutamate, which depolarizes LBPN neurons via non-NMDA and NMDA receptors. We also provide evidence that glutamate can negatively influence its own release via action on presynaptic metabotropic receptors.

Electrophysiological and Pharmacological Characteristics of Ionotropic Glutamate Receptors in Medial Vestibular Nucleus Neurons: A Whole Cell Patch Clamp Study in Acutely Dissociated Neurons

A patch clamp study was performed to determine which subtype of ionotropic glutamate receptors is involved in the glutamate-induced excitation of the medial vestibular nucleus (MVN) neurons. Whole cell recording was performed on MVN neurons that were acutely dissociated by enzymatic and mechanical treatments. Application of glutamate at a concentration of 100 fiM produced a current with a reversal potential of approximately 0 mV. The glutamate-induced current was completely blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 μM), a non-N-methyl-D-aspartate (NMDA)-receptor antagonist. Application of α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) and kainic acid (KA), non-NMDA-receptor agonists, at concentrations of 30 and 100 μM produced a concentration-dependent depolarization concomitantly with an increase in firing rates during current clamp recording. During voltage clamp recording, glutamate, AMPA and KA elicited a concentration-dependent current with an equilibrium potential of approximately 0 mV. To clarify whether NMDA receptors are present in MVN neurons, the effects of glycine on the glutamate- and NMDA-induced current were examined. Two types of NMDA receptor-mediated current (types 1 and 2) were obtained in terms of the difference in sensitivity to both magnesium ion and MK-801, which act on the NMDA-receptor channel. In the type 1 neurons, the NMDA-induced current was not apparently blocked by magnesium ion or MK-801, although a larger current was obtained in the absence of magnesium ion. In the type 2 neurons, marked blockade of the NMDA-induced current was seen in the presence of magnesium ion and MK-801, as previously reported in other neurons of the central nervous system. These findings indicate the presence of both non-NMDA and NMDA receptors, which are involved in primary afferent transmission, in the MVN neuron, and two distinct types of NMDA receptors.

Physiological properties of anatomically identified basket and bistratified cells in the CA1 area of the rat hippocampus in vitro.

Basket and bistratified cells form two anatomically distinct classes of GABAergic local-circuit neurons in the CA1 region of the rat hippocampus. A physiological comparison was made of intracellularly recorded basket (n = 13) and bistratified neurons (n = 6), all of which had been anatomically defined by their efferent target profile (Halasy et al., 1996). Basket cells had an average resting membrane potential of -64.2 +/- 7.2 vs. -69.2 +/- 4.6 mV in bistratified cells. The latter had considerably higher mean input resistances (60.2 +/- 42.1 vs. 31.3 +/- 10.9 M Ohms) and longer membrane time constants (18.6 +/- 8.1 vs. 9.8 +/- 4.5 ms) than basket cells. Differences were also apparent in the duration of action potentials, those of basket cells being 364 +/- 77 and those of bistratified cells being 527 +/- 138 microseconds at half-amplitude. Action potentials were generally followed by prominent, fast after-hyperpolarizing potentials which in basket cells were 13.5 +/- 6.7 mV in amplitude vs. 10.5 +/- 5.1 in bistratified cells. The differences in membrane time constant, resting membrane potential, and action potential duration reached statistical significance (P < 0.05). Extracellular stimulation of Schaffer collateral/commissural afferents elicited short-latency excitatory postsynaptic potentials (EPSPs) in both cell types. The average 10-90% rise time and duration (at half-amplitude) of subthreshold EPSPs in basket cells were 1.9 +/- 0.5 and 10.7 +/- 5.6 ms, compared to 3.3 +/- 1.3 and 20.1 +/- 9.7 ms in bistratified cells, the difference in EPSP rise times being statistically significant. Basket and bistratified EPSPs were highly sensitive to a bath applied antagonist of non-N-methyl-D-aspartate (NMDA) receptors, whereas the remaining slow-rise EPSP could be abolished by an NMDA receptor antagonist. Increasing stimulation intensity elicited biphasic inhibitory postsynaptic potentials (IPSPs) in both basket and bistratified cells. In conclusion, basket and bistratified cells in the CA1 area show prominent differences in several of their membrane and firing properties. Both cell classes are activated by Schaffer collateral/commissural axons in a feedforward manner and receive inhibitory input from other, as yet unidentified, local-circuit neurons.

The quantal size at retinogeniculate synapses determined from spontaneous and evoked EPSCs in guinea-pig thalamic slices.

1. To determine the quantal size at retinogeniculate synapses, spontaneous and evoked excitatory postsynaptic currents (EPSCs) were recorded in twelve neurones of the dorsal lateral geniculate nucleus in guinea-pig thalamic slices using the whole-cell patch-clamp technique. We limited our study to the fast non-N-methyl-D-aspartate (NMDA) component of the EPSCs by adding the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid to the perfusion medium. 2. Spontaneous EPSCs occurred at a frequency between 0.5 and 6.6 Hz (mean 2.5 Hz). The modal value of the peak conductance change of spontaneous excitatory events varied between cells from 102 to 179 pS. 3. EPSCs were evoked by electrical stimulation in the optic tract. The peak conductance change of EPSCs evoked by stimulation of a putative single input fibre ranged from 0.6 to 3.4 nS (mean 1.7 nS). 4. To resolve the quantal components of evoked EPSCs the external Ca2+ concentration was reduced and the external Mg2+ concentration increased for four cells. In this condition failures occurred and the amplitude histograms were multimodal with approximately equidistant peaks. 5. These multimodal histograms could be fitted by a sum of Gaussian functions with mean values corresponding to integer multiples of the modal value of the spontaneous EPSCs for the same cell. Thus, the quantal size of evoked EPSCs was the same as the modal value of spontaneous EPSCs. The mean of the apparent quantal conductance change was 138 pS. The estimated number of quanta released by stimulating a putative single input fibre in the control condition ranged from 4 to 27 (mean 13).(ABSTRACT TRUNCATED AT 250 WORDS)