Glutamatergic synaptic inputs activate neurons in the subfornical organ through non-NMDA receptors

The subfornical organ (SFO) plays an important role in central regulation of the autonomic nervous system. The synaptic transmission properties of neurons in the SFO were studied with intracellular and whole-cell patch clamp recordings in the rat slice preparations. Both the spontaneous and evoked excitatory postsynaptic potentials (EPSPs) and currents (EPSCs) were almost completely suppressed by the glutamate receptor antagonist kynurenic acid and the non-NMDA (N-methyl-D-aspartic acid) antagonist CNQX. The non-NMDA agonist kainic acid depolarized the membrane most potently, compared with NMDA and quisqualic acid. These suggest that glutamate is a main excitatory neurotransmitter in the SFO and that its action is at least partly mediated through non-NMDA receptors.     

Synaptic transmission between dorsal root ganglion and dorsal horn neurons in culture: antagonism of monosynaptic excitatory postsynaptic potentials and glutamate excitation by kynurenate

Intracellular recording techniques have been used to provide information on the identity of excitatory sensory transmitters released at synapses formed between dorsal root ganglion (DRG) and dorsal horn neurons maintained in cell culture. Explants of embryonic rat DRG were added to dissociated cultures of embryonic dorsal horn neurons and synaptic potentials were recorded intracellularly from dorsal horn neurons after DRG explant stimulation. More than 80% of dorsal horn neurons within 1 mm of DRG explants received at least one fast, DRG-evoked, monosynaptic input. In the presence of high divalent cation concentrations, the acidic amino acid receptor agonists, L-glutamate, kainate, and quisqualate excited all dorsal horn neurons which received a monosynaptic DRG neuron input, whereas aspartate and N-methyl-D-aspartate (NMDA) had little or no action. Several compounds reported to antagonize the actions of acidic amino acids were tested for their ability to block DRG-evoked synaptic potentials and glutamate-evoked responses in dorsal horn neurons. 2-Amino-5-phosphonovalerate, a selective NMDA receptor antagonist, was relatively ineffective at antagonizing DRG-evoked synaptic potentials and glutamate-evoked responses. In contrast, kynurenate was found to be a potent antagonist of amino acid-evoked responses and of synaptic transmission at all DRG-dorsal horn synapses examined. The blockade of synaptic transmission by kynurenate appeared to result from a postsynaptic action on dorsal horn neurons. These findings indicate that glutamate, or a glutamate-like compound, but not aspartate, is the excitatory transmitter that mediates fast excitatory postsynaptic potentials at the DRG-dorsal horn synapses examined in this study.     

Cyclic adenosine 3'5'-monophosphate potentiates excitatory amino acid and synaptic responses of rat spinal dorsal horn neurons.

Intracellular recordings were made from rat dorsal horn neurons in the in vitro slice preparation to study the actions of cyclic adenosine 3',5'-monophosphate (cyclic AMP). In the presence of TTX, bath application of the membrane permeable analogue of cyclic AMP, 8-Br cyclic AMP (25-100 microM) caused a small depolarization of the resting membrane potential accompanied by a variable change in membrane input resistance. In addition, 8-Br cyclic AMP caused a long-lasting increase in the spontaneous synaptic activity and the amplitude of presumed monosynaptic excitatory postsynaptic potentials evoked in the substantia gelatinosa neurons by orthodromic stimulation of a lumbar dorsal root. When the fast voltage-sensitive Na conductance was blocked by TTX, 8-Br cyclic AMP enhanced in a reversible manner, the depolarizing responses of a proportion of dorsal horn neurons to N-methyl-D-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), quisqualic acid (QA) and kainic acid (KA). The effects of 8-Br cyclic AMP on the resting membrane potential and the NMDA response of dorsal horn neurons were mimicked by reducing phosphodiesterase activity with bath application of 3-isobutyl-1-methylxanthine, but not by cyclic AMP applied extracellularly. Moreover, we have found that intracellular application of a protein inhibitor of cyclic AMP-dependent protein kinase (PKI) into dorsal horn neurons prevents the 8-Br cyclic AMP-induced potentiation of the NMDA response of these cells. These results suggest that in the rat spinal dorsal horn the activation of the adenylate cyclase-cyclic AMP-dependent protein kinase system may be involved in the enhancement of the sensitivity of postsynaptic excitatory amino acid (NMDA, AMPA, KA) receptors and modulation of primary afferent neurotransmission, including nociception.     

Actions of (-)-baclofen on rat dorsal horn neurons.

The actions of a gamma-aminobutyric acid B (GABAB) agonist, (-)-baclofen, on the electrophysiological properties of neurons and synaptic transmission in the spinal dorsal horn (laminae I-IV) were examined by using intracellular recordings in spinal cord slice from young rats. In addition, the effects of baclofen on the dorsal root stimulation-evoked outflow of glutamate and aspartate from the spinal dorsal horn were examined by using high performance liquid chromatography (HPLC) with flourimetric detection. Superfusion of baclofen (5 nM to 10 microM) hyperpolarized, in a stereoselective and bicuculline-insensitive manner, the majority (86%) of tested neurons. The hyperpolarization was associated with a decrease in membrane resistance and persisted in a nominally zero-Ca2+, 10 mM Mg(2+)- or a TTX-containing solution. Our findings indicate that the hyperpolarizing effect of baclofen is probably due to an increase in conductance to potassium ions. Baclofen decreased the direct excitability of dorsal horn neurons, enhanced accommodation of spike discharge, and reduced the duration of Ca(2+)-dependent action potentials. Baclofen depressed, or blocked, excitatory postsynaptic potentials evoked by electrical stimulation of the dorsal roots. Spontaneously occurring synaptic potentials were also reversibly depressed by baclofen. Whereas baclofen did not produce any consistent change in the rate of the basal outflow of glutamate and aspartate, the stimulation-evoked release of the amino acids was blocked. The present results suggest that baclofen, by activating GABAB receptors, may modulate spinal afferent processing in the superficial dorsal horn by at least two mechanisms: (1) baclofen depresses excitatory synaptic transmission primarily by a presynaptic mechanism involving a decrease in the release of excitatory amino acids, and (2) at higher concentrations, the hyperpolarization and increased membrane conductance may contribute to the depressant effect of baclofen on excitatory synaptic transmission in the rat spinal dorsal horn.     

Protein kinase C mediates potentiation of synaptic transmission by phorbol ester at parallel fibers in the dorsal cochlear nucleus

Many cells in the outer two layers of the dorsal cochlear nucleus (DCN) express high levels of the phospholipid-activated, calcium dependent kinase, protein kinase C (PKC), an enzyme that can phosphorylate numerous proteins involved in neurotransmission and postsynaptic signaling. We investigated the effects of stimulating PKC with phorbol esters (phorbol 12-13 diacetate; PDAc) on parallel fiber synaptic transmission in brain slices of the guinea pig DCN. Phorbol esters increased the amplitude of the postsynaptic components of the field potential, including the excitatory post-synaptic field potential (fEPSP) and the population spike following electric stimulation of parallel fibers. Phorbol esters simultaneously decreased paired-pulse facilitation, suggesting that transmitter release mechanisms were affected. Potentiation of synaptic transmission and diminished paired-pulse potentiation were also observed in intracellular recordings of DCN neurons. The effects of phorbol esters were antagonized by the specific PKC blockers bisindolylmaleimide and calphostin C. Although modulation of the synaptic potentials appears to be mediated by presynaptic PKC, the differential effects of PDAc on the fEPSP and the population spike also suggest the involvement of postsynaptic PKC and postsynaptic targets. These experiments demonstrate that protein kinase C is capable of profoundly modulating synaptic transmission at parallel fiber synapses in the DCN.     

Phorbol esters enhance transmitter release in rat hippocampal slices

Phorbol esters enhance synaptic transmission in the rat hippocampal slice preparation most likely by acting at a presynaptic locus. To more directly examine the actions of phorbol esters on neurotransmitter release we have measured their effects on the occurrence of spontaneous postsynaptic potentials as well as on the potassium stimulated release of endogenous glutamate. Both measures of transmitter release were increased by phorbol esters suggesting a functional or regulatory role for protein kinase C in controlling the release of neurotransmitter in the mammalian CNS.     

NMDA receptors in the intermediolateral column of the spinal cord mediate sympathoexcitatory cardiac responses elicited from the ventrolateral medullary pressor area.

Microinjections of L-glutamate into the intermediolateral column of the spinal cord (IML) at T1-T3 produced increases in heart rate (predominantly from the right IML) and myocardial contractility (predominantly from the left IML). Maximum responses were elicited from T2 segment. At this site, microinjections of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), quisqualic acid, kainic acid and N-methyl-D-aspartic acid (NMDA) produced dose-dependent increases in heart rate and contractility which were blocked by kynurenate (a non-selective excitatory amino acid receptor antagonist). D-2-Aminophosphonoheptanoate (DAP-7) blocked the effects of NMDA but not kainic acid, quisqualic acid and AMPA. Bilateral microinjections of kynurenate (2 nmol) and DAP-7 (5 nmol) into the IML at T1-T3 significantly decreased the baseline values for contractility index and blocked the usual increase in contractility induced by unilateral microinjections of L-glutamate (1.77 nmol) into the ventrolateral medullary pressor area (VLPA). These observations suggest that: (1) a tonic excitatory input, involving an NMDA-like amino acid as a transmitter, is present in the IML at T1-T3 and (2) the stimulation of VLPA neurons results in the release of an NMDA-like excitatory amino acid in the IML at this level.     

Interactions between excitatory amino acids and tachykinins in the rat spinal dorsal horn

Whole-cell patch-clamp technique of freshly isolated rat spinal dorsal horn (DH) neurons, intracellular recording from DH neurons in a slice preparation, and high performance liquid chromatography with fluorimetric detection of release of endogenous glutamate and aspartate from spinal cord slice following activation of primary afferent fibers were employed to investigate interactions between excitatory amino acids (EAA) and tachykinins [substance P (SP) and neurokinin A (NKA)]. Potentiation of N-methyl-D-aspartate (NMDA)-, quisqualate (QA)- and α-amino 3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, but not kainate-induced currents by SP and NKA was found. Spantide II, a claimed novel nonselective tachykinin antagonist, effectively blocked the SP (2 nM )-induced potentiation of the responses of DH neurons to NMDA. In the presence of glycine (0.1 μM), the SP-evoked increase of the NMDA-induced current was prevented. However, 7-chlorokynurenic acid (2 μM), a competitive antagonist at the glycine allosteric site of the NMDA receptor, led to the reestablishment of the SP effect. Brief high frequency electrical stimulation of primary afferent fibers produced a longlasting potentiation of presumed monosynaptic and polysynaptic excitatory postsynaptic potentials and sustained enhanced release of endogenous glutamate (218.3± 66.1 %) and aspartate (286.3 ± 58.0%). Possible functional implications of the observed phenomena are discussed in relation to transmission and integration of sensory information, including pain.     

Excitatory amino acid-evoked membrane currents and excitatory synaptic transmission in lamprey reticulospinal neurons

The characteristics of excitatory amino acid-evoked currents and of excitatory synaptic events have been examined in lamprey Müller neurons using voltage clamp and current clamp recording techniques. Application of glutamate evoked depolarizations associated with a decrease in input resistance. The reversal potential of the responses was -35 mV. Under voltage clamp conditions, a series of excitatory amino acid agonists evoked inward currents associated with little or no increase in baseline current noise. The order of potency of the excitatory amino acid agonists was quisqualate greater than kainate greater than glutamate greater than aspartate, while N-methyl-D-aspartic acid (NMDA) was inactive. Inward currents evoked by glutamate, as well as by kainate and quisqualate were attenuated reversibly by 1 mM kynurenic acid (KYN). In contrast, glutamate-evoked currents were not affected by 100 microM D(-)-2-amino-5-phosphonovaleric acid (APV), a selective NMDA antagonist. Spontaneously occurring and stimulus-evoked excitatory postsynaptic events were antagonized reversibly by 1 mM KYN. At this concentration, KYN had no effect on membrane potential, input resistance, or excitability of the cells. In contrast, excitatory postsynaptic currents were unaffected by APV. It is concluded that both glutamate responses and excitatory synaptic transmission in lamprey Müller neurons are mediated by non-NMDA-type receptors and that these receptors are associated with ionic channels with a low elementary conductance. The combined pharmacological and biophysical characteristics of these responses are therefore different from those previously reported in other preparations. Spontaneous (but not stimulus-evoked) inhibitory synaptic events in Müller neurons were blocked reversibly by 1 mM KYN but not by 100 microM APV, suggesting that excitation of interneurons inhibitory to Müller cells was also mediated by non-NMDA receptors.     

The distribution of neurons expressing calcium-permeable AMPA receptors in the superficial laminae of the spinal cord dorsal horn.

The superficial dorsal horn is a major site of termination of nociceptive primary afferents. Fast excitatory synaptic transmission in this region is mediated mainly by release of glutamate onto postsynaptic AMPA and NMDA receptors. NMDA receptors are known to be Ca2+-permeable and to provide synaptically localized Ca2+ signals that mediate short-term and long-term changes in synaptic strength. Less well known is a subpopulation of AMPA receptors that is Ca2+-permeable and has been shown to be synaptically localized on dorsal horn neurons in culture (Gu et al., 1996) and expressed by dorsal horn neurons in situ (Nagy et al., 1994; Engelman et al., 1997). We used kainate-induced cobalt uptake as a functional marker of neurons expressing Ca2+-permeable AMPA receptors and combined this with markers of nociceptive primary afferents in the postnatal rat dorsal horn. We have shown that cobalt-positive neurons are located in lamina I and outer lamina II, a region strongly innervated by nociceptors. These cobalt-positive neurons colocalize with afferents labeled by LD2, and with the most dorsal region of capsaicin-sensitive and IB4- and LA4-positive afferents. In contrast, inner lamina II has a sparser distribution of cobalt-positive neurons. Some lamina I neurons expressing the NK1 receptor, the receptor for substance P, are also cobalt positive. These neurons are likely to be projection neurons in the nociceptive pathway. On the basis of all of these observations, we propose that Ca2+-permeable AMPA receptors are localized to mediate transmission of nociceptive information.     

Substance P and neurokinin A mediate sensory synaptic transmission in young rat dorsal horn neurons

Spinal nociceptive transmission is mediated by glutamate and neuropeptides such as substance P (SP) and neurokinin A (NKA). The neuropeptide-mediated excitatory postsynaptic potentials (EPSPs) had a slow onset and long duration. Here, we demonstrate SP- and NKA-mediated excitatory postsynaptic currents (EPSCs) in dorsal horn neurons of young rats using whole-cell patch-clamp recording techniques. After complete blockade of glutamate receptor-mediated currents, we observed a small residual EPSC. The residual EPSCs exhibited temporal summation in response to a train of stimulation (six pulses delivered at 10-50 Hz). High intensity stimulation (the same or greater than the stimulation threshold for nociceptive fibers in vivo) was required for evoking these summated EPSCs. Summated EPSCs were attenuated or abolished by capsaicin pretreatment, which depletes SP and NKA from presynaptic terminals; SP and NKA pretreatment; NK(1) or NK(2) receptor antagonists; and inhibition of postsynaptic G proteins. EPSCs were neither blocked by a metabotropic glutamate receptor antagonist nor a gamma-aminobutyric acid(B) receptor antagonist. The summated EPSCs were also sensitive to voltage-gated calcium channel antagonists or mu-opioid receptor activation by DAMGO. The present study provides electrophysiological evidence that suggests the possible contribution of SP and NKA to sensory synaptic transmission between primary afferent fibers and dorsal horn neurons.     

Dual modulation of excitatory synaptic transmission by agonists at group I metabotropic glutamate receptors in the rat spinal dorsal horn

The effects of group I metabotropic glutamate (mGlu) receptors on excitatory transmission in the rat dorsal horn, but mostly substantia gelatinosa, neurons were investigated using conventional intracellular recording in slices. The broad spectrum mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S, 3R-ACPD), the group I mGlu receptor selective agonist (S)-3, 5-dihydroxyphenylglycine (DHPG), and the selective mGlu subtype 5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), all induce long-lasting depression of A primary afferent fibers-mediated monosynaptic excitatory postsynaptic potential (EPSP), and long-lasting potentiation of polysynaptic EPSP, and EPSP in cells receiving C-afferent fiber input. The DHPG potentiation of polysynaptic EPSP was partially or fully reversed by (S)-4-carboxyphenylglycine (S-4CPG), the mGlu subtype 1 preferring antagonist. 2-Methyl-6-(phenylethynyl)-pyridine, the potent and selective mGlu subtype 5 antagonist, partially reversed the CHPG potentiation of polysynaptic EPSP. The effects of DHPG on monosynaptic and polysynaptic EPSPs were reduced, or abolished, by the N-methyl-D-aspartate (NMDA) receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid (AP5). A clear and pronounced facilitation of the expression of DHPG- and CHPG-induced enhancement of polysynaptic EPSP, and EPSP evoked at C-fiber strength, was seen in the absence of gamma-aminobutyric acid subtype A receptor- and glycine-mediated synaptic inhibition. Besides dual modulation of excitatory synaptic transmission, DHPG induces depression of inhibitory postsynaptic potentials evoked by primary afferent stimulation in dorsal horn neurons. In addition, group I mGlu receptor agonists produced a direct persistent excitatory postsynaptic effect consisting of a slow membrane depolarization, an increase in input resistance, and an intense neuronal discharge. Cyclothiazide and (S)-4-CPG, the mGlu receptor subtype 1 preferring antagonists, significantly attenuated the DHPG-induced depolarization. These results demonstrate that the pharmacological activation of group I metabotropic glutamate receptors induces long-term depression (LTD) and long-term potentiation (LTP) of synaptic transmission in the spinal dorsal horn. These types of long-term synaptic plasticity may play a functional role in the generation of post-injury hypersensitivity (LTP) or antinociception (LTD).     

A₁ receptors inhibit glutamate release in rat medullary dorsal horn neurons

We have investigated the adenosine-mediated presynaptic inhibition of primary afferent-evoked glutamate release in rat substantia gelatinosa neurons of the trigeminal subnucleus caudalis using a conventional whole-cell patch clamp technique. Adenosine reversibly and concentration dependently decreased the amplitude of glutamatergic excitatory postsynaptic currents and increased the paired-pulse ratio, indicating that adenosine acts presynaptically to reduce glutamate release from primary afferents. The adenosine-induced inhibition of excitatory postsynaptic currents was occluded by a selective A? receptor antagonist, DPCPX, and was mimicked by a selective A? receptor agonist CPA. The results suggest that presynaptic A? receptors decrease action potential-dependent glutamate release from trigeminal primary afferents onto medullary dorsal horn neurons, and thus adenosine A? receptors could be a potential target for the treatment of pain of orofacial tissues.     

Neuropharmacological actions of kynurenic and quinolinic acids on horizontal cells of the isolated fish retina

We have studied the effects of naturally occurring metabolites of tryptophan, kynurenic and quinolinic acids, on the electrophysiological responses of retinal horizontal cells in the fish (Rutilus rutilus, the roach). Quinolinic acid usually hyperpolarizes the cells and reduces their light evoked responses (S-potentials) but on occasion, it causes a slight depolarization of the membrane potential. These actions are similar to those found with N-methyl-d-aspartate (NMDA) and our results are consistent with the proposal that it acts at NMDA binding sites. Kynurenic acid (1mM) invariably hyperpolarizes horizontal cells to their potassium Nernst equilibrium potential and, more significantly, blocks the depolarizing actions exerted on them by excitatory amino acids, such as kainic and quisqualic acids. We show that this action persists in the presence of the synaptic blocker, cobalt chloride, and thus is not mediated by chemical synaptic activity. Kynurenic acid does not reverse depolarization of horizontal cells by dopamine or γ-aminobutyric acid, thus its inhibitory effects are selective to the actions of excitatory amino acids. Neither xanthurenic acid, a close structural analogue of kynurenic acid, nor quinolinic acid are effective in blocking depolarizations by excitatory aino acids.     

Excitotoxicity in the embryonic chick spinal cord.

Recent evidence implicates excitatory amino acids (EAAs), acting as excitotoxic agents, in the pathogenesis of neurological disorders involving the spinal cord. In this study, we used the chick embryo spinal cord as an in vitro model for studying the sensitivity of spinal neurons to the excitotoxic effects of EAA agonists. Compounds tested include the prototypic receptor-specific agonists, N-methyl-D-aspartate (NMDA), quisqualic acid (Quis), and kainic acid (KA), and the plant-derived excitotoxic food poisons, beta-N-oxalylamino-L-alanine, beta-N-methylamino-L-alanine, and domoic acid. Each agonist induced concentration-dependent acute degeneration of neurons distributed throughout the spinal cord. These cytopathological changes consisted of acute edematous degeneration of dendrosomal structures in the dorsal horn and intermediate zone, and dark cell changes with intracytoplasmic vacuolization of motor neurons; this damage is identical to that induced by excitotoxin agonists in other regions of the central nervous system. The NMDA receptor-specific antagonist MK-801 completely blocked toxicity of NMDA, and the nonNMDA antagonist CNQX preferentially blocked the toxicity of Quis- and KA-type agonists in the spinal cord. Our findings suggest that (1) the majority of spinal neurons have all three subtypes of EAA receptors, making them acutely vulnerable to excitotoxin exposure; and (2) EAA antagonists are effective in preventing excitotoxin-induced damage of the spinal cord.     

Excitatory amino acids and synaptic transmission in embryonic rat brainstem motoneurons in organotypic culture

We used brainstem motoneurons recorded in organotypic slice co-cultures maintained for more than 18 days in vitro, together with multibarrel ionophoretic applications of glutamate receptor agonists and bath applications of specific blocking agents, to study the responses of rat brainstem motoneurons to glutamate receptor activation, and the contribution of these receptors to synaptic transmission. Differentiated brainstem motoneurons in vitro are depolarized by glutamate, N-methyl-d-aspartate (NMDA) and dl-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) iontophoresis, and express NMDA, AMPA and also specific kainate receptors, as evidenced by (+/-)2-amino-5-phosphonovaleric acid (APV)- and (-)1-(4-aminophenyl)-3-methyl-carbamoyl-4-methyl-7, 8-methylenedioxy-3,4-dihydro-5H-2,3-benzo-diazepine [GYKI 53784 (LY303070)]-resistant depolarizations. Electrical stimulations applied to the dorsal part of the explant trigger excitatory synaptic potentials with latencies distributed in three regularly spaced groups. Excitatory postsynaptic potentials (EPSPs) in the earliest group have a similar latency and time course and correspond to monosynaptic activation. EPSPs in later groups have more scattered latencies and time courses and correspond to polysynaptic activation. Monosynaptic EPSPs are insensitive to the specific NMDA blocker APV, and are completely and reversibly suppressed by the non-competitive AMPA receptor antagonist GYKI 53784 (LY303070). Detailed analysis of the spontaneous excitatory synaptic activity shows that APV decreases the frequency of spontaneous EPSPs without modifying their shape or amplitude. We conclude that excitatory synapses on brainstem motoneurons in vitro are mainly activated through AMPA receptors (AMPA-Rs). NMDA receptors (NMDA-Rs) are present in the membrane, but are located either at extrasynaptic sites or silent synapses, and are not directly involved in synaptic transmission on motoneurons. On the contrary, NMDA receptors contribute to synaptic transmission within the premotor interneuronal network.     

The effects of excitatory amino acids on intracellular calcium in single mouse striatal neurons in vitro

Using microspectrofluorimetry and the calcium-sensitive dye fura-2, we examined the effect of excitatory amino acids on [Ca2+]i in single striatal neurons in vitro. N-methyl-D-aspartic acid (NMDA) produced rapid increases in [Ca2+]i. These were blocked by DL-2-amino-5-phosphonovaleric acid (AP5), by Mg2+, by phencyclidine, and by MK801. The block produced by Mg2+ and MK801 could be relieved by depolarizing cells with veratridine. When external Ca2+ was removed, NMDA no longer increased [Ca2+]i. Furthermore, the effects of NMDA were not blocked by concentrations of La3+ that blocked depolarization induced rises in [Ca2+]i. Substitution of Na+o by Li+ did not block the effects of NMDA. Concentrations of L-glutamate greater than or equal to 10(-6) M also increased [Ca2+]i. The effects of moderate concentrations of glutamate were blocked by AP5 but not by La3+ or by substitution of Na+ by Li+. The effects of glutamate were blocked by removal of external Ca2+ but were not blocked by concentrations of Mg2+ or MK801 that completely blocked the effects of NMDA. The glutamate analogs kainic acid (KA) and quisqualic acid also increased [Ca2+]i. The effects of KA were blocked by removal of external Ca2+ but not by La3+, Mg2+, MK801, or replacement of Na+ by Li+. Although AP5 was able to block the effects of KA partially, very high concentrations were required. These results may be explained by considering the properties of glutamate-receptor-linked ionophores. Excitatory amino acid induced increases in [Ca2+]i are consistent with the possibility that Ca2+ mediates excitatory amino acid induced neuronal degeneration.     

Unmyelinated cutaneous afferent neurons activate two types of excitatory amino acid receptor in the spinal cord of Xenopus laevis embryos

The trunk and tail skin of Xenopus laevis embryos near the time of hatching is innervated by the mechanoreceptive free nerve endings of Rohon-Beard neurons, a homogeneous class of cutaneous primary afferent fibers. Rohon-Beard neurons have cell bodies and axons in the dorsal spinal cord, where they monosynaptically excite a population of dorsolaterally situated interneurons (Clarke and Roberts, 1984). EPSPs can be recorded in these dorsolateral interneurons following electrical stimulation of the unmyelinated neurites of Rohon-Beard neurons in the skin. The EPSPs are dual component, consisting of separate fast and slow potentials that are usually evoked synchronously and that closely resemble those described previously in Xenopus and lamprey motoneurons (Dale and Roberts, 1985; Dale and Grillner, 1986). The excitation of dorsolateral interneurons by Rohon-Beard neurons is reduced by the bath application of excitatory amino acid antagonists. Kynurenic acid suppresses both the fast and slow components of the EPSPs, while both (+/-)-2-amino-5-phosphonovaleric acid (APV) and 1 mM magnesium reduce the slow component but have little or no effect on the peak amplitude of the EPSPs. These data suggest that Rohon-Beard neurons release an excitatory amino acid neurotransmitter, which acts simultaneously at both N-methyl-D-aspartate (NMDA) and non-NMDA receptor types. This is the first direct demonstration of dual-component excitatory amino acid-mediated synaptic transmission from cutaneous primary afferent neurons in the vertebrate spinal cord. The bath application of the agonists NMDA, kainate, or quisqualate in salines containing 1 microM TTX depolarized the interneurons and reduced their input resistance, which suggests that the interneurons possess all 3 types of excitatory amino acid receptor. Kynurenic acid strongly inhibits responses to NMDA and kainate, but is relatively less effective against the larger responses of quisqualate in this system.     

NMDA receptor 2B subunit-mediated synaptic transmission in the superficial dorsal horn of peripheral nerve-injured neuropathic mice

Previous research has shown that peripheral inflammation and peripheral nerve injury alter the properties of NMDA receptors in the spinal dorsal horn. However, there is no direct evidence that demonstrates the influence of peripheral nerve injury on NMDA receptor-mediated synaptic transmission in the spinal dorsal horn. Using whole cell tight-seal methods, NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) were recorded from superficial dorsal horn neurons in adult mouse spinal cord slices. Peripheral nerve injury-induced changes in the pharmacological and electrophysiological properties of synaptic NMDA receptors were studied. The ratio of the amplitude of NMDA EPSCs to that of non-NMDA EPSCs was larger in nerve-ligated neuropathic mice than in sham-operated control mice. The decay phase of the NMDA EPSCs was slower in nerve-ligated neuropathic mice. The NR2B subunit-specific NMDA receptor antagonist ifenprodil (10 microM) reduced the amplitude of the NMDA EPSCs and shortened their decay phase. The sensitivity of NMDA EPSCs to ifenprodil was significantly larger in nerve-ligated neuropathic mice than in sham-operated control mice. Single-cell RT-PCR analysis performed on superficial dorsal horn neurons showed that the incidence of NR2A mRNA-expressing neurons was reduced in nerve-ligated neuropathic mice. This result, together with the electrophysiological findings, suggests that the subunit composition of the subsynaptic NMDA receptors in the superficial dorsal horn was altered by peripheral nerve injury. Pharmacological and electrophysiological changes observed in the present experiments might be the underlying causes of the hyperalgesia and allodynia induced by peripheral nerve injury and inflammation.     

Spinal dorsal horn neurons in elevated extracellular calcium: cell properties and spontaneous discharges

Recordings were made from neurons in the dorsal horn (DH), and from dorsal and ventral roots (DRs and VRs) of isolated spinal cords of infant mice. Raising calcium concentration ([Ca2+]) in the organ bath from 1.2 to 2.4 mmol/l resulted in a slight hyperpolarization, elevation of threshold current (rheobase), and augmentation of excitatory postsynaptic potentials (EPSPs). In many cells EPSPs acquired a much prolonged late phase. Orthodromic stimulation evoked in some DH neurons an action potential that had the same threshold as, and coincided in time with, the 'dorsal horn response' (DHR) recorded from DR. In spinal cords bathed in elevated [Ca2+], DR recordings showed irregularly recurring spontaneous waves, and DH neurons generated spontaneous EPSPs, often with spikes. Some neurons fired irregularly timed spontaneous action potentials that did not appear triggered by EPSPs. In less than 50% of the neurons the spontaneous EPSPs coincided in time with the spontaneous DR waves. The action potentials that appeared without EPSP were fired independently from DR activity. These observations confirm that elevation of interstitial free calcium concentration results in strong enhancement of excitatory transmission, especially of an EPSP of much extended duration. Virtually all neurons showed increased spontaneous activity in high [Ca2+], but only a minority appeared recruited into the synchronized discharges that are detectable as spontaneous waves in DR and VR recordings.