Synaptic Excitation Triggers Oscillations During NMDA Receptor Activation in Rat Abducens Motoneurons

Rat abducens motoneurons were intracellularly recorded in vivo during synaptic excitation and extracellular microionophoretic application of N -methyl- d -aspartate (NMDA). Trigeminal excitatory post-synaptic potentials (EPSPs) evoked in abducens motoneurons were studied during intracellular current injection. They were not sensitive to hyperpolarization or depolarization of the membrane potential in the range of –75 mV to –55 mV using current pulse intensities between –3 nA and + 1 nA. Microionophoretic applications of aminophosphonovalerate (APV), MK801 and i.v. injections of MK801 (1 – 3 mg/kg) or ketamine (10 mg/kg) did not modify trigeminal EPSPs, suggesting that NMDA receptors are not involved in this synaptic transmission. However, microionophoretic applications of NMDA on abducens motoneurons enhanced trigeminal EPSPs and gave rise to regenerative oscillations. The co-activation of NMDA receptors and trigeminal synapses induced these oscillations. The trigeminal EPSP may delay and reset the oscillations depending on where it was evoked in the oscillatory cycle. Depolarizing current pulses intracellularly applied to abducens motoneurons could trigger a post-hyperpolarization followed by rebound depolarization during NMDA application, confirming the activation of active membrane properties. However, depolarizing current pulses could not trigger oscillations similar to those entrained by the EPSPs. The importance of the location of trigeminal synapses in relation to those of NMDA receptors in the dendritic arborization of abducens motoneurons is discussed. Our results show that the same sensory stimulus may have different post-synaptic effects on abducens motoneurons during the co-activation of NMDA receptors. A complete modification of the motor output during NMDA receptor activation strongly supports an active role of abducens motoneurons provided that NMDA receptors are physiologically activated during motor pattern generation.     

Evidence for the Existence of a Functional Polysynaptic Pathway From Trigeminal Afferents to Lumbar Motoneurons in the Neonatal Rat

Stimulation of trigeminal afferents has been reported to have powerful effects on the spinal cord in adult animals of several species. In the present study, the pathway transmitting these influences was investigated in the neonatal rat. Experiments were performed on in vitro brainstem/spinal cord preparations. Stimulation of the trigeminal nerve evoked bilateral polysynaptic discharges in lumbar ventral roots. Intracellular recordings from lumbar motoneurons showed mainly excitatory responses, although a few inhibitory responses were also observed. Experiments with perfusion of different parts of the preparation with general or selective synaptic blockers revealed a synaptic relay under GABAergic control in the brainstem, and at least one synapse in the cervical and in the thoracic spinal cord. The involvement of lumbar interneurons was established by perfusing the lumbar enlargement with saline containing either a high concentration of divalent ions or mephenesin in order to reduce transmission along polysynaptic pathways. The contribution of excitatory amino acid transmission was evaluated and was found to evoke mixed receptor responses. The course of the pathway was traced by using different lesions to the brainstem and spinal cord. The pathway was found to be ipsilateral in the brainstem and to become bilateral in the spinal cord. The results of the present study demonstrate that polysynaptic sensorimotor pathways are present at birth. The results are discussed in relation to the pontomedullary locomotor strip, which has been thought to share many features with the trigeminal system.     

Expression of NMDA Receptor mRNAs in Rat Motoneurons is Down-regulated after Axotomy

The cytotoxic effects of glutamate via the N -methyl-D-aspartate (NMDA) receptor have been suggested to take part in the events leading to death of motoneurons after neonatal axotomy. By the use of in situ hybridization and immunohistochemistry we have investigated motoneuron mRNA expression of the NMDA receptor subunits NR1, NR2B and NR2D and of the NR1 subunit protein in two lesion models leading to partial motoneuron death: sciatic nerve transection early postnatally in the rat and ventral root avulsion in the adult rat. The results were compared with a lesion model with no subsequent death of motoneurons, i.e. sciatic nerve transection in the adult rat. All lesions were followed by down-regulation of the mRNAs for all studied subunits in severed motoneuron populations; down-regulation was detectable already at early stages postoperatively before any significant death had taken place. The strongest down-regulation was in fact seen in the lesion with the largest loss of motoneurons (ventral root avulsion). The reduction in the expression of NR1 mRNA was paralleled by a decrease in NR1 subunit protein. We conclude that down-regulation of NMDA receptor subunit expression is part of the acute response to axonal injury in motoneurons, whether or not neuronal death follows, and that the susceptibility of lesioned motoneurons to excitotoxic effects should be highest early after axonal injury.     

Modulation of GABA-mediated Synaptic Potentials by Glutamatergic Agonists in Neonatal CA3 Rat Hippocampal Neurons

Intracellular recordings were made from slices of adult and neonatal hippocampal neurons. During the first 2 weeks of life the majority of pyramidal cells exhibited spontaneous gamma-aminobutyric acid (GABA)-mediated synaptic potentials, which were depolarizing at birth and became hyperpolarizing by the end of the first postnatal week. These synaptic potentials were reduced in frequency or blocked by the N-methyl-d-aspartate (NMDA) receptor antagonist d(-)2-amino-5-phosphonovalerate (AP-5, 50 microM) (13/15 cells). The non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 - 10 microM) abolished the GABA-mediated synaptic potentials in all the cells tested (n=12), Superfusion of l-glutamate (up to 100 microM) increased the frequency of both depolarizing and hyperpolarizing GABA-mediated synaptic potentials. This effect was reduced by AP-5 or dl-2-amino-7-phosphonoheptanoate (AP-7, 50 microM) and fully blocked by concomitant application of AP-5 (50 microM) and CNQX (5 - 10 microM). NMDA (0.5 - 2 microM) increased the frequency of the GABA-mediated synaptic potentials. These effects were blocked by AP-5 (50 microM) and by bicuculline (10 microM). Quisqualate (100 - 300 nM), (RS)-alpha-amino-3-hydroxy-5-methyl-4-izopropionate (AMPA, 100 - 300 nM) and kainate (100 nM) also increased the frequency of the GABA-mediated synaptic potentials. These effects were blocked by CNQX (5 - 10 microM) and by bicuculline (10 microM) but not by AP-5 (50 microM). In the presence of tetrodotoxin (TTX, 1 microM), quisqualate (up to 300 nM), AMPA (up to 500 nM) and kainate (100 nM) had no effect on membrane potential or input resistance. In conclusion, our experiments suggest that, in early postnatal life, NMDA and non-NMDA receptors located on GABAergic interneurons modulate GABAergic synaptic potentials.     

MK 801, an OBS N-methyl-D-aspartate channel blocker, does not improve the functional recovery nor spinal cord blood flow after spinal cord compression in rats

Damage to the central nervous system is followed by local release of excitatory amino acids, e.g. glutamate. These have been claimed to increase the metabolic need of already hypoxic neurons, and thereby to promote cell death. To investigate whether N-methyl-D-aspartate (NMDA) receptor-mediated mechanisms are involved in the damage consequent to spinal cord injury, 20 rats were exposed to 5-min compression of the thoracic spinal cord produced with a load of 35 g on a 2.2 x 5 mm sized plate. One group of animals was given a noncompetitive NMDA channel blocker, MK-801, in a dose of 10 mg/kg b.w and one group saline alone. The neurologic function was evaluated on the inclined plane for 4 days when spinal cord blood flow (SCBF) was measured with the 14C-iodoantipyrine autoradiographic technique. One day after trauma the animals in both groups were paraparetic and exhibited a significantly decreased capacity angle at the inclined plane test (about 35 degrees compared with about 63 degrees before compression). Thereafter, the motor function improved slightly, but to a similar extent in the two groups. On Day 4, gray and white matter SCBF was similar in the two groups. The results indicate that MK 801 in the dose used does not prevent the development of neurologic dysfunction or the reduction in SCBF after spinal cord compression.     

Spinal Cord Injury Alters Spinal Shox2 Interneurons by Enhancing Excitatory Synaptic Input and Serotonergic Modulation While Maintaining Intrinsic Properties in Mouse

Neural circuitry generating locomotor rhythm and pattern is located in the spinal cord. Most spinal cord injuries (SCIs) occur above the level of spinal locomotor neurons; therefore, these circuits are a target for improving motor function after SCI. Despite being relatively intact below the injury, locomotor circuitry undergoes substantial plasticity with the loss of descending control. Information regarding cell type-specific plasticity within locomotor circuits is limited. Shox2 interneurons (INs) have been linked to locomotor rhythm generation and patterning, making them a potential therapeutic target for the restoration of locomotion after SCI. The goal of the present study was to identify SCI-induced plasticity at the level of Shox2 INs in a complete thoracic transection model in adult male and female mice. Whole-cell patch-clamp recordings of Shox2 INs revealed minimal changes in intrinsic excitability properties after SCI. However, afferent stimulation resulted in mixed excitatory and inhibitory input to Shox2 INs in uninjured mice which became predominantly excitatory after SCI. Shox2 INs were differentially modulated by serotonin (5-HT) in a concentration-dependent manner in uninjured conditions but following SCI, 5-HT predominantly depolarized Shox2 INs. 5-HT₇ receptors mediated excitatory effects on Shox2 INs from both uninjured and SCI mice, but activation of 5-HT_2B/2C receptors enhanced excitability of Shox2 INs only after SCI. Overall, SCI alters sensory afferent input pathways to Shox2 INs and 5-HT modulation of Shox2 INs to enhance excitatory responses. Our findings provide relevant information regarding the locomotor circuitry response to SCI that could benefit strategies to improve locomotion after SCI.SIGNIFICANCE STATEMENT Current therapies to gain locomotor control after spinal cord injury (SCI) target spinal locomotor circuitry. Improvements in therapeutic strategies will require a better understanding of the SCI-induced plasticity within specific locomotor elements and their controllers, including sensory afferents and serotonergic modulation. Here, we demonstrate that excitability and intrinsic properties of Shox2 interneurons, which contribute to the generation of the locomotor rhythm and pattering, remain intact after SCI. However, SCI induces plasticity in both sensory afferent pathways and serotonergic modulation, enhancing the activation and excitation of Shox2 interneurons. Our findings will impact future strategies looking to harness these changes with the ultimate goal of restoring functional locomotion after SCI.     

Roles of Glycinergic Inhibition and N-Methyl-D-Aspartate Receptor Mediated Excitation in the Locomotor Rhythmicity of One Half of the Xenopus Embryo Central Nervous System

Recent investigations into the neural basis for swimming in Xenopus embryos have pointed to central roles played by N-methyl-D-aspartate (NMDA) receptor-mediated excitation acting within and glycinergic reciprocal inhibition acting between motor systems for the muscular antagonists on the two sides of the CNS. A 'reduced' preparation consisting of only one half of the CNS divided sagittally along its midline is used here to examine the basis for rhythmicity within each side in the absence of reciprocal connections. Divided preparations transected rostrally at levels between the otic capsule and the obex can all generate a rhythmic pattern of motor discharge similar to that which underlies swimming. All rhythm generation is blocked by the NMDA antagonist (+/-)-2-amino-5-phosphonovaleric acid (AP5) at 20 microM. However, neither glycinergic nor GABAergic inhibition is required for a basic rhythmicity since some rhythm persists in the presence of 10 microM strychnine and 50 microM bicuculline, though it is no longer sustained. In the divided spinal cord alone, rhythm generation requires extracellular Mg2+. If the most caudal segment of the divided hindbrain is left attached, extracellular Mg2+ is required only if strychnine is present. If more of the hindbrain is included, extracellular Mg2+ is no longer necessary for rhythm generation even in the presence of strychnine. It seems that rhythm generation by a single side of the spinal cord requires NMDA receptor-mediated excitation together with the voltage dependency conferred on it by extracellular Mg2+, but not inhibition in order to occur. As more of the hindbrain is left attached, the requirement for extracellular Mg2+ becomes progressively less strong. For sustained rhythm generation, one side of the CNS requires both excitation and glycinergic inhibition.     

Microinfusion of the Metabotropic Glutamate Receptor Agonist 1S, 3R–1-Aminocyclopentane-1, 3-dicarboxylic Acid Into the Nucleus Accumbens Induces Dopamine-dependent Locomotor Activation in the Rat

Although the striatum has one of the highest densities of metabotropic glutamate receptor (mGluR) binding sites in the brain, little is known about their physiological role. In this study we characterized the contribution of mGluRs located in the ventral part of the striatum (the nucleus accumbens) to the control of extrapyramidal motor function. Activation of mGluRs by local infusion of the selective agonist 1S, 3R–1-aminocyclopentane-1, 3-dicarboxylic acid (1 S, 3R-ACPD; 25, 50 and 100 nmol/0.5 μl) into the nucleus accumbens induced a dose-dependent increase in locomotor activity in rats. Intra-accumbens infusion of a selective antagonist of mGluRs, a-methyl-4-carboxyphenylglycine (MCPG) did not modify spontaneous locomotion but decreased the locomotor response to 1S, 3R-ACPD. This effect appeared to be mediated by dopamine, since blockade of dopamine receptors with haloperidol (0.05 and 0.1 mg/kg i.p.) dose-dependently reduced 1 S3R-ACPD-induced locomotor activation. Furthermore, D-amphetamine (0.5 mg/kg, i.p.) combined with intra-accumbens infusion of 1S, 3R-ACPD (100 nmol) potentiated the locomotor hyperactivity response to a higher level than that seen with a single treatment with either drug. In contrast, D-amphetamine-induced hypermotility was abolished by infusion of MCPG (100 nmol) into the nucleus accumbens. These results demonstrate that glutamate may control extrapyramidal motor function through metabotropic receptors. Furthermore, activation of metabotropic glutamate receptors appears to act in synergy with the dopamine system at the level of the nucleus accumbens to produce a motor stimulant response.     

Galanin Receptor Binding Sites in Adult Rat Spinal Cord Respond Differentially to Neonatal Capsaicin, Dorsal Rhizotomy and Peripheral Axotomy

The discrete distribution and possible changes in specific [¹²⁵I]galanin binding sites were evaluated in the rat spinal cord following neonatal capsaicin treatment, dorsal rhizotomy and sciatic nerve section. The highest density of [¹²⁵I]galanin binding sites in the normal rat spinal cord was particularly evident in the superficial layers of the dorsal horn whereas moderate to low amounts of labelling were associated with the deeper dorsal horn, areas around the central canal and the ventral horn. Capsaicin-treated rats, compared to littermate controls, showed a significant bilateral increase in [¹²⁵I]galanin binding in the superficial laminae of the dorsal horn. Similarly, unilateral dorsal rhizotomy evoked a significant increase in the density of [¹²⁵I]galanin binding sites in the superficial dorsal horn ipsilateral to surgery. Section of the sciatic nerve, on the other hand, induced a significant depletion in [¹²⁵I]galanin binding in laminae I and II of the ipsilateral dorsal horn. These results, in parallel to those reported for galanin immunoreactivity under similar conditions, suggest that [¹²⁵I]galanin binding sites are preferentially located postsynaptically to the primary afferent fibre terminals in the dorsal horn of the spinal cord. Thus it seems that galanin, at the level of the dorsal spinal cord, regulates the processing of nociceptive information by acting on its own class of specific receptors located postsynaptically to primary sensory terminals.     

NMDA antagonists and potentiation of NMDA-induced motoneuron depolarizations in the isolated frog spinal cord

The action of N-methyl-D-aspartate (NMDA) antagonists on motoneurons was studied in the isolated, hemisected frog spinal cord using sucrose gap techniques. NMDA-evoked motoneuron depolarizations were depressed by application of APV, APH, kynurenate, Mg2+ ions, ketamine, and MK-801. Upon returning to normal Ringer's solution after exposure to all antagonists (except MK-801). NMDA responses were significantly potentiated. Kainate- and quisqualate-induced depolarizations were unchanged. The facilitation appeared to result, at least in part, from a direct action on motoneuron membranes since it persisted in the presence of tetrodotoxin which eliminated interneuronal firing. However, indirect actions involving interneurons also contributed to the potentiation because NMDA-evoked changes in K+ release were increased following exposure to NMDA antagonists and return to normal medium. Reduction of temperature (7 degrees C) which should reduce amino acid uptake did not affect results with APV. In addition, desensitization of NMDA responses was not altered by application of APV. The results indicate that NMDA antagonists have complex and long-lasting effects on the function of the NMDA receptor complex.     

Colocalization of fixative-modified glutamate and glutaminase but not GAD in rubrospinal neurons

In an attempt to identify putative neurotransmitters of rubrospinal neurons, immunocytochemical procedures were utilized in combination with retrograde tracing techniques in 15 adult male rats. Following injections of horseradish peroxidase (HRP) or wheat germ agglutinin conjugated to HRP (WGA-HRP) into the spinal cord, midbrain sections were processed with a combined procedure that allowed visualization of both the retrograde tracer and one or more antigens including glutamate, glutaminase, and glutamatic acid decarboxylase (GAD). Initial colocalization studies demonstrated that glutamatelike and glutaminaselike immunoreactivities were cocontained within the same neurons. Following injections of HRP or WGA-HRP into the spinal cord approximately 53% of retrogradely labeled neurons contained glutamate immunoreactivity. Triple-labeling experiments indicated that glutamatelike immunoreactivity was colocalized with glutaminase immunoreactivity in retrogradely labeled rubrospinal neurons. Retrogradely labeled neurons did not contain GAD immunoreactivity. Moreover, triple labeling experiments verified that glutamatelike immunoreactive retrogradely labeled cells did not cocontain GAD immunoreactivity. These studies demonstrate that glutamate and its synthesizing enzyme, glutaminase, are present in some rubrospinal neurons and raise the possibility that a component of the rubrospinal projection may be glutamatergic. GAD, on the other hand, is not present in rubrospinal neurons. This finding supports the hypothesis that GABAergic neurons play a role as interneurons in the red nucleus.     

Pre- and Post-synaptic Actions of 5-Hydroxytryptamine in the Rat Lumbar Dorsal Horn In Vitro: Implications for Somatosensory Transmission

Since the relative contribution of pre- versus post-synaptic actions of 5-hydroxytryptamine (5-HT) to modulation of somatosensory processing in the dorsal horn is not known, recordings fro m primary afferents and dorsal horn neurons from in vitro rat spinal cord were used to address this issue. 5-HT produced a depression of spontaneous dorsal root potentials and a slow primary afferent depolarization (PAD): the PAD versus 5-HT concentration-response curve was bell shaped (maximum at 5 μM; 250±C 41.5 μV). In 28/40 dorsal horn neurons, 5-HT elicited a slow depolarization not clearly associated with a specific input resistance change. Excitatory synaptic transmission from primary afferents to dorsal horn neurons was depressed by 5-HT in 40/45 neurons. 5-HT ≥ 5 μM significantly ( P ≤ 0.05) decreased the amplitude, shortened the total duration and half-decay time of the excitatory post-synaptic potential (EPSP). A dominant effect of 5-HT on longer latency EPSP components was evident. There was no direct relationship between the magnitude of PAD and the reduction of the EPSP by 5-HT. 5-Carboxamidotryptamine, an agonist for 5-HT¹ receptors, mimicked the depression of neurotransmission in the dorsal horn without producing PAD. A sample of dorsal horn neurons ( n = 8) was injected with biocytin and their morphology described. All had somata within laminae III-VI. In five of these neurons 5-HT depressed the EPSP but in one interneuron-like and one unclassed neuron the EPSP was potentiated. These data suggest that whilst depression of synaptic transmission is the predominant effect of 5-HT in the deep dorsal horn, this is not easily related to PAD or cellular actions of 5-HT on dorsal horn neurons.     

Cellular and Subcellular Distribution of NMDAR1 Splice Variant mRNA in the Rat Lumbar Spinal Cord

The regional distribution of alternatively spliced messenger RNA encoding the N -methyl-D-aspartate (NMDA) receptor R1 subunit (NMDAR1) variants was examined by in situ hybridization in the rat lumbar spinal cord. Splice-specific oligonucleotide probes [recognizing full-length mRNA (NMDAR1-1), deletion exon 21 (NMDAR1-2), deletion exon 22 (NMDAR1-3), combined deletion exons 21 and 22 (NMDAR1-4) and mRNA which lacks (NMDAR1-a) or contains exon 5 (NMDAR1-b)] detected marked differences in abundance and distribution of N- and C-terminal spliced variants. The NMDAR1-a, NMDAR1-2 and NMDAR1-4 mRNAs were evenly distributed throughout all laminae of the dorsal and ventral horns. In the superficial dorsal horn NMDAR1-b mRNA was preferentially detected in laminae II inner and III, while NMDAR1-1 mRNA was restricted to laminae I to III. Large neurons in laminae IV and V contained mainly NMDAR1-a, NMDAR1-2 and NMDAR1-4 mRNAs and occasionally NMDAR1-b. The NMDAR1-3 variant was only detected in very low abundance, being restricted to occasional cells in lamina I and II. In the ventral horn, motor neurons showed strong signals for NMDAR1-a, NMDAR1-b, NMDAR1-2 and NMDAR1-4 mRNAs. Serial sectioning through large motor neurons permitted the detection of multiple splice variants in single neurons. Analysis of the subcellular distribution of the mRNAs revealed that the NMDAR1-1 mRNA was almost exclusively found in the cell nucleus, NMDAR1-a mRNA was largely in the cytoplasm, while all other splice variants showed a homogeneous distribution between nucleus and cytoplasm. Comparison of the in situ hybridization images with functional analyses of heteromeric recombinant receptors will be necessary to ascertain whether splice variants of the NMDAR1 receptor subunit can account for some of the known electrophysiological properties of spinal cord neurons under physiological and pathophysiological conditions.     

GAP-43, aFGF, CCK and α- and β-CGRP in Rat Spinal Motoneurons Subjected to Axotomy and/or Dorsal Root Severance

The mRNA levels for growth-associated protein 43 (GAP-43), acidic fibroblast growth factor (aFGF), α and β-calcitonin gene-related peptide (CGRP), cholecystokinin (CCK) and choline acetyltransferase (ChAT) in rat lumbar spinal motoneurons were studied by in situ hybridization 1, 5 and 21 days and 20 weeks following unilateral peripheral nerve sectioning, ventral rhizotomy or dorsal rhizotomy. Furthermore, CGRP- and aFGF-like immunoreactivities in the ventral horn were studied using immunohistochemistry. One to 21 days after axotomy, GAP-43 and α-CGRP mRNAs increased in lesioned motoneurons, while the aFGF mRNA levels were marginally higher in motoneurons on the lesion side as compared to the control side. β-CGRP, CCK and ChAT mRNA levels, on the other hand, decreased during the short-term response (1 – 21 days) to axotomy. After ventral rhizotomy, but not peripheral axotomy, there was complete disappearance of aFGF-like immunoreactivity in the ventral root proximal to the lesion. In animals subjected to long-term survival (20 weeks) after peripheral axotomy, the expression of all studied substances had returned to normal levels. Unilateral dorsal rhizotomy did not induce any substantial short- or long-term shifts in the cellular expression of the GAP-43, aFGF, CGRP and CCK peptides or their mRNAs in motoneurons of lesioned segments. These results indicate that peptides/proteins in motoneurons are expressed differentially after axotomy. Whereas α-CGRP and GAP-43 are up-regulated, CCK and β-CGRP become down-regulated and aFGF is largely unaffected.     

CPP, a new potent and selective NMDA antagonist. Depression of central neuron responses, affinity for [H]d-AP5 binding sites on brain membranes and anticonvulsant activity

Properties of a new potent antagonist acting selectively at N-methyl-D-aspartate (NMDA) type excitatory amino acid receptors are described. This compound, 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) is more potent than all previously reported NMDA antagonists in depressing mammalian spinal neuronal responses (cat and immature rat), in its affinity for [3H]D-AP5 (a radiolabelled NMDA antagonist) binding sites on rat brain membranes, and as an anticonvulsant in mice.     

The role of putative excitatory amino acid neurotransmitters in the initiation of locomotion in the lamprey spinal cord. I. The effects of excitatory amino acid antagonists

The activation of N-methyl-D-aspartate (NMDA) and kainate receptors will evoke fictive locomotion in the appropriate motor pattern for locomotion in the isolated lamprey spinal cord, but not a selective activation of quisqualate receptors. The present experiments test whether the initiation of locomotion in response to sensory stimulation depends on these types of receptors. An in vitro preparation of the lamprey spinal cord with part of its tailfin left innervated has been used. In this preparation a sequence of fictive locomotion (i.e. alternating bursts in the segmental ventral roots with a rostrocaudal phase lag) can be elicited by continual sensory stimulation of the tailfin. The effects of excitatory amino acid antagonists were studied by recordings from ventral roots (extracellularly) and motoneurones (intracellularly). It was found that the strong initial bursts of each swimming sequence induced by sensory stimulation were depressed by combined NMDA/kainate antagonists (cis-2,3-piperidine dicarboxylate (PDA) and gamma-D-glutamylglycine (gamma-DGG] whereas the less intense burst activity, occurring particularly towards the end of each swimming sequence, was depressed by a selective NMDA antagonist, 2-amino-5-phosphonovalerate (2-APV). This condition could be mimicked in an isolated spinal cord preparation by an application of L-glutamate; the low-level fictive locomotion induced by low doses of L-Glu (less than 100 microM) was depressed by a NMDA antagonist (2-APV), and, if higher doses were applied, the activity was only depressed by PDA/gamma-DGG. The mode and time course of the depression (by excitatory amino acid antagonists) of fictive locomotion, induced by sensory stimulation, shows that the putative excitatory amino acid neurotransmitter directly or indirectly acts at the pattern generating circuitry within the spinal cord.     

A conformationally restricted analogue of -glutamate, the (2S,3R,4S) isomer of -α(carboxycyclopropyl)glycine, activates the NMDA-type receptor more markedly than NMDA in the isolated rat spinal cord

Depolarizing actions of 4 conformationally restricted L-glutamate analogues, (2S,3S,4S) isomer (L-CCG-I), (2S,3R,4R) isomer (L-CCG-II), (2S,3S,4R) isomer (L-CCG-III) and (2S,3R,4S) isomer (L-CCG-IV) of L-alpha-(carboxycyclopropyl)-glycine (L-CCG), were investigated in the isolated rat spinal cord by extracellular recordings of potential changes of motoneurones from the ventral roots, in order to study the interaction between the conformation of glutamate and its receptor subtype. The order of the depolarizing activity was quisqualate greater than L-CCG-IV = kainate greater than NMDA greater than L-CCG-I greater than L-CCG-III greater than L-CCG-II. The depolarization caused by L-CCG-IV was effectively blocked by the NMDA antagonists and Mg2+ ions, while the L-CCG-I response was not affected by these blockers. These results suggest that the NMDA-type receptor is activated by a folded form of L-glutamate.