EFFECTS OF AMINO ACID ANTAGONISTS ON SPONTANEOUS DORSAL ROOT ACTIVITY AND EVOKED DORSAL HORN FIELD POTENTIALS IN AN ISOLATED PREPARATION OF RAT SPINAL CORD

Fast and slow dorsal horn field potentials and spontaneous dorsal root activity were recorded from 19–23-day-old rat isolated spinal cord preparations. The effects of GABA, glycine, and glutamate antagonists were tested on these recordings. CNQX, an AMPA/kainate antagonist, reduced all 3 components of the dorsal horn field potential whereas MK801, an NMDA ion channel antagonist, reduced the fast S2 component and the slow wave. Both reduced spontaneous dorsal root activity. NMDA antagonists, D-AP5, 7-chlorokynurenic acid and arcaine, and the metabotropic glutamate antagonists L-AP3 and ethylglutamic acid, while having little effect on the fast components of the field potential, all reduced the slow component. The GABA antagonist, bicuculline, and the glycine antagonist, strychnine, while having no effect on the fast S1 and slow components of the field potential, reduced both the fast S2 component of the field potential and spontaneous dorsal root activity. These results suggest that non-NMDA glutamate receptors are involved in low and high threshold transmission to dorsal horn neurones while NMDA and metabotropic glutamate receptors are primarily involved in high threshold transmission and both GABA and glycine have roles in the transmission or modulation of sensory information within the dorsal horn of the cord.     

Multiple effects of phorbol esters in the rat spinal dorsal horn

Spinal cord slice preparation and intracellular recording techniques were used to examine the effects of phorbol esters on the sodium- and calcium-dependent action potentials, the excitatory synaptic transmission, the basal (resting) and the dorsal root stimulation-evoked release of 9 endogenous amino acids, including glutamate and aspartate, and the responsiveness of the rat dorsal horn neurons to excitatory amino acids (glutamic, kainic, quisqualic, and N-methyl-D-aspartic). 4-beta-Phorbol-12, 13-dibutyrate and 4-beta-phorbol-12, 13-diacetate produced minor alterations in membrane potential and resistance, but they broadened the sodium-dependent action potential and reduced the duration of the calcium-dependent action potential. In addition, phorbol esters caused a marked and long-lasting increase in the amplitude and the duration of excitatory postsynaptic potentials (EPSPs) evoked in dorsal horn neurons by orthodromic stimulation of a lumbar dorsal root. Phorbol esters produced a brief increase in the basal and electrically evoked release of endogenous excitatory (glutamic, aspartic) and inhibitory amino acids (glycine, GABA). In addition, the rates of release of alanine, serine, and threonine were also elevated. In the presence of TTX, phorbol esters selectively enhanced, in a reversible manner, the depolarizing responses of dorsal horn neurons to N-methyl-D-aspartic acid and L-glutamate but not the responses to kainic or quisqualic acids. The potentiation of the NMDA response was blocked by APV, a specific NMDA receptor antagonist. Thus, phorbol esters appear to enhance excitatory synaptic transmission in the rat spinal dorsal horn slice preparation by acting both at pre- and postsynaptic sites. Phorbol esters could potentiate excitatory synaptic transmission by acting predominantly at a postsynaptic site (NMDA receptor), since the duration of the increased responsiveness of dorsal horn neurons to glutamate and NMDA correlates better with the enhancement of EPSPs than with the increased release of the stimulation-evoked glutamate and aspartate. The increased release of endogenous amino acids is consistent with a presynaptic (terminal) site of action, but it could also be explained by enhanced interneuronal activity. Although our results suggest that in the rat spinal dorsal horn protein kinase C may have a role in controlling the release of putative excitatory and inhibitory neurotransmitters and may also be involved in the regulation of postsynaptic NMDA receptors, the identity of endogenous substance(s) participating in these effects is presently unknown.     

Role of kainate receptors in nociception

Nociceptive nerve fibers use -glutamate as a fast excitatory neurotransmitter and it is therefore not surprising that both, ionotropic and metabotropic glutamate receptors play pivotal roles for transmission of nociceptive information in spinal cord. A subtype of ionotropic glutamate receptors, the kainate receptor, is present in spinal dorsal horn. However, its role has remained obscure as specific antagonists and agonists have become available only recently. Kainate receptors are present on small, including nociceptive, dorsal root ganglion cells and on intrinsic dorsal horn neurons, and those two locations can be targeted separately by appropriate agonists and antagonists. Postsynaptic kainate receptors on spinal dorsal horn neurons are activated by high intensity electrical stimulation of the dorsal root entry zone that activates nociceptive primary afferent fibers. In contrast, low intensity stimulation that activates only non-nociceptive fibers is ineffective. Selective blockade of kainate receptors may produce analgesia. Here, we review what is known about localization of kainate receptors in dorsal root ganglia and spinal dorsal horn and their physiological and pathophysiological importance with special reference to nociceptive pathways. A short overview on molecular biology and agonist and antagonist pharmacology is included.     

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).     

Comparison of primary afferent and glutamate excitation of neurons in the mammalian spinal dorsal horn

The actions of L-glutamate and agonists, agents blocking their membrane receptors and dorsal root afferent volleys, were compared on intracellularly recorded neuronal activity in an in vitro horizontal slice preparation of the hamster spinal dorsal horn. Bath-applied L-glutamate or L-aspartate (less than or equal to 1 mM) rapidly depolarized and excited less than a third of the dorsal horn neurons sampled. Bathing solutions containing low Ca2+ eliminated synaptic transmission in the slices but failed to block the excitatory effects of L-glutamate for the majority of the neurons tested. N-Acetylaspartylglutamate had no effect on dorsal horn neurons at concentrations up to 1 mM. Neurons excited by L-glutamate were most commonly located in the superficial dorsal horn (laminae I and II). Neurons insensitive to L-glutamate were more broadly distributed, with a number being located in laminae III-V. Kynurenic acid, 2-amino-4-phosphonobutyric acid, and 2,3-piperidine dicarboxylic acid selectively antagonized rapid, short-lasting synaptic components of the dorsal cord potentials. Kynurenic acid reversibly antagonized intracellularly recorded L-glutamate-induced excitation, spontaneous synaptic potentials, and fast synaptic potentials evoked by dorsal root volleys. Compounds with strong antagonist actions at the NMDA receptor, 2-amino-5-phosphonovaleric acid and D-alpha-aminoadipic acid, were much less effective in suppressing the effects of L-glutamate or in blocking synaptic potentials. We conclude that a subset of spinal neurons directly excited by dorsal root fibers have excitatory membrane receptors activated by L-glutamate. This conclusion is consistent with the concept that L-glutamate or a substance binding to the receptors it activates is released from the central terminals of some primary afferent fibers and mediates fast synaptic transmission from them to certain spinal neurons in the dorsal horn.     

Visualizing sensory transmission between dorsal root ganglion and dorsal horn neurons in co-culture with calcium imaging

Sensory information is conveyed to the central nervous system by primary afferent neurons within dorsal root ganglia (DRG), which synapse onto neurons of the dorsal horn of the spinal cord. This synaptic connection is central to the processing of both sensory and pain stimuli. Here, we describe a model system to monitor synaptic transmission between DRG neurons and dorsal horn neurons that is compatible with high-throughput screening. This co-culture preparation comprises DRG and dorsal horn neurons and utilizes Ca(2+) imaging with the indicator dye Fura-2 to visualize synaptic transmission. Addition of capsaicin to co-cultures stimulated DRG neurons and led to activation of dorsal horn neurons as well as increased intracellular Ca(2+) concentrations. This effect was dose-dependent and absent when DRG neurons were omitted from the culture. NMDA receptors are a critical component of synapses between DRG and dorsal horn neurons as MK-801, a use-dependent non-competitive antagonist, prevented activation of dorsal horn neurons following capsaicin treatment. This model system allows for rapid and efficient analysis of noxious stimulus-evoked Ca(2+) signal transmission and provides a new approach both for investigating synaptic transmission in the spinal cord and for screening potential analgesic compounds.     

Long-range oscillatory Ca2+ waves in rat spinal dorsal horn

Synchronous activity of large populations of neurons shapes neuronal networks during development. However, re-emergence of such activity at later stages of development could severely disrupt the orderly processing of sensory information, e.g. in the spinal dorsal horn. We used Ca2+ imaging in spinal cord slices of neonatal and young rats to assess under which conditions synchronous activity occurs in dorsal horn. No spontaneous synchronous Ca2+ transients were detected. However, increasing neuronal excitability by application of 4-aminopyridine after pretreatment of the slice with blockers of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate, gamma-aminobutyric acid (GABA)(A) and glycine receptors evoked repetitive Ca2+ waves in dorsal horn. These waves spread mediolaterally with a speed of 1.0 +/- 0.1 mm/s and affected virtually every dorsal horn neuron. The Ca2+ waves were associated with large depolarizing shifts of the membrane potential of participating neurons and were most likely synaptically mediated because they were abolished by blockade of action potentials or N-methyl-D-aspartate (NMDA) receptors. They were most pronounced in the superficial dorsal horn and absent from the ventral horn. A significant proportion of the Ca2+ waves spread to the contralateral dorsal horn. This seemed to be enabled by disinhibition as primary afferent-induced dorsal horn excitation crossed the midline only when GABA(A) and glycine receptors were blocked. Interestingly, the Ca2+ waves occurred under conditions where AMPA/kainate receptors were blocked. Thus, superficial dorsal horn NMDA receptors are able to sustain synchronous neuronal excitation in the absence of functional AMPA/kainate 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.     

Substance P release in the dorsal horn assessed by receptor internalization: NMDA receptors counteract a tonic inhibition by GABA(B) receptors

Inhibitory amino acids have antinociceptive actions in the spinal cord that may involve inhibition of neurotransmitter release from primary afferents. Rat spinal cord slices with dorsal roots were used to study the effect of GABA and glycine on substance P release, assessed by the internalization of neurokinin 1 receptors. After electrical stimulation of the dorsal root at 100 Hz, about half of neurokinin 1 receptor-immunoreactive neurons in laminae I-IIo showed internalization. This internalization was inhibited by GABA (100 microM) and the GABA(B) agonist R-baclofen (10 microM), but not by the GABA(A) agonist muscimol (20 microM) or glycine (100 microM). The GABA(B) antagonist 2-hydroxysaclofen (100 microM) reversed the inhibitory effect of GABA, but not the GABA(A) antagonist bicuculline (100 microM). These findings demonstrate that GABA(B) receptors, but not GABA(A) or glycine receptors, inhibit substance P release induced by dorsal root stimulation. In contrast, R-baclofen did not inhibit the internalization produced by NMDA (100 microM), indicating that the stimulatory effect of NMDA receptors on substance P release is able to surmount the inhibitory effect of GABA(B) receptors. In the presence of the GABA(B) antagonist 2-hydroxysaclofen (100 microM), but not in its absence, stimulation of the dorsal root at 1 or 10 Hz was able to elicit internalization, which was not inhibited by the NMDA receptor antagonist AP-5 (50 microM) or the channel blocker MK-801 (10 microM). Therefore, inhibition of substance P release by GABA(B) receptors is tonic, and in its absence SP release no longer requires NMDA receptor activation.     

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.     

Excitatory and inhibitory transmission from dorsal root afferents to neonate rat motoneurons in vitro

Intracellular recordings were made from antidromically identified motoneurons in neonate (12-22 days) rat transverse spinal cord slices and the transmitters and receptors probably involved in initiating the excitatory (EPSP) and inhibitory (IPSP) postsynaptic potentials were investigated. Stimulation of dorsal roots elicited in motoneurons an EPSP, an IPSP, or an EPSP followed by an IPSP. EPSPs in 70% of motoneurons had a short latency (less than or equal to 1 ms) and in the remaining cells a latency longer than 1 ms. The IPSPs had a long latency (greater than or equal to 1 ms). Short- and long-latency EPSPs were enhanced by the acidic amino acid uptake inhibitor L-aspartic acid-beta-hydroxamate (AAH) and depressed by the non-selective glutamate receptor antagonists gamma-D-glutamylglycine (DGG) and kynurenic acid. Short-latency EPSPs were suppressed by the quisqualate/kainate (QA/KA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) but not by the N-methyl-D-aspartate (NMDA) receptor antagonists D-(-)-2-amino-5-phosphonovaleric acid (APV) and ketamine. Long-latency EPSPs were reduced by DNQX as well as by APV and ketamine. Superfusion of the slices with a Mg-free solution increased the EPSPs and unmasked a late, APV-sensitive component. The IPSP was reduced by the glycine antagonist strychnine as well as by APV and ketamine but resistant to DNQX. The results indicate that stimulation of dorsal roots elicited in motoneurons a monosynaptic EPSP mediated by glutamate/aspartate acting predominantly on the QA/KA subtype of glutamate receptors; an NMDA component can be unveiled in Mg-free solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution and depression of the GABA(B) receptor in the spinal dorsal horn of adult rat.

gamma-Aminobutyric acid (GABA) is a principal inhibitory neurotransmitter in vertebrate nervous system. The metabotropic receptor for GABA, GABA(B) receptor, is characterized as a G protein-coupled receptor subtype. In the present study, GABA(B) receptor-like immunoreactivity (GABA(B)R-LI) in the rat spinal cord and dorsal root ganglion (DRG), as well as GABA(B) receptor-mediated depression in the spinal dorsal horn were examined by using immunohistochemistry and whole-cell voltage-clamp recording technique, respectively. Under light microscope, GABA(B)R-LI was densely found in laminae I and II of the dorsal horn. DRG cells of various diameters also showed GABA(B)R-LI. Electron microscopy further revealed that GABA(B)R-LI was also localized in terminals of myelinated, unmyelinated fibers as well as the somatodendritic sites of dorsal horn neurons. Bath application of a GABA(B) receptor agonist, baclofen (10 microM, 30 s), induced a slow outward (inhibitory) current in dorsal horn neurons. This slow current was depressed when the postsynaptic G protein-coupled receptor was inhibited, indicating the postsynaptic action of baclofen. Under the condition of postsynaptic GABA(B) receptor being inhibited, baclofen (10 microM, 60 s) depressed large (Abeta) and fine (C, Adelta) afferent fiber-evoked monosynaptic excitatory postsynaptic currents, indicating presynaptic inhibition of GABA(B) receptor on elicited neurotransmitter release. Taken together, the results suggest that baclofen-sensitive GABA(B) receptor is expressed pre- and postsynaptically on primary afferent fibers and neurons in the spinal dorsal horn; activation of GABA(B) receptor in the dorsal horn postsynaptically hyperpolarizes dorsal horn neurons and presynaptically inhibits primary afferents.     

NMDA receptor-independent mechanisms responsible for the rate of rise of cumulative depolarization evoked by trains of dorsal root stimuli on rat spinal motoneurones

The mechanisms responsible for the rate of rise (RR) of cumulative depolarization induced by dorsal root stimulus trains were investigated with intracellular recordings from motoneurones of the rat isolated spinal cord. The NMDA receptor antagonists CPP or APV depressed the cumulative depolarization but not its RR which could still be fast enough to elicit action potential wind-up. RR size was correlated with a slow synaptic potential (detected in CPP or APV solution) with which it shared similar voltage dependence. The NK1 antagonist SR 140333 depressed cumulative depolarization, RR and slow synaptic potentials. It appears that the RR (and the ability to express wind-up) was determined by summation of slow synaptic potentials partly mediated via activation of NK1 receptors.     

Participation of AMPA- and NMDA-type excitatory amino acid receptors in the spinal reflex transmission,in rat

Classical in vitro and in vivo models and electrophysiological techniques were used to investigate the role of AMPA- and NMDA-type glutamate receptors in various components of spinal segmental reflex potentials. In the rat hemisected spinal cord preparation, the AMPA antagonists NBQX and GYKI 52466 abolished the monosynaptic reflex (MSR) potential but caused only partial inhibition of the motoneuronal population EPSP. NMDA antagonists had no noticeable effect on the MSR in normal medium, but markedly depressed the late part of EPSP. However, an NMDA receptor antagonist sensitive monosynaptic response was recorded in magnesium-free medium at complete blockade of the AMPA receptors. In spinalized rats, the AMPA antagonists completely blocked all components of the dorsal root stimulation evoked potential. MK-801 (2mg/kg, i.v.) reduced monosynaptic responses in a frequency dependent way, with no effect at 0.03 Hz and 22% inhibition at 0.25 Hz. The reduction of the di- and polysynaptic reflex components was about 30% and did not depend on stimulation frequency. Long-latency reflex discharge responses, especially when evoked by train stimulation, were more sensitive to MK-801 than the polysynaptic reflex.These results suggest that glutamate activates MSR pathways through AMPA receptors. However, under certain conditions, NMDA receptors can modulate this transmission through plastic changes in the underlying neuronal circuits. AMPA and NMDA receptors play comparable roles in the mediation of longer latency reflex components.     

Activation of NMDA receptors drives action potentials in superficial dorsal horn from neonatal rats

We have investigated the role of NMDA receptors in mediating synaptic transmission in spinal cord lamina II over the first 2 weeks of postnatal development. High intensity root stimulation evoked D-APV-sensitive slow synaptic activity in lamina II neurons that drove action potential firing. This NMDA receptor-mediated activity was enhanced when bicuculline and strychnine were used to block synaptic inhibition. When activated by repetitive focal stimulation, synaptic activity mediated by NMDA receptors alone drove action potential firing. NMDA receptors were also able to drive action potential firing at synapses where AMPA receptors were present but blocked. Our data show that in lamina II of the dorsal horn, NMDA receptors significantly affect neuronal excitability even in the absence of co-activation of AMPA receptors.     

The acute and chronic effects of capsaicin on slow excitatory transmission in rat dorsal horn

The acute and chronic effects of capsaicin on rat spinal dorsal horn neurons and the excitatory transmission in the dorsal horn were investigated by means of intracellular recording techniques in the spinal cord slice preparation. Bath application of capsaicin (1–2 × 10⁻⁵M) produced in a majority of cells a prolonged depolarization associated with an increase in synaptic activity and intense neuronal discharges. During and immediately following the capsaicin depolarization, repetitive stimulation of a dorsal root failed to elicit the slow depolarization.

After neonatal capsaicin treatment the proportion of dorsal horn neurons exhibiting the slow excitatory transmission was markedly reduced, however, the fast excitatory postsynaptic potentials were present in all examined cells. In addition, the proportion and sensitivity of the cells responding with a slow depolarization to substance P increased.     

Ionotropic glutamate receptors contribute to maintained neuronal hyperexcitability following spinal cord injury in rats

In this study, we examined whether topical treatment of glutamate receptor antagonists attenuate hyperexcitability of lumbar spinal dorsal horn neurons following low thoracic hemisection spinal cord injury in rats. Four weeks after spinal hemisection, neuronal activity in response to mechanical stimuli applied on the peripheral receptive field was significantly increased in three different phenotypes of lumbar spinal dorsal horn neurons: wide dynamic range (WDR), low threshold (LT) and high threshold (HT). Topical application of MK-801 (NMDA receptor antagonist, 50 µg) significantly attenuated the activity of WDR, but not LT and HT neurons; whereas, NBQX (AMPA receptor antagonist, 0.5 and 1 µg) significantly attenuated neuronal activity in all three phenotypes of neurons (*p < 0.05). However, MCPG (group I/II metabotropic glutamate receptor antagonist, 100 µg) had no effect. The present study, in the context of previous work, suggests that ionotropic glutamate receptor activation play critical roles in the maintenance of neuronal hyperexcitability and neuropathic “below-level” pain behavior following spinal hemisection injury.     

Slow excitatory transmission in rat dorsal horn: possible mediation by peptides

Repetitive stimulation of a dorsal root elicited a slow depolarization in about half of the dorsal horn neurons examined in the rat spinal cord slice preparation. The response was markedly depressed or abolished in the presence of substance P, substance P antagonists and capsaicin. In some dorsal horn neurons a slow hyperpolarization was also observed.     

In vitro sacral cord preparation and motoneuron recording from adult mice

We report the development of an intracellular recording technique for adult mouse motoneurons in sacral spinal cord. Based on a similar preparation for adult rat, we modified the cord preparation solution and filled the sharp electrode with a solution that has physiological osmolarity and pH. The viability of the preparation was examined by recording root reflexes. Short-latency reflexes mediated through monosynaptic transmission between S1 and S3 ventral root were reliably produced by dorsal root electrical stimuli and were stably recorded for more than eight hours. Long-lasting potentiation of the root reflex was observed by bath application of methoxamine, a noradrenergic alpha1 receptor agonist. Bath application of strychnine and picrotoxin, antagonists for glycine and GABA(A) receptors respectively, unmasked long-lasting reflexes that may contain polysynaptic components. In addition, on the background of strychnine and picrotoxin, adding methoxamine induced spontaneous ventral root activity. For intracellular recording, the motoneurons could be reliably penetrated and held for up to 30 min. In all 16 motoneurons recorded, resting membrane potential, input resistance, action potentials and repetitive firing were comparable to those of rat motoneurons. Thus, this preparation is viable and provides a new method for combined electrophysiological and genetic studies of the adult mouse spinal cord.     

Substance P and NMDA receptors mediate a slow nociceptive ventral root potential in neonatal rat spinal cord

Substance P and glutamate actions have separately been implicated in the generation of nociceptive-related slow ventral root potentials (slow VRPs). We report that slow VRPs are dependent on both substance P and NMDA receptor-mediated neurotransmission. Slow VRPs of 10-40 s duration were evoked by electrically stimulating a lumbar dorsal root and recorded at the corresponding ipsilateral ventral root in spinal cords isolated from 1- to 5-day-old rats; the monosynaptic reflex was also recorded. The NMDA receptor antagonist APV (5-20 microM) and the substance P antagonist spantide (10-20 microM) both reversibly depressed the slow VRP without affecting the monosynaptic reflex; spantide and APV applied together nearly abolished the slow VRP. The quisqualate-kainate receptor antagonist CNQX (1-5 microM) reduced the monosynaptic reflex and an early component of the slow VRP. A slow VRP could be elicited by brief (0.1-1.0 s) focal applications of either substance P (2-20 microM) or NMDA (10 microM), and also by CGRP (2-20 microM). Substance P-evoked and NMDA-evoked responses were blocked by their respective antagonists spantide and APV. Each was also cross-sensitive to the other antagonist. Both excitatory amino acids, acting on an NMDA receptor, and substance P, acting on a tachykinin receptor, thus appear to be involved in generating this slow potential. Both NMDA and tachykinin receptors are necessary to generate a full response.