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.     

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.     

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.     

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).     

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.     

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.     

Glutamate and metabotropic glutamate receptors associated with innervation of the uterine cervix during pregnancy: receptor antagonism inhibits c-Fos expression in rat lumbosacral spinal cord at parturition

Dorsal root ganglia (DRG) neurons connect the spinal cord and uterine cervix, and are activated at parturition with subsequent stimulation of secondary neurons in the spinal dorsal horn and autonomic areas. Neuropeptide neurotransmitters and receptors have been studied in these areas, but amino acid transmitters, e.g., glutamate, an excitatory neurotransmitter involved in sensory and nociceptive processing, have not been characterized. To determine if glutamate is involved in innervation of the cervix, rats were examined for markers of glutamatergic neurons in the L6-S1 spinal cord, DRG and cervix. Metabotropic glutamate receptors mGluR5 in the spinal dorsal horn and their expression over pregnancy were examined in pregnant rats and pregnant rats treated continuously with an antagonist of mGluR5, 2-methyl-6-(phenylethynyl) pyridine (MPEP). Rats were allowed to deliver pups to determine if the antagonist altered the expression of an early response gene protein, Fos, in the L6-S1 cord. Immunohistochemistry showed glutamate- and vesicular glutamate transporter1 (VGluT1)-positive fibers in the cervix, glutamate- and VGluT1-expressing neurons in the DRG, some of which also exhibited retrograde tracer from cervical injections, and VGluT1 and mGluR5 immunoreactivities in the L6-S1 spinal dorsal horns. Expression of mGluR5 receptors increased over pregnancy. Fos-positive neurons were present among mGluR5-immunoreactivity in the spinal dorsal horn. Parturition-induced Fos-positive neurons in the spinal cords were abundant in control rats, but were reduced by 70% in MPEP-treated animals. These results suggest that glutamate is likely involved in the transmission of sensory signals, possibly pain, from the cervix to the spinal cord at parturition.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Role of glutamate receptors in the dorsal reticular nucleus in formalin‐induced secondary allodynia

The role of glutamate receptors present in the medullary dorsal reticular nucleus (DRt) in the formalin test and formalin‐induced secondary nociception was studied in rats. Secondary mechanical allodynia was assessed with von Frey filaments applied to the rat's hindpaw, and secondary thermal hyperalgesia was evaluated with the tail‐immersion test. The selective glutamate receptor antagonists MK801 (N‐methyl‐d ‐aspartate receptor antagonist), 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) (AMPA/KA receptor antagonist) and A841720 (metabotropic glutamate 1 receptor antagonist) were injected into the DRt before or 6 days after formalin injection in the rat. In the formalin test, the three antagonists significantly reduced the number of flinches in both phases of the test. DRt microinjection of MK801 or A841720, but not of CNQX, reduced both secondary nociceptive behaviors. Moreover, pre‐treatment with the three antagonists injected into the DRt prevented the development of secondary mechanical allodynia and secondary thermal hyperalgesia. Similarly, in these rats, the number of c‐Fos‐like immunoreactive neurons were markedly reduced in both the superficial and deep lamina of the dorsal horn. Our findings support the role of DRt as a pain facilitator in acute and chronic pain states, and suggest a key role of glutamate receptors during the development and maintenance of formalin‐induced secondary allodynia.     

Lateral habenula and hippocampus: A complex interaction raphe cells-mediated

Summary The study has shown an excitatory influence exerted by lateral habenula (LH) on hippocampal pyramidal cells. The modulatory influence is paradoxically serotonine-mediated; in fact all LH stimulation effects were abolished by intrahippocampal iontophoretic methysergide application. The data suggest the involvement of dorsal raphe nucleus. In fact, the dorsal raphe nucleus stimulation caused on hippocampus an expected inhibitory effect antagonized by intrahippocampal iontophoretic methysergide application. In the context of this neural structure we have highlighted a disinhibitory relation between two types of cells: slow serotonergic efferent neurones and fast GABAergic interneurones. The disinhibitory hypothesis is also supported by the following experimental tests performed on both slow and fast raphe cells: a) LH stimulation at low and high frequencies; b) iontophoretic administration of NMDA and GABA; c) LH stimulation during intraraphe iontophoretic injection of 2-APV (NMDA antagonist) and bicuculline (GABA antagonist).     

Coexistence of NADPH diaphorase with GABA,glycine, and acetylcholine in rat spinal cord

The enzyme NADPH diaphorase is present in many spinal neurons, and is thought to correspond to nitric oxide synthase. In order to determine which types of neuron in the spinal cord contain this enzyme, we have carried out a combined enzyme histochemical and immunocytochemical study with antibodies to GABA, glycine, and choline acetyltransferase. Two hundred and twenty-four NADPH diaphorase-positive neurons in midlumbar spinal cord from four rats were tested for GABA- and glycine-like immunoreactivity. The majority of these neurons (207/224) were GABA-immunoreactive and 139 were also glycine-immunoreactive. NADPH diaphorase-positive neurons in laminae I and II generally showed both types of immunoreactivity, while those in deeper laminae of the dorsal horn and around the central canal either showed both types or else were only GABA-immunoreactive. Since GABA and acetylcholine are thought to coexist in spinal neurons, NADPH diaphorase staining was combined with immunostaining for choline acetyltransferase. Immunoreactive neurons in laminae III and IV were all NADPH diaphorase-positive, while only some of those around the central canal and in the deeper laminae of the dorsal horn were positive. Choline acetyltransferase-immunoreactive neurons in the intermediolateral cell column (presumed sympathetic preganglionic neurons) were often NADPH diaphorase-positive, whereas those in the ventral horn (presumed motorneurons) were not. NADPH diaphorase-positive cells in the intermediolateral cell column were not immunoreactive with GABA or glycine antibodies. These results suggest that NADPH diaphorase is largely restricted to GABAergic neurons in the lumbar spinal cord, and that it is mainly present in those neurons in which GABA coexists with glycine or acetylcholine. Since nitric oxide has been implicated in pain processing and hyperalgesia, while GABA, glycine, and acetylcholine are thought to be involved in analgesia and prevention of hyperalgesia, it is likely that nitric oxide synthase-containing GABAergic neurons in dorsal horn have dual actions in transmission of nociceptive information. © 1993 Wiley-Liss, Inc.     

An NK1 Receptor-dependent Component of the Slow Excitation Recorded Intracellularly from Rat Motoneurons Following Dorsal Root Stimulation

Intracellular recording from lumbar motoneurons of the neonatal rat spinal cord in vitro was used to study how recently developed non-peptide antagonists such as SR-140333 and SR-48698, known to block distinct subtypes of tachykinin receptors peripherally, might affect synaptic transmission elicited by electrical stimulation of dorsal root fibres. SR-140333 (1 μM) preferentially antagonized responses mediated by an exogenously applied agonist acting on the NK₁ receptor subclass, while SR-48968 (0.5 μM) preferentially reduced responses mediated by an exogenously applied agonist acting on the NK₂ receptor subclass. SR-48968 did not affect fast or slow excitatory postsynaptic potentials (EPSPs) or ‘wind-up’responses induced by repetitive, low-frequency stimulation (mimicking certain types of nociceptive input); binding studies using this radiolabelled ligand disclosed specific binding activity (21 fmol/mg protein) selectively displaced by an NK₂ receptor agonist. SR-140333 reduced the late component of fast and slow EPSPs, and of wind-up. Pharmacological block of ionotropic glutamate receptors abolished all dorsal root-evoked EPSPs. In comparison to glutamate receptor blockers, SR-140333 was a weaker antagonist of slow synaptic responses, though it displayed preferential antagonism towards some components of the wind-up phenomenon. The present results provide evidence obtained with a novel NK₁ antagonist that a neuropeptide (presumably substance P), although not directly released by primary afferents onto motoneurons, is a neurotransmitter (acting via NK₁ receptors) in the pathway mediating slow synaptic responses of motoneurons, and is presumably involved in signalling nociceptive inputs from the periphery.