Terminals Of Primary Afferent Fibers Research Articles

Peripheral GABA receptors regulate colonic afferent excitability and visceral nociception.

While the presence of GABA receptors on primary afferents has been well described, most functional analyses have focused on the regulation of transmitter release from central terminals and/or signalling in the sensory neuron cell body. Evidence that GABA receptors are transported to peripheral terminals and that there are several sources of GABA in the colon raise the possibility that GABA signalling in the periphery may influence colonic afferent excitability. GABAA and GABAB are present and functional in the colon, where exogenous agonists decrease the excitability of colonic afferents and suppress visceral nociception. Endogenous GABA release within the colon is sufficient to establish the resting excitability of colonic afferents as well as the behavioural response to noxious stimulation of the colon, primarily via GABAA receptors. Peripheral GABA receptors may serve as a viable target for the treatment of visceral pain. It is well established that GABA receptors at the central terminals of primary afferent fibres regulate afferent input to the superficial dorsal horn. However, the extent to which peripheral GABA signalling may also regulate afferent input remains to be determined. The colon was used to explore this issue because of the numerous endogenous sources of GABA that have been described in this tissue. The influence of GABA signalling on colonic afferent excitability was assessed in an ex vivo mouse colorectum pelvic nerve preparation where test compounds were applied to the receptive field. The visceromotor response (VMR) evoked by noxious colorectal distension was used to assess the impact of GABA signalling on visceral nociception, where test compounds were applied directly to the colon. Application of either GABAA or GABAB receptor agonists attenuated the colonic afferent response to colon stretch. Conversely, GABAA and GABAB receptor antagonists increased the stretch response. However, while the noxious distension-induced VMR was attenuated in the presence of GABAA and GABAB receptor agonists, the VMR was only consistently increased by GABAA receptor antagonists. These results suggest that GABA receptors are present and functional in the peripheral terminals of colonic afferents and activation of these receptors via endogenous GABA release contributes to the establishment of colonic afferent excitability and visceral nociception. These results suggest that increasing peripheral GABA receptor signalling could be used to treat visceral pain.

Combined Changes in Chloride Regulation and Neuronal Excitability Enable Primary Afferent Depolarization to Elicit Spiking without Compromising its Inhibitory Effects.

The central terminals of primary afferent fibers experience depolarization upon activation of GABAA receptors (GABAAR) because their intracellular chloride concentration is maintained above electrochemical equilibrium. Primary afferent depolarization (PAD) normally mediates inhibition via sodium channel inactivation and shunting but can evoke spikes under certain conditions. Antidromic (centrifugal) conduction of these spikes may contribute to neurogenic inflammation while orthodromic (centripetal) conduction could contribute to pain in the case of nociceptive fibers. PAD-induced spiking is assumed to override presynaptic inhibition. Using computer simulations and dynamic clamp experiments, we sought to identify which biophysical changes are required to enable PAD-induced spiking and whether those changes necessarily compromise PAD-mediated inhibition. According to computational modeling, a depolarizing shift in GABA reversal potential (EGABA) and increased intrinsic excitability (manifest as altered spike initiation properties) were necessary for PAD-induced spiking, whereas increased GABAAR conductance density (ḡGABA) had mixed effects. We tested our predictions experimentally by using dynamic clamp to insert virtual GABAAR conductances with different EGABA and kinetics into acutely dissociated dorsal root ganglion (DRG) neuron somata. Comparable experiments in central axon terminals are prohibitively difficult but the biophysical requirements for PAD-induced spiking are arguably similar in soma and axon. Neurons from naïve (i.e. uninjured) rats were compared before and after pharmacological manipulation of intrinsic excitability, and against neurons from nerve-injured rats. Experimental data confirmed that, in most neurons, both predicted changes were necessary to yield PAD-induced spiking. Importantly, such changes did not prevent PAD from inhibiting other spiking or from blocking spike propagation. In fact, since the high value of ḡGABA required for PAD-induced spiking still mediates strong inhibition, we conclude that PAD-induced spiking does not represent failure of presynaptic inhibition. Instead, diminished PAD caused by reduction of ḡGABA poses a greater risk to presynaptic inhibition and the sensory processing that relies upon it.

Differential distribution of PI3K isoforms in spinal cord and dorsal root ganglia: Potential roles in acute inflammatory pain

PI3-kinases (PI3Ks) participate in nociception within spinal cord, dorsal root ganglion (DRG), and peripheral nerves. To extend our knowledge, we immunohistochemically stained for each of the 4 class I PI3K isoforms along with several cell-specific markers within the lumbar spinal cord, DRG, and sciatic nerve of naive rats. Intrathecal and intraplantar isoform specific antagonists were given as pretreatments before intraplantar carrageenan; pain behavior was then assessed over time. The α-isoform was localized to central terminals of primary afferent fibers in spinal cord laminae IIi to IV as well as to neurons in ventral horn and DRG. The PI3Kβ isoform was the only class I isoform seen in dorsal horn neurons; it was also observed in DRG, Schwann cells, and axonal paranodes. The δ-isoform was found in spinal cord white matter oligodendrocytes and radial astrocytes, and the γ-isoform was seen in a subpopulation of IB4-positive DRG neurons. No isoform co-localized with microglial markers or satellite cells in naive tissue. Only the PI3Kβ antagonist, but none of the other antagonists, had anti-allodynic effects when administered intrathecally; coincident with reduced pain behavior, this agent completely blocked paw carrageenan-induced dorsal horn 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (AMPA) receptor trafficking to plasma membranes. Intraplantar administration of the γ-antagonist prominently reduced pain behavior. These data suggest that each isoform displays specificity with regard to neuronal type as well as to specific tissues. Furthermore, each PI3K isoform has a unique role in development of nociception and tissue inflammation.

A novel role of prostaglandin E2 in neuropathic pain

Neuropathic pain produced by damage to or dysfunction of the nervous system is a common and severely disabling state that affects millions of people worldwide. Recent evidence indicates that activated microglia are key cellular intermediaries in the pathogenesis of neuropathic pain and that ATP serves as the mediator. However, the in vivo mechanism underlying the retention of activated microglia in the injured region has not yet been completely elucidated. Prostaglandin E(2) (PGE(2)) is the principal proinflammatory prostanoid and plays versatile roles by acting via four PGE receptor subtypes, EP1-EP4. In the present study, we investigated the role of PGE(2) in spinal microglial activation in relation to neuropathic pain by using genetic and pharmacological methods. Mice deficient in microsomal prostaglandin E synthase-1 impaired the activation of microglia and the NMDA-nitric oxide (NO) cascade in spinal neurons in the dorsal horn and did not exhibit mechanical allodynia after peripheral nerve injury. The intrathecal injection of indomethacin, a nonsteroidal anti-inflammatory drug, ONO-8713, a selective EP1 antagonist, or 7-nitroindole, a neuronal NO synthase inhibitor, attenuated mechanical allodynia and the increase in activated microglia observed in the established neuropathic-pain state. We further demonstrated that ATP-induced microglial migration was blocked in vitro by PGE(2) via EP2 and by S-nitrosoglutathione, an NO donor. Taken together, the present study suggests that PGE(2) participated in the maintenance of neuropathic pain in vivo not only by activating spinal neurons, but also by retaining microglia in the central terminals of primary afferent fibers via EP2 subtype and via EP1-mediated NO production.

Protein Kinase C Mediates the Synergistic Interaction Between Agonists Acting at α2-Adrenergic and Delta-Opioid Receptors in Spinal Cord

Coactivation of spinal alpha(2)-adrenergic receptors (ARs) and opioid receptors produces antinociceptive synergy. Antinociceptive synergy between intrathecally administered alpha(2)AR and opioid agonists is well documented, but the mechanism underlying this synergy remains unclear. The delta-opioid receptor (DOP) and the alpha(2A)ARs are coexpressed on the terminals of primary afferent fibers in the spinal cord where they may mediate this phenomenon. We evaluated the ability of the DOP-selective agonist deltorphin II (DELT), the alpha(2)AR agonist clonidine (CLON) or their combination to inhibit calcitonin gene-related peptide (CGRP) release from spinal cord slices. We then examined the possible underlying signaling mechanisms involved through coadministration of inhibitors of phospholipase C (PLC), protein kinase C (PKC) or protein kinase A (PKA). Potassium-evoked depolarization of spinal cord slices caused concentration-dependent release of CGRP. Coadministration of DELT and CLON inhibited the release of CGRP in a synergistic manner as confirmed statistically by isobolograpic analysis. Synergy was dependent on the activation of PLC and PKC, but not PKA, whereas the effect of agonist administration alone was only dependent on PLC. The importance of these findings was confirmed in vivo, using a thermal nociceptive test, demonstrating the PKC dependence of CLON-DELT antinociceptive synergy in mice. That inhibition of CGRP release by the combination was maintained in the presence of tetrodotoxin in spinal cord slices suggests that synergy does not rely on interneuronal signaling and may occur within single subcellular compartments. The present study reveals a novel signaling pathway underlying the synergistic analgesic interaction between DOP and alpha(2)AR agonists in the spinal cord.

Transient receptor potential vanilloid type 1 receptor regulates glutamatergic synaptic inputs to the spinothalamic tract neurons of the spinal cord deep dorsal horn

The spinothalamic tract (STT) neurons in the spinal dorsal horn play an important role in transmission and processing of nociceptive sensory information. Although transient receptor potential vanilloid type 1 (TRPV1) receptors are present in the spinal cord dorsal horn, their physiological function is not fully elucidated. In this study, we examined the role of TRPV1 in modulating neuronal activity of the STT neurons through excitatory and inhibitory synaptic inputs. Whole-cell patch-clamp recordings were performed on STT neurons labeled by a retrograde fluorescent tracer injected into the ventral posterior lateral (VPL) nucleus of the thalamus. Capsaicin (1 μM) increased the frequency of miniature excitatory postsynaptic currents (mEPSC) without changing the amplitude or decay time constant of mEPSC. In contrast, capsaicin had no distinct effect on GABAergic miniature inhibitory postsynaptic currents (mIPSC). Capsazepine (10 μM), a TRPV1 receptor antagonist, abolished the effect of capsaicin on mEPSCs. Capsazepine itself did not affect the baseline amplitude and frequency of mEPSC. The effect of capsaicin on mEPSC was also abolished by removal of external Ca 2+, but not by treatment with Cd 2+. Furthermore, capsaicin increased the firing activity of the STT neurons and this increase in neuronal activity by capsaicin was abolished in the presence of non– N-methyl- d-aspartic acid (NMDA) and NMDA receptor antagonists, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX) and (R)-amino-5-phosphonovaleric acid (APV). These data suggest that activation of TRPV1 potentiates the glutamate release from excitatory terminals of primary afferent fibers and subsequently increases the neural activity of STT neurons of the rat spinal cord deep dorsal horn.

Vesicular glutamate transporters type 1 and 2 expression in axon terminals of the rat nucleus of the solitary tract

The nucleus of the solitary tract is the site of termination of primary afferent fibers running in the facial, glossopharyngeal and vagus nerves. The present study was performed to map the distribution of glutamatergic axons terminals in the rat nucleus of the solitary tract using immunodetection of vesicular glutamate transporter 1 and vesicular glutamate transporter 2. The two vesicular glutamate transporters were differentially distributed among nucleus of the solitary tract subdivisions. Vesicular glutamate transporter 1 immunoreactivity was mostly found in the lateral part of the nucleus (ventrolateral, interstitial and intermediate subdivisions) whereas vesicular glutamate transporter 2 labeling was distributed throughout the nucleus of the solitary tract. Electron microscope examination indicated that vesicular glutamate transporter immunoreactivity was localized in axon terminals filled with round synaptic vesicles. After injection of cholera toxin B subunit in sensory ganglia, anterograde labeling was found in vesicular glutamate transporter 1, as well as vesicular glutamate transporter 2-immunoreactive boutons. Double immunolabeling experiments allowed distinctions between terminals expressing either vesicular glutamate transporter 1 or vesicular glutamate transporter 2 or both vesicular glutamate transporter 1 and vesicular glutamate transporter 2 immunoreactivities. The latter population, expressing both transporters immunolabeling, completely disappeared after deafferentation induced by removal of sensory ganglia.This study indicates that vesicular glutamate transporter content identifies three different subpopulations of glutamatergic boutons in the nucleus of the solitary tract and provides definitive evidence that primary afferent neurons contribute glutamatergic terminals to the nucleus of the solitary tract.

Presynaptic NMDA receptors modulate glutamate release from primary sensory neurons in rat spinal cord dorsal horn.

NMDA receptors have the potential to produce complex activity-dependent regulation of transmitter release when localized presynaptically. In the somatosensory system, NMDA receptors have been immunocytochemically detected on presynaptic terminals of primary afferents, and these have been proposed to drive release of substance P from central terminals of a subset of nociceptors in the spinal cord dorsal horn. Here we report that functional NMDA receptors are indeed present at or near the central terminals of primary afferent fibers. Furthermore, we show that activation of these presynaptic receptors results in an inhibition of glutamate release from the terminals. Some of these NMDA receptors may be expressed in the preterminal axon and regulate the extent to which action potentials invade the extensive central arborizations of primary sensory neurons.

Basal and activity-induced release of substance P from primary afferent fibres in NK 1 receptor knockout mice: evidence for negative feedback

The concept that NK 1 receptors are located pre-junctionally on substance P (SP)-containing nerves, acting as autoreceptors to inhibit SP release, has been suggested, but remains a controversial issue. To further investigate the existence of this receptor on central and peripheral terminals of primary afferent fibres, NK 1 receptor knockout mice and an NK 1 receptor antagonist were used in nerve-attached tissue preparations. These were the isolated dorsal horn of the spinal cord with dorsal roots attached, and the hairy skin of the hind paw with attached saphenous nerve. The results reveal that in the dorsal horn preparation, basal release of SP is significantly higher in NK 1 −/− mice than NK 1 +/+ mice ( P<0.05, n=7 mice/strain). However, a difference in SP release evoked in the dorsal horn by electrical stimulation of the dorsal roots or capsaicin application was not observed. In contrast, antidromic electrical stimulation of the saphenous nerve caused a substantially greater release of SP in the skin of NK 1 −/− mice than in NK 1 +/+ mice ( P<0.05, n=5 to 6 mice/strain). These results provide evidence for the existence of NK 1 autoreceptors on sensory nerves in skin, which may be relevant to the modulation of their peripheral pathophysiological effector functions.

Expression of vesicular glutamate transporters, VGluT1 and VGluT2, in axon terminals of nociceptive primary afferent fibers in the superficial layers of the medullary and spinal dorsal horns of the rat.

We examined immunohistochemically whether the vesicular glutamate transporters (VGluTs), VGluT1 and VGluT2, might be expressed in synaptic terminals of nociceptive primary afferent fibers within laminae I and II of the medullary and spinal dorsal horns of the rat. VGluT1 immunoreactivity (IR) was intense in the inner part of lamina II but weak in lamina I and the outer part of lamina II. VGluT2-IR was most intense in lamina I and the outer part of lamina II. Expression of VGluTs in synaptic terminals was confirmed by dual immunofluorescence histochemistry for VGluTs and synaptophysin. Expression of VGluTs in axon terminals of primary afferent fibers terminating in laminae I and II was also confirmed immunohistochemically after unilateral dorsal rhizotomy. The dual immunofluorescence histochemistry indicated expression of VGluTs in substance P (SP)-containing axon terminals in lamina I and the outer part of lamina II. Electron microscopy confirmed the coexpression of VGluTs and SP in axon terminals within laminae I and II; VGluTs was associated with round synaptic vesicles at the asymmetric synapses. It was further observed that isolectin IB4, a marker for unmyelinated axons, often bound with VGluT2-immunopositive structures but rarely with VGluT1-immunopositive structures in lamina II. Thus, the results indicated in laminae I and II of the medullary and spinal dorsal horns that both VGluT1 and VGluT2 were expressed in axon terminals of primary afferent fibers, including SP-containing nociceptive fibers and that VGluT in unmyelinated primary afferent fibers terminating in lamina II was primarily VGluT2.

GABAergic modulation of primary gustatory afferent synaptic efficacy.

Modulation of synaptic transmission at the primary sensory afferent synapse is well documented for the somatosensory and olfactory systems. The present study was undertaken to test whether GABA impacts on transmission of gustatory information at the primary afferent synapse. In goldfish, the vagal gustatory input terminates in a laminated structure, the vagal lobes, whose sensory layers are homologous to the mammalian nucleus of the solitary tract. We relied on immunoreactivity for the GABA-transporter, GAT-1, to determine the distribution of GABAergic synapses in the vagal lobe. Immunocytochemistry showed dense, punctate GAT-1 immunoreactivity coincident with the layers of termination of primary afferent fibers. The laminar nature and polarized dendritic structure of the vagal lobe make it amenable to an in vitro slice preparation to study early synaptic events in the transmission of gustatory input. Electrical stimulation of the gustatory nerves in vitro produces synaptic field potentials (fEPSPs) predominantly mediated by ionotropic glutamate receptors. Bath application of either the GABA(A) receptor agonist muscimol or the GABA(B) receptor agonist baclofen caused a nearly complete suppression of the primary fEPSP. Coapplication of the appropriate GABA(A) or GABA(B) receptor antagonist bicuculline or CGP-55845 significantly reversed the effects of the agonists. These data indicate that GABAergic terminals situated in proximity to primary gustatory afferent terminals can modulate primary afferent input via both GABA(A) and GABA(B) receptors. The mechanism of action of GABA(B) receptors suggests a presynaptic locus of action for that receptor.

Functional Expression of AMPA Receptors on Central Terminals of Rat Dorsal Root Ganglion Neurons and Presynaptic Inhibition of Glutamate Release

No direct evidence has been found for expression of functional AMPA receptors by dorsal root ganglion neurons despite immunocytochemical evidence suggesting they are present. Here we report evidence for expression of functional AMPA receptors by a subpopulation of dorsal root ganglion neurons. The AMPA receptors are most prominently located near central terminals of primary afferent fibers. AMPA and kainate receptors were detected by recording receptor-mediated depolarization of the central terminals under selective pharmacological conditions. We demonstrate that activation of presynaptic AMPA receptors by exogenous agonists causes inhibition of glutamate release from the terminals, possibly via primary afferent depolarization (PAD). These results challenge the traditional view that GABA and GABA A receptors exclusively mediate PAD, and indicate that PAD is also mediated by glutamate acting on presynaptically localized AMPA and kainate receptors.

Release of immunoreactive brain-derived neurotrophic factor in the spinal cord of the rat following sciatic nerve transection.

Using the antibody microprobe method, the sites of spinal release of immunoreactive brain-derived neurotrophic factor (BDNF) was studied in normal rats, and rats with prior sciatic nerve transection. In normal rats, a significant basal release of immunoreactive BDNF was found in the superficial dorsal horn. Following sciatic nerve transection (performed 14 days previously), release of BDNF was found throughout the whole of the dorsal horn, extending into deeper laminae. Electrical stimulation of the ipsilateral sciatic nerve at a strength adequate to excite either A fibres (20 Hz at 2×threshold voltage) or A and C fibres (2 Hz at 20×threshold voltage) did not alter the basal release of immunoreactive BDNF in normal or in nerve-injured rats. The results suggest that BDNF is released from the central terminals of primary afferent fibres, but such release is not solely dependent upon action potential invasion of these terminals. The increased extent of release following nerve transection is consistent with the hypothesis that BDNF plays a role in the central response to peripheral nerve injury.

Distribution of immunoreactivity for the ?2 and ?3 subunits of the GABAA receptor in the mammalian spinal cord

The localization of GABAA receptors in cat and rat spinal cord was analyzed using two monoclonal antibodies specific for an epitope shared by the beta 2 and beta 3 subunits of the receptor. beta 2/beta 3-subunit immunoreactivity was the most intense in inner lamina II, lamina III, and lamina X, and it was the least intense in lamina IX. In laminae I-III, generally, the staining had a rather diffuse appearance, but the surfaces of small cell bodies in these laminae were outlined clearly by discrete labeling, as were many cell bodies and dendrites in deeper laminae. Rhizotomy experiments and ultrastructural observations indicated that beta 2/beta 3-subunit immunoreactivity in the dorsal horn was largely localized in intrinsic neuropil elements rather than in the terminals of primary afferent fibers, even though labeling overlapped with the terminal fields of different types of primary afferents and was also detected on the membranes of dorsal root ganglion neurons. With few exceptions (most notably, a highly immunoreactive group of dorsolaterally located cells in the cat lumbar ventral horn), motoneurons expressed low levels of beta 2/beta 3-subunit immunoreactivity. Labeling of neuronal membranes was fairly continuous, but focal accumulations of beta 2/beta 3-subunit immunoreactivity were also detected using immunofluorescence. Focal "hot spots" correlated ultrastructurally with the presence of synaptic junctions. Dual-color immunofluorescence revealed that focal accumulations of beta 2/beta 3-subunit immunoreactivity were frequently apposed by glutamic acid decarboxylase (GAD)-immunoreactive terminals. However, the density of continuous-membrane beta 2/beta 3 immunolabeling and GAD terminal density were not correlated in many individual neurons. The results suggest the existence of "classical" (synaptic) and "nonclassical" (paracrine) actions mediated via spinal cord GABAA receptors. The study also revealed the relative paucity of beta 2/beta 3-subunit immunoreactivity postsynaptic to certain GABAergic terminals, particularly those presynaptic to motoneurons or primary afferent terminals.

Distribution of immunoreactivity for the beta 2 and beta 3 subunits of the GABAA receptor in the mammalian spinal cord.

The localization of GABAA receptors in cat and rat spinal cord was analyzed using two monoclonal antibodies specific for an epitope shared by the beta 2 and beta 3 subunits of the receptor. beta 2/beta 3-subunit immunoreactivity was the most intense in inner lamina II, lamina III, and lamina X, and it was the least intense in lamina IX. In laminae I-III, generally, the staining had a rather diffuse appearance, but the surfaces of small cell bodies in these laminae were outlined clearly by discrete labeling, as were many cell bodies and dendrites in deeper laminae. Rhizotomy experiments and ultrastructural observations indicated that beta 2/beta 3-subunit immunoreactivity in the dorsal horn was largely localized in intrinsic neuropil elements rather than in the terminals of primary afferent fibers, even though labeling overlapped with the terminal fields of different types of primary afferents and was also detected on the membranes of dorsal root ganglion neurons. With few exceptions (most notably, a highly immunoreactive group of dorsolaterally located cells in the cat lumbar ventral horn), motoneurons expressed low levels of beta 2/beta 3-subunit immunoreactivity. Labeling of neuronal membranes was fairly continuous, but focal accumulations of beta 2/beta 3-subunit immunoreactivity were also detected using immunofluorescence. Focal "hot spots" correlated ultrastructurally with the presence of synaptic junctions. Dual-color immunofluorescence revealed that focal accumulations of beta 2/beta 3-subunit immunoreactivity were frequently apposed by glutamic acid decarboxylase (GAD)-immunoreactive terminals. However, the density of continuous-membrane beta 2/beta 3 immunolabeling and GAD terminal density were not correlated in many individual neurons. The results suggest the existence of "classical" (synaptic) and "nonclassical" (paracrine) actions mediated via spinal cord GABAA receptors. The study also revealed the relative paucity of beta 2/beta 3-subunit immunoreactivity postsynaptic to certain GABAergic terminals, particularly those presynaptic to motoneurons or primary afferent terminals.

Can the α2-adrenoceptor agonist-mediated suppression of nocifensive reflex responses be due to action on motoneurons or peripheral nociceptors?

To exclude the possibility, that the suppression of nocifensive reflex responses induced by α 2-adrenergic agents is due to action on α-motoneurons or peripheral nociceptors, we studied the effect of medetomidine, an α 2-adrenoceptor agonist, on the monosynaptic reflex and on the primary afferent nociceptor-mediated antidromic vasodilator response in rats. Additionally, the effect on the dorsal root potential, an index of a transient excitability change in the central terminals of primary afferent fibers, was determined. Medetomidine was applied systemically at doses (100 and 300 μg/kg which have proven strongly antinociceptive in previous studies. The amplitudes of a submaximal monosynaptic reflex volley or a submaximal dorsal root potential were not changed by medetomidine. Medetomidine induced a decrease of cutaneous blood flow but did not abolish the vasodilatatory response to antidromic stimulation of the sciatic nerve at C-fiber intensity as determined by the laser Doppler flow method. The results indicate that the α 2-adrenoceptor-mediated suppression of nocifensive reflex responses is not caused by a decreased excitability of motoneurons or peripheral nociceptors. An α 2-adrenoceptor agonist does not modulate the transient stimulus-evoked change in the excitability of central terminals of primary afferent fibers.

Afferent volley patterns and the spinal release of immunoreactive substance P in the dorsal horn of the anaesthetized spinal cat.

Microprobes bearing immobilized antibodies to the C-terminus of substance P were used to measure release of this neuropeptide in the spinal cord of the anaesthetized spinal cat in response to peripheral nerve stimulation. Release of substance P was just detectable in laminae I, II with 150 stimuli (0.5 Hz, 5 min) and was near maximal with 300 stimuli. Using two periods of stimulation of 10 min separated by 15 min, greater levels of substance P were detected during the second period. Fifteen to 25 min after two periods of peripheral nerve stimulation levels of substance P detected by microprobes were still elevated above those present prior to stimulation. Stimulation with bursts of three impulses when delivering a fixed number of stimuli resulted in detection of increased levels of substance P at sites adjacent to the areas of maximal release. The results suggest that maximal release of substance P from the central terminals of primary afferent fibres occurs with relatively few impulses and at low frequencies in agreement with what is known of release from the peripheral terminals of these fibres.

Morphology, physiology and in vivo activity of cuticular stress detector afferents in crayfish

Cuticular Stress Detector afferents (CSD1 and CSD2) in the 5th walking legs of crayfish (Pacifastacus leniusculus, Procambarus clarkii) have been studied in an in vitro preparation allowing intracellular recordings to be made from the central terminals of primary afferent fibres during mechanical stimulation of the sense organs. Biocytin anterograde fills and transverse sections of the sensory nerves showed CSD1 to comprise fewer and more heterogeneous fibres than CSD2. Lucifer yellow filling of single fibres showed branching patterns compatible with monosynaptic projections to some motorneuronal groups. Whole nerve recordings during sinusoidal or ramp stimuli showed an important contribution from units with phasic properties. The intracellular recordings identified three features unique to CSD1: 1. Many ‘on-off’ units have a phasic response to both increases and decreases of force. 2. Many ‘high threshold’ units respond only to high amplitude vibratory stimuli. 3. A few sensory fibres have a main branch projecting rostrally within the interganglionic connectives, possibly as far as the brain. In vivo recordings of CSD1 activity during forward locomotion on a treadmill showed a discharge occurring in advance of, as well as during, the power stroke. It is therefore suggested that at least some CSD1 fibres encode active as well as passive force during locomotion.

Localization of 5-HT3 receptors in the rat spinal cord: immunohistochemistry and in situ hybridization.

Specific antibodies raised against a fusion protein containing the amino acid sequence of the putative second intracellular loop of the cloned 5-HT3-A receptor subunit were used for the immunohistochemical visualization of 5-HT3 receptors in the rat spinal cord. A dense 5-HT3-like immunoreactivity was found in the superficial layers of the dorsal horn, which closely matched the labelling of 5-HT3 binding sites by [125I]iodo-zacopride. This immunostaining was markedly decreased following unilateral rhizotomy, consistently with a preferential location of 5-HT3 receptors on terminals of primary afferent fibres, and with the presence of 5-HT3 mRNA in dorsal root ganglia. However, a significant proportion of 5-HT3 receptors persisted after rhizotomy, and the corresponding mRNA was found in the dorsal horn of the spinal cord. 5-HT3 receptors are therefore also located on intrinsic neurones of the spinal cord.

Evidence for presynaptic N-methyl-D-aspartate autoreceptors in the spinal cord dorsal horn.

The N-methyl-D-aspartate (NMDA) receptor has been implicated in a variety of systems that undergo plastic changes in the central nervous system. We used electron microscopic immunocytochemistry with an antibody directed against an alternatively spliced exon near the C terminus of NMDAR1, the essential functional subunit of the NMDA receptor, to study the distribution of the NMDA receptor in the spinal cord and CA1 region of the hippocampus, two regions where NMDA-mediated long-term plasticity has been demonstrated. In CA1, we found that the NMDA receptor is exclusively expressed on postsynaptic structures. By contrast, in the spinal cord we found that in about one-third of labeled synapses, the receptor is located in the presynaptic terminal, immediately adjacent to the vesicle release site at the active zone. Using combined postembedding immunocytochemistry, we also showed that > 70% of the NMDA receptor immunoreactive terminals are glutamate positive, which suggests that the presynaptic NMDA receptor is an autoreceptor. Nerve ligation studies demonstrated that the receptor is transported in dorsal roots and sciatic nerve to the spinal cord and periphery, respectively. These data indicate that an NMDA autoreceptor is located in terminals of primary afferent fibers, where it could facilitate the transmission of inputs to the spinal cord by increasing the release of neurotransmitter from the primary afferent terminal.