Small-diameter Primary Afferent Fibers Research Articles

Reactive oxygen species increase neuronal excitability via activation of nonspecific cation channel in rat medullary dorsal horn neurons.

The caudal subnucleus of the spinal trigeminal nucleus (medullary dorsal horn; MDH) receives direct inputs from small diameter primary afferent fibers that predominantly transmit nociceptive information in the orofacial region. Recent studies indicate that reactive oxygen species (ROS) is involved in persistent pain, primarily through spinal mechanisms. In this study, we aimed to investigate the role of xanthine/xanthine oxidase (X/XO) system, a known generator of superoxide anion (O2·−), on membrane excitability in the rat MDH neurons. For this, we used patch clamp recording and confocal imaging. An application of X/XO (300 µM/30 mU) induced membrane depolarization and inward currents. When slices were pretreated with ROS scavengers, such as phenyl N-tert-butylnitrone (PBN), superoxide dismutase (SOD), and catalase, X/XO-induced responses decreased. Fluorescence intensity in the DCF-DA and DHE-loaded MDH cells increased on the application of X/XO. An anion channel blocker, 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), significantly decreased X/XO-induced depolarization. X/XO elicited an inward current associated with a linear current-voltage relationship that reversed near −40 mV. X/XO-induced depolarization reduced in the presence of La3+, a nonselective cation channel (NSCC) blocker, and by lowering the external sodium concentration, indicating that membrane depolarization and inward current are induced by influx of Na+ ions. In conclusion, X/XO-induced ROS modulate the membrane excitability of MDH neurons, which was related to the activation of NSCC.

Brain imaging signatures of the relationship between epidermal nerve fibers and heat pain perception

Although the small-diameter primary afferent fibers in the skin promptly respond to nociceptive stimuli and convey sensory inputs to the central nervous system, the neural signatures that underpin the relationship between cutaneous afferent fibers and pain perception remain elusive. We combined skin biopsy at the lateral aspect of the distal leg, which is used to quantify cutaneous afferent fibers, with fMRI, which is used to assess brain responses and functional connectivity, to investigate the relationship between cutaneous sensory nerves and the corresponding pain perception in the brain after applying heat pain stimulation to the dorsum of the right foot in healthy subjects. During painful stimulation, the degree of cutaneous innervation, as measured by epidermal nerve fiber density, was correlated with individual blood oxygen level-dependent (BOLD) signals of the posterior insular cortex and of the thalamus, periaqueductal gray, and rostral ventromedial medulla. Pain perception was associated with the activation of the anterior insular cortex and with the functional connectivity from the anterior insular cortex to the primary somatosensory cortex during painful stimulation. Most importantly, both epidermal nerve fiber density and activity in the posterior insular cortex showed a positive correlation with the strength of coupling under pain between the anterior insular cortex and the primary somatosensory cortex. Thus, our findings support the notion that the neural circuitry subserving pain perception interacts with the cerebral correlates of peripheral nociceptive fibers, which implicates an indirect role for skin nerves in human pain perception.

Differential regulation of peripheral IL-1β-induced mechanical allodynia and thermal hyperalgesia in rats

This study examined the differential mechanisms of mechanical allodynia and thermal hyperalgesia after injection of interleukin (IL) 1β into the orofacial area of male Sprague-Dawley rats. The subcutaneous administration of IL-1β produced both mechanical allodynia and thermal hyperalgesia. Although a pretreatment with iodoresiniferatoxin (IRTX), a transient receptor potential vanilloid 1 (TRPV1) antagonist, did not affect IL-1β-induced mechanical allodynia, it significantly abolished IL-1β-induced thermal hyperalgesia. On the other hand, a pretreatment with D-AP5, an N-methyl-d-aspartate (NMDA) receptor antagonist, and NBQX, an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, blocked IL-1β-induced mechanical allodynia. Pretreatment with H89, a protein kinase A (PKA) inhibitor, blocked IL-1β-induced mechanical allodynia but not thermal hyperalgesia. In contrast, pretreatment with chelerythrine, a protein kinase C (PKC) inhibitor, inhibited IL-1β-induced thermal hyperalgesia. Subcutaneous injections of 2% lidocaine, a local anesthetic agent, blocked IL-1β-induced thermal hyperalgesia but not IL-1β-induced mechanical allodynia. In the resiniferatoxin (RTX)-pretreated rats, a subcutaneous injection of IL-1β did not produce thermal hyperalgesia due to the depletion of TRPV1 in the primary afferent fibers. Double immunofluorescence revealed the colocalization of PKA with neurofilament 200 (NF200) and of PKC with the calcitonin gene-related peptide (CGRP) in the trigeminal ganglion. Furthermore, NMDA receptor 1 (NR1) and TRPV1 predominantly colocalize with PKA and PKC, respectively, in the trigeminal ganglion. These results suggest that IL-1β-induced mechanical allodynia is mediated by sensitized peripheral NMDA/AMPA receptors through PKA-mediated signaling in the large-diameter primary afferent nerve fibers, whereas IL-1β-induced thermal hyperalgesia is mediated by sensitized peripheral TRPV1 receptors through PKC-mediated signaling in the small-diameter primary afferent nerve fibers.

Enhanced LTP of primary afferent neurotransmission in AMPA receptor GluR2-deficient mice

Ca 2+-permeable-AMPA receptors (AMPARs) are expressed in the superficial dorsal horn (SDH, laminae I/II) of the spinal cord, the area involved in transmission and modulation of sensory information, including nociception. A possible role of Ca 2+-permeable-AMPARs in synaptic strengthening has been suggested in postnatal DH cultures, but their role in the long-lasting activity-dependent synaptic plasticity of primary afferent neurotransmission in the adult mouse SDH has not been investigated. In the present study the role of Ca 2+-permeable-AMPARs in the regulation of long-lasting synaptic plasticity, specifically long-term potentiation (LTP) and long-term depression (LTD) in the SDH, was investigated using mice deficient in AMPAR GluR2 subunit. We show here that the GluR2 mutants exhibited no changes in passive membrane properties, but a significant increase in rectification of excitatory postsynaptic currents, the finding suggesting increased expression of Ca 2+-permeable-AMPARs. In the absence of GluR2, high-frequency stimulation (HFS) of small-diameter primary afferent fibers induced LTP that is enhanced and non-saturating in the SDH at both primary afferent A δ- and/or C-fibers monosynaptic and polysynaptic pathways, whereas neuronal excitability and paired-pulse depression were normal. The LTP could be induced in the presence of the NMDA receptor antagonist d-AP5, and L-type Ca 2+ channel blockers, suggesting that Ca 2+-permeable-AMPARs are sufficient to induce LTP in the SDH neurons of adult mouse spinal cord. In contrast, the induction of HFS-LTD is reduced in the SDH of GluR2 mutants. These results suggest an important role for AMPAR GluR2 subunit in regulating synaptic plasticity with potential relevance for long-lasting hypersensitivity in pathological states.

Changes in nociceptive sensory innervation in the epidermis of the rat lower lip skin in a model of neuropathic pain

The epidermis is innervated by fine nerve endings that are important in the perception of nociceptive stimuli. However, their role in neuropathic pain is controversial. In this paper, changes in the innervation patterns of epidermal sensory afferent fibres in the rat lower lip have been studied following bilateral chronic constriction injury (CCI) of the mental nerve—a purely sensory branch of the trigeminal nerve. Sections of the lower lip were processed for immunocytochemistry using antibodies against Protein Gene Product (PGP) 9.5 and Calcitonin Gene-Related Peptide (CGRP) to identify the non-peptidergic and the peptidergic populations of nociceptive small diameter primary sensory afferent fibres. Peptidergic fibres co-localised both markers and the non-peptidergic fibres only stained for PGP 9.5 and not for CGRP. We quantified the total fibre length per 6000 μm 2 in the epidermis at several time points following CCI. Our data indicate that both fibre populations were significantly decreased at 2 weeks post-CCI, followed by fibre re-growth at levels above those seen in sham-operated animals at 4 weeks; however, this increase was only statistically significant for the non-peptidergic population. At 8 weeks post-CCI, the fibre lengths of both populations did not differ significantly from shams. This transient hyper-innervation of the epidermis by one subpopulation of nociceptive fibres coincided with the occurrence of spontaneous pain or dysesthetic sensations which we detected in a previous study in the same animal model. Therefore, we speculate that this transient hyper-innervation of the epidermis following injury could play a role in nociception in these animals.

Autonomic dysreflexia and primary afferent sprouting after clip-compression injury of the rat spinal cord.

Spinal cord injury leads to many forms of autonomic dysfunction including autonomic dysreflexia, a condition involving recurrent episodes of paroxysmal hypertension and associated bradycardia. This hypertension may reach intensities that are life-threatening. We investigated autonomic dysreflexia and the sprouting of central processes of primary afferent neurons (a potential mechanism for autonomic dysreflexia) in a clinically-relevant calibrated clip-compression model of spinal cord injury in the rat. Autonomic dysreflexia was induced by colon distension in the conscious rats 2 weeks after severe (50-g) clip compression injury of the spinal cord at the 4th thoracic segment. The central arbor of small-diameter primary afferent fibers in laminae III-VII of the spinal cord dorsal horn was also assessed at 2 weeks after cord injury by quantitative morphometry, using calcitonin gene-related peptide as a marker. In response to colon distension, arterial pressure increased by 41 +/- 3 mmHg from a resting value of 109 +/- 4 mmHg, and heart rate decreased by 124 +/- 13 beats/min from a value of 515 +/- 16 beats/min (n = 7). Minimal locomotor function was recovered by these rats: by 2 weeks after injury they attained scores of only 3.1 +/- 1.3 on the Basso, Beattie and Bresnahan scale. Histopathology of the clip-compression lesion site in the cord consisted of extensive central necrosis extending several segments rostral and caudal to the lesion. Quantitative measures of the small-diameter afferent arbors revealed significant increases in area ranging from 20-27% in thoracolumbar segments caudal to the injury (n = 5) in comparison to sham-injured rats (n = 6). A second study was done to assess the impact of severity of injury on the relationship between the size of the primary afferent arbors and autonomic dysreflexia. At 2 weeks after milder (20-g) clip injury at T4, rats exhibited responses to colon distension that were not those associated with autonomic dysreflexia (n = 5). Arterial pressure increased by only 16 +/- 3 mmHg and heart rate tended to increase (+19 +/- 12 beats/min). These rats attained a locomotor score of 7.1 +/- 0.4 by 2 weeks. The lesions at the injury site also contained necrosis and mild cavitation within the gray matter. No change in the small-diameter afferent arbor was detected at 2 weeks after the 20-g clip injury at T4 (n = 6 rats). These findings suggest that after severe but not mild clip compression injury of the spinal cord, sprouting of the afferent component of the spinal reflex are contributes to the development of autonomic dysreflexia. Neither dysreflexia, nor changes in the afferent arbor size occurred after mild cord injury. This clinically relevant clip compression cord injury model, studied more frequently for locomotor function, is excellent for investigating mechanisms for the development of autonomic dysreflexia and strategies for its prevention.

Crossed and uncrossed projections to the cat sacrocaudal spinal cord: III. Axons expressing calcitonin gene-related peptide immunoreactivity.

We have investigated the projection patterns of peptidergic small-diameter primary afferent fibers to the cat sacrocaudal spinal cord, a region associated with midline structures of the lower urogenital system and of the tail. Calcitonin gene-related peptide (CGRP)-immunoreactive (CGRP-IR) primary afferent fibers were observed within the superficial laminae, rostrally as the typical inverted U-shaped band that capped the separate dorsal horns (S1 to rostral S2) and caudally as a broad band that spanned the entire mediolateral extent of the fused dorsal horns (caudal S2 and caudal). Within the dorsal gray commissure, labeling was seen as a periodic vertical, midline band. CGRP-IR labeling was prevalent in an extensive mediolateral distribution at the base of the dorsal horn, originating from both lateral and medial collateral bundles that extend from the superficial dorsal horn. Some bundles, in part traveling within the dorsal commissure, conspicuously crossed the midline. In addition to the robust projection to the superficial dorsal horn, there was a more extensive distribution of CGRP-IR fibers within the deeper portions of the cat sacrocaudal dorsal horn than has been reported for other regions of the cat spinal cord. Presumably, these deep projections convey visceral information to projection or segmental neurons at the neck of the dorsal horn and in the region of the central canal. This deep distribution overlaps the reported projections of the pelvic and pudendal nerves. In addition, the contralateral projections of CGRP-IR fibers may form an anatomical substrate of the bilateral receptive fields for selective dorsal horn neurons. The density and variety of CGRP-IR projection patterns is a reflection of the functional attributes of the innervated structures.

Neutralizing Intraspinal Nerve Growth Factor Blocks Autonomic Dysreflexia Caused By Spinal Cord Injury

Autonomic dysreflexia is a condition that develops after spinal cord injury in which potentially life-threatening episodic hypertension is triggered by stimulation of sensory nerves in the body below the site of injury. Central sprouting of small-diameter primary afferent fibers in the dorsal horn of the spinal cord occurs concurrently with the development of this condition. We propose a model for the development of autonomic dysreflexia in which increased nerve growth factor (NGF) in the injured cord stimulates small-diameter primary afferent fiber sprouting, thereby magnifying spinal sympathetic reflexes and promoting dysreflexia. We identified this population of afferent neurons using immunocytochemistry for calcitonin gene-related peptide. Blocking intraspinal NGF with an intrathecally-delivered neutralizing antibody to NGF prevented small-diameter afferent sprouting in rats 2 weeks after a high thoracic spinal cord transection. In the same rats, this anti-NGF antibody treatment significantly decreased (by 43%) the hypertension induced by colon stimulation. The extent of small-diameter afferent sprouting after cord transection correlated significantly with the magnitude of increases in arterial pressure during the autonomic dysreflexia. Neutralizing NGF in the spinal cord is a promising strategy to minimize the life-threatening autonomic dysreflexia that develops after spinal cord injury.

In vivo effects of the NMDA receptor agonist tetrazolyl-glycine related to the function of small-diameter primary afferents.

The NMDA receptor agonist tetrazolyl-glycine (TG; 100 microg kg(-1), i.v.) caused a depressor reflex in anaesthetized rats. The NMDA receptor antagonist MK-801 (300 microg kg(-1), i.v.) inhibited this depressor reflex, but not that induced by pentylenetetrazol (PTZ; 100 mg kg(-1), i.v.), indicating a selective effect of TG on NMDA receptors in vivo. Capsaicin pretreatment, which excludes the function of small-diameter primary afferent fibres, caused only a reduction of the TG-induced depressor reflex, suggesting a reduction of NMDA receptors. The absence of effects of TG and PTZ on the blood pressure in pithed rats excluded any peripheral vascular actions of TG and PTZ. The depressor reflex evoked by afferent nerve stimulation was also inhibited by MK-801 (300 microg kg(-1), i.v.), but not by the tachykinin antagonist L-742694 (10 mg kg(-1), i.v.), confirming the essential role of glutamate in the neurotransmission of signals at central terminals of small-diameter afferent neurons. Plasma protein extravasation in the rat hind paw, induced by neurotransmitters released at peripheral terminals of small diameter afferent neurons by antidromic nerve stimulation, was not influenced by MK-801, indicating that glutamate is either not released or has no effect there. It is concluded that the NMDA agonist TG is a valuable tool to study the functions of primary afferents in vivo.

Differential sensitivities of TTX-resistant and TTX-sensitive sodium channels to anesthetic concentrations of ethanol in rat sensory neurons.

Ethanol at concentration of 200 mM induces anesthesia in experimental animals and depresses neurotransmission in isolated spinal cords. To determine whether actions on primary afferent nerve terminals contribute to ethanol's depressant effects on spinal cord, a study was undertaken to test whether ethanol blocks sodium currents (I(Na)) in dorsal root ganglion neurons (DRGn). Whole-cell patch clamp was used to examine I(Na) in DRGn isolated from 1- to 15-day-old rats. At a holding potential of -80 mV ethanol (200 mM) decreased peak tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) I(Na) by 19.0% +/- 2.7 (mean +/- SEM) and 8.5% +/- 2.2, respectively. Maximal available I(Na) was reduced to 82 +/- 4% (TTX-R) and 93 +/- 1% (TTX-S) of control. Steady-state inactivation curves were shifted in the hyperpolarizing direction by 2.1 +/- 0.2 mV (TTX-R) and 1.1 +/- 0.1 mV (TTX-S). At prepulse potentials of -30 mV (TTX-R) and -70 mV (TTX-S), these shifts contributed an additional 17 +/- 1% (TTX-R) and 7 +/- 1% (TTX-S) reduction in available I(Na). Ethanol thus selectively induced both voltage-independent and voltage-dependent block of TTX-R I(Na) in DRGn. Because DRGn TTX-R sodium channels are associated with small-diameter primary afferent fibers, these results are consistent with a role for ethanol actions on sodium channels in depression of nociceptive-related neurotransmission in spinal cord.

The Cloned Capsaicin Receptor Integrates Multiple Pain-Producing Stimuli

Capsaicin, the main pungent ingredient in “hot” chili peppers, elicits burning pain by activating specific (vanilloid) receptors on sensory nerve endings. The cloned vanilloid receptor (VR1) is a cation channel that is also activated by noxious heat. Here, analysis of heat-evoked single channel currents in excised membrane patches suggests that heat gates VR1 directly. We also show that protons decrease the temperature threshold for VR1 activation such that even moderately acidic conditions (pH ≤ 5.9) activate VR1 at room temperature. VR1 can therefore be viewed as a molecular integrator of chemical and physical stimuli that elicit pain. Immunocytochemical analysis indicates that the receptor is located in a neurochemically heterogeneous population of small diameter primary afferent fibers. A role for VR1 in injury-induced hypersensitivity at the level of the sensory neuron is presented.

Immunoreactivity for endomorphin-2 occurs in primary afferents in rats and monkey.

Antisera were raised against endomorphin-2, a recently isolated endogenous opioid peptide that binds potently and selectively to the mu-opioid receptor. When sections of spinal cord were stained immunocytochemically, a dense plexus of fibres and varicosities was visualized in the superficial dorsal horn of rats and one monkey. Following unilateral multiple dorsal rhizotomy, labeling for endomorphin-2 was markedly reduced ipsilateral to the lesion. In sections stained for both endomorphin-2 and CGRP, double-labeling was observed. Taken together, these data suggest that endomorphin-2 occurs in small diameter primary afferent fibres in rodents and primates. It appears possible that the release of neurotransmitters from nociceptive primary afferents might be regulated by release of endomorphin-2 from primary afferent terminals.

Diminished Inflammation and Nociceptive Pain with Preservation of Neuropathic Pain in Mice with a Targeted Mutation of the Type I Regulatory Subunit of cAMP-Dependent Protein Kinase

To assess the contribution of PKA to injury-induced inflammation and pain, we evaluated nociceptive responses in mice that carry a null mutation in the gene that encodes the neuronal-specific isoform of the type I regulatory subunit (RIbeta) of PKA. Acute pain indices did not differ in the RIbeta PKA mutant mice compared with wild-type controls. However, tissue injury-evoked persistent pain behavior, inflammation of the hindpaw, and ipsilateral dorsal horn Fos immunoreactivity was significantly reduced in the mutant mice, as was plasma extravasation induced by intradermal injection of capsaicin into the paw. The enhanced thermal sensitivity observed in wild-type mice after intraplantar or intrathecal (spinal) administration of prostaglandin E2 was also reduced in mutant mice. In contrast, indices of pain behavior produced by nerve injury were not altered in the mutant mice. Thus, RIbeta PKA is necessary for the full expression of tissue injury-evoked (nociceptive) pain but is not required for nerve injury-evoked (neuropathic) pain. Because the RIbeta subunit is only present in the nervous system, including small diameter trkA receptor-positive dorsal root ganglion cells, we suggest that in inflammatory conditions, RIbeta PKA is specifically required for nociceptive processing in the terminals of small-diameter primary afferent fibers.

Differential activities of intrathecal MK-801 or morphine to alter responses to thermal and mechanical stimuli in normal or nerve-injured rats

Nerve ligation injury in rats results in reduced nociceptive and non-nociceptive thresholds, similar to some aspects of clinical conditions of neuropathic pain. Since underlying mechanisms of hyperalgesia and allodynia may differ, the present study investigated the pharmacology of morphine and MK-801 in rats subjected to a tight ligation of the L5 and L6 nerve roots or to a sham-operation procedure. Response to acute nociception was measured by (a) withdrawal of a hindpaw from a radiant heat source, (b) withdrawal of the tail from a radiant heat source or (c) the latency to a rapid flick of the tail following immersion in water at different noxious temperatures. Mechanical thresholds were determined by measuring response threshold to probing the hindpaw with von Frey filaments. Nerve ligation produced a significant, stable and long-lasting decrease in threshold to mechanical stimulation (i.e., tactile allodynia) when compared to sham-operated controls. Standardization of the diameter of the filaments (to that of the largest filament) did not alter the response threshold in nerve-injured animals. Nerve ligation produced decreased response latency of the ipsilateral paw (i.e., hyperalgesia) when compared to that of sham-operated rats. Tail-flick latencies to thermal stimuli induced by water at constant temperatures (48°, 52° or 55°C) or by radiant heat were not significantly different between nerve-injured and sham-operated groups. At doses which were not behaviorally toxic, MK-801 had no effect on tactile allodynia. At these doses, MK-801 blocked decreased paw withdrawal latency to radiant heat in nerve-injured rats, but did not significantly elevate the response threshold of sham-operated rats. Systemic (i.p.) or intracerebroventricular (i.c.v.) doses of morphine previously shown to be antiallodynic in nerve-ligated rats did not affect the response to probing with von Frey filaments in sham-operated controls. Intrathecal (i.t.) morphine did not change paw withdrawal thresholds elicited by von Frey filaments of either nerve-ligated rats (as previously reported) or of sham-operated rats at doses maximally effective against thermal stimuli applied to the tail or foot. Spinal morphine produced dose-dependent antinociception in both nerve-injured and sham-operated groups in the foot-flick test but was less potent in the nerve-injured group. Presuppression of hyperalgesia of the foot with i.t. MK-801 in nerve-injured animals did not alter the potency of i.t. morphine. I.t. morphine was also active in the tail-flick tests with decreased potency in nerve-injured animals and, at some stimulus intensities, with a decreased efficacy as well. These data emphasize the distinction between the inactivity of morphine to suppress mechanical withdrawal thresholds (as elicited by von Frey filaments) and the activity of this compound to block the response to an acute thermal nociceptive stimulus in sham-operated or nerve-injured rats. It appears that nerve ligation injury produces a thermal allodynia/hyperalgesia which is likely dependent upon opioid-sensitive small-diameter primary afferent fibers and a mechanical allodynia which may be largely independent of small-fiber input.

NK1-tachykinin receptors and prolonged, stimulus-evoked alterations in the excitability of withdrawal reflexes in the decerebrated and spinalized rabbit

Intense natural or electrical stimulation of afferents from the toes or the heel results in prolonged changes in the excitability of the heel withdrawal reflex pathway in the rabbit. This study has investigated the roles played by tachykinin NK1 receptors in mediating these effects. Reflexes were evoked by electrical stimulation of the sural nerve and recorded from the gastrocnemius medialis muscle nerve. High-intensity electrical stimulation of the common peroneal nerve, or application of a crush stimulus to the toes, resulted in suppression of gastrocnemius reflex responses to between 30 and 50% of controls, from which recovery was complete in 15-25 min. In contrast, intense electrical stimulation of the sural nerve, or application of mustard oil to the heel, facilitated the sural to gastrocnemius reflex to two to four times control values. Recovery was rarely complete within 30 min of these stimuli. Administration of the NK1 receptor antagonist CP-96,345, but not its enantiomer CP-96,344, reduced gastrocnemius reflex responses to sural nerve stimulation per se; significantly decreased the time to recovery after common peroneal nerve stimulation and toe crush (but did not affect maximum inhibition); and significantly reduced the facilitation of reflexes resulting from sural nerve stimulation or mustard oil applied to the heel in the first 3-5 min after the application of the stimuli. Both CP-96,345 and CP-96,344 reduced blood pressure and heart rate. These data show that: (i) blockade of NK1-receptors reduces excitatory drive from sural nerve afferents to GM motoneurones; (ii) NK1-receptors are involved in the generation of the early excitatory events which follow stimulation of nociceptive afferents from the heel; and (iii) have a role in the later stages of prolonged, opioid-mediated inhibition of reflexes resulting from activation of fine afferents from the toes. We believe that (ii) and (iii) reflect a role for tachykinins as transmitters from small diameter primary afferent fibres.

Co-injection of wheat germ agglutinin-HRP and choleragenoid-HRP into the sciatic nerve of the rat blocks transganglionic transport.

We report on the surprising loss of transganglionic and retrograde labeling in the spinal cord of the rat after co-injection of the tracers wheat germ agglutinin-HRP (WGA-HRP) and choleragenoid toxin-HRP (CTB-HRP) into the sciatic nerve. Injection of WGA-HRP alone produced a pattern of transganglionic label consistent with transport by small-diameter primary afferent fibers. Small cell bodies were labeled in the ipsilateral dorsal root ganglion (DRG) and there was dense terminal labeling in the superficial dorsal horn of the lumbar spinal cord. Injection of CTB-HRP alone produced a pattern of transganglionic labeling consistent with transport by large-diameter primary afferent fibers. Large cell bodies were labeled in the DRG and there was dense terminal labeling in the nucleus proprius (Laminae III-V) in the spinal cord. CTB-HRP also produced extensive retrograde labeling of ventral horn motor neurons. When the two tracers were co-injected, we found few labeled cells in the ipsilateral DRG and there was almost complete loss of transganglionic terminal labeling in the lumbar spinal cord. Retrograde labeling of motor neurons was also significantly reduced. Even when one of the tracers (e.g., WGA-HRP) was injected 24 hr after and up to 10 mm proximal to the site of the first tracer (e.g., CTB-HRP), an inhibitory interaction was detected. The labeling pattern was always characteristic of the first tracer injected.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of effect of microinjection of noradrenaline or medetomidine on stimulus-evoked release of substance P in the spinal cord of the cat: a study with antibody microprobes.

1. Experiments were performed on barbiturate anaesthetized, spinalized cats to investigate the effect of microinjected noradrenaline or medetomidine on the release of immunoreactive substance P in the dorsal spinal cord following peripheral nerve stimulation. The presence of immunoreactive substance P was assessed with microprobes bearing C-terminus-directed antibodies to substance P. 2. Noradrenaline or medetomidine were microinjected into the grey matter of the spinal cord, near microprobe insertion sites, at depths of 2.5, 2.0, 1.5 and 1.0 mm below the spinal cord surface with volumes of approximately 0.125 microliters and a concentration of 10(-3) M. 3. In the untreated spinal cord, electrical stimulation of the ipsilateral tibial nerve (suprathreshold for C-fibres) elicited release of immunoreactive substance P which was centred in and around lamina II. Neither noradrenaline nor medetomidine administration in the manner described produced significant alterations in this pattern of nerve stimulus-evoked release. 4. In agreement with recent ultrastructural studies these results do not support a control of substance P release by catecholamines released from sites near to the central terminals of small diameter primary afferent fibres.

Capsazepine, a novel capsaicin antagonist, selectively antagonises the effects of capsaicin in the mouse spinal cord in vitro

The mouse hemisected spinal cord with attached dorsal roots and spinal ganglia in vitro preparation was used to investigate the effects of the capsaicin antagonist, capsazepine (2-[2-(4-chlorophenyl)ethylamino-thiocarbonyl]-7,8-dihydroxy-2,3,4,5-tetrahydro-1 H-2-benzazepine). The spinal cord and the ganglia were separated by a perspex gap, allowing application of drugs separately to each compartment. Intracellular recordings were made from 37 cells in laminae II–VI of 12 to 20-day-old mice. Brief applications (30 s) of capsaicin (0.8 μM) excited dorsal horn neurones by activating small diameter primary afferent fibres. The response to capsaicin administered to the spinal cord or to the spinal ganglia was antagonised by the capsaicin antagonist, capsazepine (1.5 μM), administered to the same site. Excitatory postsynaptic potentials (EPSPs) evoked by electrical stimulation of the dorsal root were not affected by capsazepine. Capsazepine itself (5 μM) did not affect the membrane potential of the dorsal horn cells. Capsazepine did not depress the depolarization evoked by substance P. When capsazepine was applied to the spinal cord and capsaicin to the dorsal root ganglion the capsaicin effect was not antagonised. These data suggest that capsaicin-induced depolarization of spinal dorsal horn neurones was mediated via activation of a specific receptor on primary afferent neurones.

The contribution of the nervous system to inflammation and inflammatory disease

Recent studies have identified a major contribution of the nervous system to inflammation and to inflammatory disease. In particular, substances released from the peripheral terminals of small diameter primary afferent fibers and from sympathetic postganglionic nerve (SPGN) terminals have been implicated in several of the major components of acute inflammation (e.g., vasodilatation and plasma extravasation) as well as in the regulation of tissue injury in an inflammatory disease model, experimental arthritis in the rat. Although the release of peptides from primary afferent terminals has received the most attention, our studies have established an important contribution of mast cells and the SPGN terminals to acute inflammation. We describe studies which indicate that plasma extravasation provoked by activation of small diameter primary afferents in the knee joint of the rat involves a cascade of events in which the mast cell and then the sympathetic terminal are sequentially activated. Our studies indicate that release of prostaglandins, but neither norepinephrine nor neuropeptide Y, from the SPGN terminal contributes to increased plasma extravasation. Although activation of the SPGN terminal (via the mast cell) or more directly, via injection of bradykinin, increased plasma extravasation, surgical or pharmacological sympathectomy decreased the severity of experimental arthritis. In related studies we demonstrated that adrenal medullary-derived epinephrine can exacerbate arthritis through a beta-receptor-mediated regulation of the release of an as yet unidentified substance(s) from the SPGN terminal. Our results raise important questions as to whether acute inflammation contributes to tissue repair or to further injury in the setting of disease.

The induction and maintenance of central sensitization is dependent on [formula omitted] acid receptor activation; implications for the treatment of post-injury pain hypersensitivity states

Repetitive stimulation of small diameter primary afferent fibres produces a progressive increase in action potential discharge (windup) and a prolonged increase in the excitability of neurones in the spinal cord following the stimulus. Previous studies have demonstrated that windup is the consequence of the temporal summation of slow synaptic potentials and that the slow potentials and windup are reduced by pretreatment with N- methyl- d-aspartic acid (NMDA) antagonists. We have now examined whether primary afferent induced hypersensitivity states in flexor motoneurones are also dependent on the activation of NMDA receptors and whether windup is a possible trigger for the production of the central hypersensitivity. Both a non-competitive (MK-801) and a competitive (D-CPP) NMDA antagonist, at doses that did not modify the baseline reflex, reduced the facilitation of the flexor reflex produced by either brief electrical stimulation of the sural nerve (1 Hz for 20 sec at C-fibre strength), or by the cutaneous application of the chemical irritant mustard oil. These antagonists also prevented windup from occurring in the motoneurones. When the the MK-801 and the D-CPP were administered once a state of central facilitation had been induced by prior treatment with mustard oil, they returned the facilitated reflex to its pretreatment level. These results indicate that NMDA receptors are involved in the induction and maintenance of the central sensitization produced by high threshold primary afferent inputs. Because central sensitization is likely to contribute to the post-injury pain hypersenstivity states in man, these data have a bearing both on the potential role of NMDA antagonists for pre-emptive analgesia and for treating established pain states.