Peripheral Receptive Fields Research Articles

5beta-reduced neuroactive steroids are novel voltage-dependent blockers of T-type Ca2+ channels in rat sensory neurons in vitro and potent peripheral analgesics in vivo.

T-type Ca(2+) channels are believed to play an important role in pain perception, and anesthetic steroids such as alphaxalone and allopregnanolone, which have a 5alpha-configuration at the steroid A, B ring fusion, are known to inhibit T-type Ca(2+) channels and cause analgesia in a thermal nociceptive model (Soc Neurosci Abstr 29:657.9, 2003). To define further the structure-activity relationships for steroid analgesia, we synthesized and examined a series of 5beta-reduced steroids for their ability to induce thermal antinociception in rats when injected locally into the peripheral receptive fields of the nociceptors and studied their effects on T-type Ca(2+) channel function in vitro. We found that most of the steroids completely blocked T-type Ca(2+) currents in vitro with IC(50) values at a holding potential of -90 mV ranging from 2.8 to 40 microM. T current blockade exhibited mild voltage-dependence, suggesting that 5beta-reduced neuroactive steroids stabilize inactive states of the channel. For the most potent steroids, we found that other voltage-gated currents were not significantly affected at concentrations that produce nearly maximal blockade of T currents. All tested compounds induced dose-dependent analgesia in thermal nociceptive testing; the most potent effect (ED(50), 30 ng/100 microl) obtained with a compound [(3beta,5beta,17beta)-3-hydroxyandrostane-17-carbonitrile] that was also the most effective blocker of T currents. Compared with previously studied 5alpha-reduced steroids, these 5beta-reduced steroids are more efficacious blockers of neuronal T-type Ca(2+) channels and are potentially useful as new experimental reagents for understanding the role of neuronal T-type Ca(2+) channels in peripheral pain pathways.

Corticothalamic modulation on formalin-induced change of VPM thalamic activities.

Spontaneous activities of single cells were extracellularly recorded in ventral posterior medial (VPM) thalamus of anesthetized rats to characterize the corticothalamic modulation on formalin-induced changes of spontaneous thalamic firing. Formalin injected into the peripheral receptive field, dose-dependently induced the reversible facilitation of spontaneous activities of VPM. However, when the primary somatosensory (SI) cortex was inactivated by muscimol, the pattern of formalin-induced changes of VPM firing was altered. This altered responsiveness included both first and second phase of facilitated spontaneous activities. Bicuculline infused into SI cortex did not alter the pattern of formalin-induced thalamic changes. These results suggest that the pain reactivity of VPM thalamus may be modulated by cortex via corticothalamic pathway during the generation of inflammatory pain.

Redox modulation of peripheral T-type Ca 2+ channels in vivo: alteration of nerve injury-induced thermal hyperalgesia

We reported recently that redox agents, including the endogenous amino acid l-cysteine, modulate T-type Ca 2+ currents in primary sensory neurons in vitro, and alter mechanical and thermal nociception in peripheral nociceptors in vivo in intact animals [Neuron 31 (2001) 75]. Here, we studied the effects of locally applied redox agents ( l-cysteine and 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) on thermal hyperalgesia in animals with neuropathic pain due to chronic constrictive injury (CCI) of the sciatic nerve. We found that, following injection into the peripheral receptive fields, the endogenous reducing agent l-cysteine increased thermal hyperalgesia in a dose-dependent manner in rats with CCI of the sciatic nerve as well as in sham-operated rats. However, the magnitude of the increase was smaller and duration of effect was shorter in rats with CCI of the sciatic nerve compared to sham-operated animals. DTNB, an exogenous oxidizing agent, induced dose-dependent alleviation of thermal hyperalgesia in rats with CCI of the sciatic nerve and caused analgesia in sham-operated rats. DTNB completely blocked l-cysteine-induced thermal hyperalgesia in both animal groups. Mibefradil, a potent and preferential T-type Ca 2+ channel blocker, abolished l-cysteine-induced increase in thermal hyperalgesia in both animal groups suggesting the involvement of T-type Ca 2+ channels in peripheral nociception. These results indicate for the first time that redox modulation of T-type Ca 2+ channels in rat peripheral nociceptors is operational in pain states caused by peripheral axonal injury. Since thermal hyperalgesia is a common symptom of axonal injury, locally applied oxidizing agents could be used as a novel treatment to ameliorate neuropathic pain.

Potent analgesic effects of anticonvulsants on peripheral thermal nociception in rats.

1. Anticonvulsant agents are commonly used to treat neuropathic pain conditions because of their effects on voltage- and ligand-gated channels in central pain pathways. However, their interaction with ion channels in peripheral pain pathways is poorly understood. Therefore, we studied the potential analgesic effects of commonly used anticonvulsant agents in peripheral nociception. 2. We injected anticonvulsants intradermally into peripheral receptive fields of sensory neurons in the hindpaws of adult rats, and studied pain perception using the model of acute thermal nociception. Commonly used anticonvulsants such as voltage-gated Na+ channel blockers, phenytoin and carbamazepine, and voltage-gated Ca2+ channel blockers, gabapentin and ethosuximide, induced dose-dependent analgesia in the injected paw, with ED50 values of 0.30, 0.32 and 8, 410 microg per 100 microl, respectively. 3. Thermal nociceptive responses were not affected in the contralateral, noninjected paws, indicating a lack of systemic effects with doses of anticonvulsants that elicited local analgesia. 4. Hill slope coefficients for the tested anticonvulsants indicate that the dose-response curve was less steep for gabapentin than for phenytoin, carbamazepine and ethosuximide. 5. Our data strongly suggest that cellular targets like voltage-gated Na+ and Ca2+ channels, similar to those that mediate the effects of anticonvulsant agents in the CNS, may exist in the peripheral nerve endings of rat sensory neurons. Thus, peripherally applied anticonvulsants that block voltage-gated Na+ and Ca2+ channels may be useful analgesics.

Neither Spinal γ-Aminobutyric Acid-A nor Strychnine-Sensitive Glycine Receptor Systems Are the Sole Mediators of Halothane Depression of Spinal Dorsal Horn Sensory Neurons

Inhaled anesthetics depress the response of spinal dorsal horn low-threshold (LT) neurons to peripheral receptive field stimulation. Part of that depression may be mediated by anesthetic interactions with gamma-aminobutyric acid type A (GABA(A)) and strychnine-sensitive glycine inhibitory neurotransmitter systems. In this electrophysiological study, we attempted to antagonize halothane depression of LT neurons by administering bicuculline (a competitive GABA(A) antagonist) and/or strychnine (a competitive glycine antagonist) systemically, alone or in combination, to decerebrate, spinal cord-transected rats. We observed that both bicuculline and strychnine, alone or in combination, significantly but only partially reversed halothane depression of LT neuronal responses to receptive field stimulation. The inability of bicuculline and strychnine, alone or in combination, to completely reverse halothane depression suggests that although GABA(A) and glycine systems are involved in the observed halothane depression, additional mechanisms of action are also required for halothane depression of LT spinal sensory neurons. The results of this study support the hypothesis that halothane depression of spinal sensory neurons is mediated, but not completely, by the anesthetic effects on gamma-aminobutyric acid type A and strychnine-sensitive glycine neurotransmitter systems.

Spinal interleukin-6 (IL-6) inhibits nociceptive transmission following neuropathy

Interleukin-6 (IL-6) is a neuropoietic cytokine which is dramatically upregulated following peripheral nerve injury at the site of injury, in the dorsal root ganglion (DRG) and in the spinal cord. The functional effects of IL-6 in nociception in normal conditions and following nerve injury are unclear. Thus the aim of this study was to assess the effect of spinal IL-6 administration on nociceptive transmission in naive, sham-operated and neuropathic (spinal nerve ligation, SNL) rats using in vivo electrophysiology to elucidate the possible role of IL-6 in neuropathic pain. In anaesthetised rats, extracellular recordings were made from individual convergent dorsal horn neurones following electrical and natural (mechanical and thermal) stimulation of peripheral receptive fields. Exogenous spinal IL-6 (100–500 ng) had no significant effect on electrically evoked neuronal responses in naive rats. In contrast, following neuropathy, spinal IL-6 produced a dose-related inhibition of the electrically evoked C-fibre, initial C-fibre and measures of neuronal hyperexcitability (post discharge and wind-up). In addition, spinal IL-6 markedly inhibited mechanical neuronal responses in neuropathic rats. Higher doses of spinal IL-6 also inhibited, to a lesser degree, the initial C-fibre, post discharge and wind-up responses in sham-operated rats. These studies show that following nerve injury the actions of the cytokine alter so that spinal administration of IL-6 elicits anti-nociceptive effects not observed under normal conditions. Moreover, the inhibitory effects of IL-6 on C-fibre activity and neuronal hyperexcitability, suggest IL-6 to be a potential modulator of neuropathic pain.

Intraplantar injection of anandamide inhibits mechanically-evoked responses of spinal neurones via activation of CB 2 receptors in anaesthetised rats

Anti-nociceptive effects of the endocannabinoid anandamide are well established. Anandamide has, however, also been shown to activate pro-nociceptive vanilloid 1 (VR1) receptors present on primary afferent nociceptors. The aim of the present study was to determine the effect of intraplantar injection of anandamide on dorsal spinal neuronal responses in control rats and rats with hindpaw carrageenan-induced inflammation. Effects of intraplantar administration of anandamide (50 μg in 50 μl) on peripheral mechanically-evoked responses of spinal neurones were studied in halothane-anaesthetised rats in vivo. Responses of spinal neurones to mechanical punctate stimulation (von Frey filaments, 8–80 g) of the peripheral receptive field were similar in non-inflamed rats and rats with hindpaw carrageenan-induced inflammation. Intraplantar injection of anandamide, but not vehicle, significantly ( P<0.05) inhibited innocuous and noxious mechanically-evoked responses of spinal neurones in rats with hindpaw inflammation, but not in non-inflamed rats. Co-administration of the cannabinoid 2 (CB 2) receptor antagonist, SR144528 (10 μg in 50 μl), but not the cannabinoid 1 (CB 1) receptor antagonist, SR141716A (10 μg in 50 μl), significantly blocked inhibitory effects of anandamide on peripheral evoked neuronal responses in rats with hindpaw inflammation. This study demonstrates inhibitory effects of exogenous anandamide on mechanically-evoked responses under inflammatory conditions in vivo, which are mediated by peripheral CB 2 receptors.

Similar electrophysiological changes in axotomized and neighboring intact dorsal root ganglion neurons.

We investigated electrophysiological changes in chronically axotomized and neighboring intact dorsal root ganglion (DRG) neurons in rats after either a peripheral axotomy consisting of an L5 spinal nerve ligation (SNL) or a central axotomy produced by an L5 partial rhizotomy (PR). SNL produced lasting hyperalgesia to punctate indentation and tactile allodynia to innocuous stroking of the foot ipsilateral to the injury. PR produced ipsilateral hyperalgesia without allodynia with recovery by day 10. Intracellular recordings were obtained in vivo from the cell bodies (somata) of axotomized and intact DRG neurons, some with functionally identified peripheral receptive fields. PR produced only minor electrophysiological changes in both axotomized and intact somata in L5 DRG. In contrast, extensive changes were observed after SNL in large- and medium-sized, but not small-sized, somata of intact (L4) as well as axotomized (L5) DRG neurons. These changes included (in relation to sham values) higher input resistance, lower current and voltage thresholds, and action potentials with longer durations and slower rising and falling rates. The incidence of spontaneous activity, recorded extracellularly from dorsal root fibers in vitro, was significantly higher (in relation to sham) after SNL but not after PR, and occurred in myelinated but not unmyelinated fibers from both L4 (9.1%) and L5 (16.7%) DRGs. We hypothesize that the changes in the electrophysiological properties of axotomized and intact DRG neurons after SNL are produced by a mechanism associated with Wallerian degeneration and that the hyperexcitability of intact neurons may contribute to SNL-induced hyperalgesia and allodynia.

Convergence of selected inputs from sensory afferents to trigeminal premotor neurons with possible projections to masseter motoneurons in the rabbit

Peripheral input convergence on trigeminal premotor neurons in the vicinity of trigeminal motor nucleus has been investigated. Thirty neurons were identified by their antidromic responses to microstimulation of the masseteric subnucleus of trigeminal motor nucleus (NVmot-mass). Peripheral receptive fields were found in the buccal mucosae, periodontal ligaments, palate, tongue and vibrissae for 16 neurons located in the intertrigeminal area (NVint), supratrigeminal area (NVs), main sensory trigeminal nucleus (NVsnpr) and subnucleus γ of the oral nucleus of the spinal trigeminal tract (NVspo-γ). Eleven neurons in the NVint, NVs and NVspo-γ responded to passive jaw opening: nine neurons were activated and two were inhibited. None of the neurons responded to both the orofacial mechanical stimulation and passive jaw opening. Forty-six percent of neurons (13 out of 28 tested) received inputs from the inferior alveolar nerve (IAN) and 53% of neurons (8 out of 15 tested) received inputs from the infraorbital nerve (ION). Out of 15 neurons tested for inputs from the IAN and ION, 7 neurons in the NVsnpr and NVspo-γ received input from both. Sixteen percent of neurons (4 out of 25) received inputs from the masseteric nerve (MassN). None of the neurons with inputs from IAN and/or ION also received inputs from the MassN. We suggest that trigeminal premotor interneurons with projections to the NVmot-mass fall into two broad categories, those with inputs from the IAN and/or ION and those with inputs from the MassN, possibly muscle spindle afferents, and no neuron receiving inputs from both.

Nerve injury induces plasticity that results in spinal inhibitory effects of galanin

Galanin is a 29-amino-acid neuropeptide that has been implicated in the processes of nociception. This study examines the effect of exogenous galanin on dorsal horn neurone activity in vivo in the spinal nerve ligation (SNL) model of neuropathic pain. SNL rats but not naive or sham-operated rats exhibited behaviour indicative of allodynia. In anaesthetized rats, extracellular recordings were made from individual convergent dorsal horn neurones following stimulation of peripheral receptive fields electrically or with natural (innocuous mechanical, noxious mechanical and noxious thermal) stimuli. Spinal administration of galanin (0.5–50 μg) caused a slight facilitation of the neuronal responses to natural and electrical stimuli in naive rats and up to a 65% inhibition of neuronal responses in sham-operated rats following 50 μg galanin. In contrast, there was a marked inhibition of up to 80% of responses to both natural and electrical stimuli in SNL rats following spinal galanin administration. These results suggest that following peripheral nerve injury, there is plasticity in the levels of galanin and/or its receptors at spinal cord level so that the effect of exogenous galanin favours inhibitory function.

Dorsal root sensitivity to interleukin-1 beta, interleukin-6 and tumor necrosis factor in rats.

The release of inflammatory cytokines caused by a disrupted disc may play a critical role in pain production at nerve endings, axons, and nerve cell bodies. Herniated disc tissue has been shown to release inflammatory cytokines such as interleukin-1 beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor (TNF), and other algesic chemicals. This study was designed to characterize the effects of these proinflammatory cytokines on the somatosensory neural response at the dorsal root level in rats. It is hypothesized that their effects on nerve endings in disc and adjacent tissue contribute to low-back pain, and the effects on dorsal root axons and ganglia contribute to radiculopathy and sciatica. Surgically isolated sacral dorsal roots were investigated by electrophysiologic techniques. IL-1beta, IL-6, or TNF (100 ng, each) were applied onto the dorsal roots. Neural responses and mechanosensitivity of the receptive fields were evaluated over time. The results showed that 3 h after each cytokine application, the neural activity was statistically decreased. The mechanical sensitivity of the receptive fields increased at 90 min following IL-1beta or TNF application, and returned to normal more than 3 h after IL-1beta application. IL-1beta, IL-6, and TNF may be neurotoxic to dorsal root axons. Furthermore IL-1beta and TNF may sensitize the peripheral receptive fields. This study suggests that dorsal roots may be impaired by these proinflammatory cytokines.

Physiological properties of periodontal mechanosensitive neurones in the posteromedial ventral nucleus of rat thalamus

Unitary discharges of periodontal mechanosensitive (PM) neurones responding to mechanical tooth stimulation were recorded from the posteromedial ventral nucleus (VPM) of rat thalamus. PM neurones are distributed in the ventromedial area in the rostral two-thirds of the VPM nucleus. Maxillary and mandibular tooth-sensitive neurones are arranged in dorsoventral sequence. Of the PM neurones, 36% were slowly adapting to pressure applied to the tooth and 67% were rapidly adapting. The majority of PM units were sensitive to the contralateral incisor tooth. Response magnitudes of the slowly adapting neurones varied with stimulus direction and were directionally selective to mechanical tooth stimulation. The optimal stimulus direction was labiolingual or linguolabial. Rapidly adapting neurones were directionally non-selective to tooth stimulation. The threshold for mechanical stimulation was <0.05 N. Mean response latencies evoked by electrical stimulation of the peripheral receptive fields were 4.6 ms in the slowly adapting neurones and 5.8 ms in the rapidly adapting neurones.

Response properties of periodontal mechanosensitive neurones in the rat trigeminal sensory complex projecting to the posteromedial ventral nucleus of the thalamus

Unitary discharges from periodontal mechanosensitive (PM) neurones responding to mechanical stimulation of the tooth were recorded from the trigeminal sensory complex in the rat brainstem. Of the PM units recorded, 22% were activated by antidromic stimulation of the contralateral (20%) or ipsilateral (2%) posteromedial ventral nucleus of the thalamus. Although thalamic-projecting neurones were recorded extensively throughout the trigeminal sensory complex, they originated most often in the region from the caudal main sensory nucleus to the rostral subnucleus oralis of the trigeminal spinal tract nucleus. The response latencies of the rostral nucleus units to orthodromic stimulation of peripheral receptive fields and antidromic stimulation of the thalamus were significantly shorter than those of the caudal nucleus units. More than half were single-tooth units originating from incisor teeth. They responded continuously when pressure was applied to the tooth. The magnitude of the response varied with the direction of the stimulus. Maximal responses were obtained when the stimulus was applied labiolingually or vice versa. The threshold for mechanical stimulation of the tooth was less than 0.05 N. The rostrocaudal distribution and response properties of thalamic-projecting PM neurones were very similar to those of non-thalamic-projecting PM units that were not activated by antidromic stimulation of the thalamus.

Redox Modulation of T-Type Calcium Channels in Rat Peripheral Nociceptors

Although T-type calcium channels were first described in sensory neurons, their function in sensory processing remains unclear. In isolated rat sensory neurons, we show that redox agents modulate T currents but not other voltage- and ligand-gated channels thought to mediate pain sensitivity. Similarly, redox agents modulate currents through Ca v3.2 recombinant channels. When injected into peripheral receptive fields, reducing agents, including the endogenous amino acid L-cysteine, induce thermal hyperalgesia. This hyperalgesia is blocked by the oxidizing agent 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) and the T channel antagonist mibefradil. DTNB alone and in combination with mibefradil induces thermal analgesia. Likewise, L-cysteine induces mechanical DTNB-sensitive hyperalgesia in peripheral receptive fields. These data strongly suggest a role for T channels in peripheral nociception. Redox sites on T channels in peripheral nociceptors could be important targets for agents that modify pain perception.

GABA(A) and 5-HT(3) receptors are involved in dorsal root reflexes: possible role in periaqueductal gray descending inhibition.

The dorsal root reflex (DRR) is a measure of the central excitability of presynaptic inhibitory circuits in the spinal cord. Activation of the periaqueductal gray (PAG), a center for descending inhibition of spinal cord nociceptive transmission, induces release of variety of neurotransmitters in the spinal cord, including GABA and serotonin (5-HT). GABA has been shown to be involved in generation of DRRs. In this study, pharmacological agents that influence DRRs and their possible mechanisms were investigated. DRRs were recorded in anesthetized rats from filaments teased from the cut central stump of the left L(4) or L(5) dorsal root, using a monopolar recording electrode. Stimulating electrodes were placed either on the left sciatic nerve or transcutaneously in the left foot. Animals were paralyzed and maintained by artificial ventilation. Drugs were applied topically to the spinal cord. A total of 64 units were recorded in 34 Sprague-Dawley rats. Peripheral receptive fields were found for nine of these units. In these units, DRRs were evoked by brush, pressure, and pinch stimuli. Nine units were tested for an effect of electrical stimulation in the periaqueductal gray on the DRRs. In eight cases, DRR responses were enhanced following PAG stimulation. The background activity was 4.2 +/- 1.9 spikes/s (mean +/- SE; range: 0-97.7; n = 57). The responses to agents applied to the spinal cord were (in spikes/s): artificial cerebrospinal fluid, 7.1 +/- 3.6 (range: 0-86.9; n = 25); 0.1 mM GABA, 16.8 +/- 8.7 (range: 0-191.0; n = 22); 1.0 mM GABA, 116.0 +/- 26.5 (range: 0.05-1001.2; n = 50); and 1.0 mM phenylbiguanide (PBG), 68.1 +/- 25.3 (range: 0-1,073.0; n = 49). Bicuculline (0.5 mM, n = 27) and ondansetron (1.0 mM, n = 10) blocked the GABA and PBG effects, respectively (P < 0.05). Significant cross blockade was also observed. It is concluded that GABA(A) receptors are likely to play a key role in the generation of DRRs, but that 5-HT(3) receptors may also contribute. DRRs can be modulated by supraspinal mechanisms through descending systems.

Tactile Responses in the Granule Cell Layer of Cerebellar Folium Crus IIa of Freely Behaving Rats

We recorded activity from the granule cell layer (GCL) of cerebellar folium Crus IIa as freely moving rats engaged in a variety of natural behaviors, including grooming, eating, and free tactile exploration. Multiunit responses in the 1000-4500 Hz range were found to be strongly correlated with tactile stimulation of lip and whisker (perioral) regions. These responses occurred regardless of whether the stimulus was externally or self-generated and during both active and passive touch. In contrast, perioral movements that did not tactually stimulate this region of the face (e.g., chewing) produced no detectable increases in GCL activity. In addition, GCL responses were not correlated with movement extremes. When rats used their lips actively for palpation and exploration, the tactile responses in the GCL were not detectably modulated by ongoing jaw movements. However, active palpation and exploratory behaviors did result in the largest and most continuous bursts of GCL activity: responses were on average 10% larger and 50% longer during palpation and exploration than during grooming or passive stimulation. Although activity levels differed between behaviors, the position and spatial extent of the peripheral receptive field was similar over all behaviors that resulted in tactile input. Overall, our data suggest that the 1000-4500 Hz multiunit responses in the Crus IIa GCL of awake rats are correlated with tactile input rather than with movement or any movement parameter and that these responses are likely to be of particular importance during the acquisition of sensory information by perioral structures.

Peripheral administration of low pH solutions causes activation and sensitisation of convergent dorsal horn neurones in the anaesthetised rat

This is the first study to examine the effects of peripheral administration of acid on the activity of dorsal horn neurones in vivo. Extracellular recordings from convergent neurones revealed increases in neuronal activity evoked by administration of low pH solutions into the peripheral receptive field. Threshold for activity ranged from pH 5.85 to 2.5. The magnitude of responses increased with decreasing pH; maximum effects were achieved with pH 2.5 (648±181 action potentials/60 s, as compared to control-evoked activity of 86.3±29 action potentials/60 s). Activity lasted for up to 60 s, likely to represent the time for which the solutions were able to surmount the buffering capacity of the intact hindpaw. Significant sensitisation of the neurones to both innocuous (von Frey filament 9 g) and noxious (30 g) mechanical punctate stimuli was also observed.

MGlu5 receptors and nociceptive function II. mGlu5 receptors functionally expressed on peripheral sensory neurones mediate inflammatory hyperalgesia

Previous studies have demonstrated that the metabotropic glutamate receptor subtype 5 (mGlu5 receptor) is expressed in the cell bodies of rat primary afferent neurones. We have further investigated the function and expression of mGlu5 receptors in primary afferent neurones, and their role in inflammatory nociception. Freund's complete adjuvant-induced inflammatory hyperalgesia of the rat hind paw was significantly reduced by intraplantar, but not by intracerebroventricular or intrathecal microinjection of the selective mGlu5 receptor antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP). Pharmacological comparison in vivo of the nociceptive effects of glutamate, and ionotropic and metabotropic glutamate (mGlu) receptor agonists applied to the rat hind paw, indicated that group I mGlu receptor agonists induce a dose-dependent decrease in paw withdrawal threshold (mechanical hyperalgesia). Group I mGlu agonist-induced hyperalgesia was inhibited by co-microinjection of MPEP, but not by the mGlu1 receptor antagonist ( S)-4-carboxy-phenylglycine (4-CPG). Carrageenan-induced inflammatory hyperalgesia was inhibited by pre-treatment of the inflamed hind paw with MPEP, but not following MPEP injection into the contralateral hind paw. Dorsal horn neurones receiving peripheral nociceptive and non-nociceptive afferent input were recorded in anaesthetized rats following microinjection of CHPG into their peripheral receptive fields. CHPG significantly increased the frequency and duration of firing of dorsal horn wide dynamic range (WDR) neurones and this activity was prevented by co-administration of CHPG and MPEP into their receptive fields. Immunohistochemical experiments revealed the co-expression of mGlu5 receptor protein and βIII tubulin in skin from naive rats, indicating the constitutive expression of mGlu5 receptors on peripheral neurones. Double-labelling of adult rat DRG cells with mGlu5 receptor and vanilloid receptor subtype 1 antisera also supports the expression of mGlu5 receptors on peripheral nociceptive afferents. These results suggest that mGlu5 receptors expressed on the peripheral terminals of sensory neurones are involved in nociceptive processes and contribute to the hyperalgesia associated with inflammation.

Unaltered peripheral excitatory actions of nociceptin contrast with enhanced spinal inhibitory effects after carrageenan inflammation: an electrophysiological study in the rat

Nociceptin (orphanin FQ) is the endogenous agonist of the opioid receptor-like (ORL-1) receptor. The actions of this peptide have been studied extensively at a number of sites with diverse actions being reported. Here, in a rat model of peripheral inflammation, we examine the effects of nociceptin on the responses of dorsal horn neurones when applied directly to the spinal cord and, in separate studies, into the peripheral receptive fields in the hindpaw of the halothane anaesthetized rat. As changes in the receptor density and expression of the message for nociceptin have been reported after inflammation we have compared these actions to previously reported effects in normal animals. The dose-dependent inhibitory actions of nociceptin on C-fibre evoked responses and input (measures of presumed pre-synaptic excitability) are increased 3–4 h after inflammation whereas its inhibitory effects on post-synaptic mechanisms (wind-up) remain unchanged. These inhibitory effects were partly reversible by high doses of naloxone. This increased potency of nociceptin after inflammation is consistent with an increased receptor density in the superficial spinal cord. In contrast, the peripheral administration of nociceptin produced dose-dependent excitations of dorsal horn neurones and a degree of sensitization to mechanical stimuli. This peripheral action was unchanged after inflammation. These diverse site-dependent actions of nociceptin further emphasize the complexities of this novel opioid system.

Electrophysiological assessment of the cutaneous arborization of Adelta-fiber nociceptors.

Little is known about the relationship between the branching structure and function of physiologically identified cutaneous nociceptor terminals. The axonal arborization itself, however, has an impact on the afferent signal that is conveyed along the parent axon to the CNS. We therefore developed electrophysiological techniques to investigate the branching structure of cutaneous nociceptors. Single-fiber recordings were obtained from physiologically identified nociceptors that innervated the hairy skin of the monkey. Electrodes for transcutaneous stimulation were fixed at two separate locations inside the receptive field. For 32 Adelta-fiber nociceptors, distinct steps in latency of the recorded action potential were observed as the intensity of the transcutaneous electrical stimulus increased, indicating discrete sites for action potential initiation. The number of discrete latencies at each stimulation location ranged from 1 to 9 (3.7 +/- 0. 2; mean +/- SE) and the mean size of the latency step was 9.9 +/- 1. 0 ms (range: 0.4-89.1 ms). For seven Adelta fibers, collision techniques were used to locate the position of the branch point where the daughter fibers that innervated the two locations within the receptive field join the parent axon. To correct for changes in electrical excitability at the peripheral terminals, collision experiments between the two skin locations and between each skin location and a nerve trunk electrode were necessary. Nine branch points were studied in the seven Adelta fibers; the mean propagation time from the action potential initiation site to the branch point was 31 +/- 5 ms corresponding to a distance of 54 +/- 10 mm. Almost half of the daughter branches were unmyelinated. These results demonstrate that collision techniques can be used to study the functional anatomy of physiologically identified nociceptive afferent terminals. Furthermore these results indicate that some nociceptive afferents branch quite proximal to their peripheral receptive field. Occlusion of action potential activity can occur in these long branches such that the shorter branches dominate in the response to natural stimuli.