C-fibre Nociceptors Research Articles

Differential Roles of Galanin on Mechanical and Cooling Responses at the Primary Afferent Nociceptor

BackgroundGalanin is expressed in a small percentage of intact small diameter sensory neurons of the dorsal root ganglia and in the afferent terminals of the superficial lamina of the dorsal horn of the spinal cord. The neuropeptide modulates nociception demonstrating dose-dependent pro- and anti-nociceptive actions in the naïve animal. Galanin also plays an important role in chronic pain, with the anti-nociceptive actions enhanced in rodent neuropathic pain models. In this study we compared the role played by galanin and its receptors in mechanical and cold allodynia by identifying individual rat C-fibre nociceptors and characterising their responses to mechanical or acetone stimulation.ResultsMechanically evoked responses in C-fibre nociceptors from naive rats were sensitised after close intra-arterial infusion of galanin or Gal2-11 (a galanin receptor-2/3 agonist) confirming previous data that galanin modulates nociception via activation of GalR2. In contrast, the same dose and route of administration of galanin, but not Gal2-11, inhibited acetone and menthol cooling evoked responses, demonstrating that this inhibitory mechanism is not mediated by activation of GalR2. We then used the partial saphenous nerve ligation injury model of neuropathic pain (PSNI) and the complete Freund’s adjuvant model of inflammation in the rat and demonstrated that close intra-arterial infusion of galanin, but not Gal2-11, reduced cooling evoked nociceptor activity and cooling allodynia in both paradigms, whilst galanin and Gal2-11 both decreased mechanical activation thresholds. A previously described transgenic mouse line which inducibly over-expresses galanin (Gal-OE) after nerve injury was then used to investigate whether manipulating the levels of endogenous galanin also modulates cooling evoked nociceptive behaviours after PSNI. Acetone withdrawal behaviours in naive mice showed no differences between Gal-OE and wildtype (WT) mice. 7-days after PSNI Gal-OE mice demonstrated a significant reduction in the duration of acetone-induced nociceptive behaviours compared to WT mice.ConclusionsThese data identify a novel galaninergic mechanism that inhibits cooling evoked neuronal activity and nociceptive behaviours via a putative GalR1 mode of action that would also be consistent with a TRP channel-dependent mechanism.

The Cav3.2 T‐type calcium channel regulates temporal coding in mouse mechanoreceptors

In mammals there are three types of low-voltage-activated (LVA) calcium channels,Cav3.1, Cav3.2 and Cav3.3, which all give rise to T-type Ca2+currents. T-type Ca2+currents have long been known to be highly enriched in a sub-population of medium-sized sensory neurones in the dorsal root ganglia (DRG). However, the identity of the T-type-rich sensory neurones has remained controversial and the precise physiological role of the Cav3.2 calcium channel in these sensory neurones has not been directly addressed. Here we show, using Cav3.2−/− mutant mice,that these channels are essential for the normal temporal coding of moving stimuli by specialized skin mechanoreceptors called D-hair receptors.We show that D-hair receptors from Cav3.2−/− fire approximately 50% fewer spikes in response to ramp-and-hold displacement stimuli compared to wild type receptors. The reduced sensitivity of D-hair receptors in Cav3.2−/− mice is chiefly due to an increase in the mechanical threshold and a substantial temporal delay in the onset of high-frequency firing to moving stimuli.We examined the receptive properties of other cutaneous mechano receptors and Aδ- and C-fibre nociceptors in Cav3.2−/− mice, but found no alteration in their mechanosensitivity compared to Cav3.2+/+mice. However, C-fibre nociceptors recorded in Cav3.2−/− mutant mice displayed a small but statistically significant reduction in their spiking rate during noxious heat ramps when compared to C-fibres in control mice. The T-type calcium channel Cav3.2 is thus not only a highly specific marker of D-hair receptors but is also required to maintain their high sensitivity and above all to ensure ultra rapid temporal detection of skin movement.

CS03-01 - Learning and memory in pain pathways

Hyperalgesia frequently results from injuries or inflammation of peripheral tissues, including nervous tissue and paradoxically also from the treatment with μ-opioid receptor agonists. Compelling evidence indicates that signal amplification in central pain pathways plays an important role for the maintenance of hyperalgesia1. In superficial spinal dorsal horn synaptic transmission between nociceptive C-fibres and lamina I projection neurons can be potentiated for prolonged periods of time in an activity dependent manner. These forms of synaptic long-term potentiation (LTP) can be securely prevented when opioids are applied during afferent stimulation. The blockage of LTP induction by opioids is a likely mechanism or pre-emptive analgesia. Upon withdrawal from high doses of opioids, however, LTP may develop at C-fibre synapses. During the latter form of LTP induction presynaptic activity at C-fibres is depressed rather than enhanced. Despite these fundamental differences in the induction, activity dependent- and opioidergic LTP share signalling pathways. This includes the activation of NMDA receptors, the rise in postsynaptic Ca2+ concentration and the activation of protein kinase C. Induction of opioidergic LTP further requires postsynaptic G-protein coupling which is in contrast to the presynaptic inhibition by opioids. LTP induction is abolished by blocking the Ca2+ rise upon withdrawal from the opioids. It is likely that the potentiation in synaptic strength translates into enhanced pain behaviour1. Plasticity at the first synapse in pain pathways is a promising target for the prevention and treatment of hyperalgesia of various origins.

Differentiation between Aδ and C fibre evoked nociceptive reflexes by TTX resistance and opioid sensitivity in the cat

The contribution of nociceptive Aδ-fibres and C-fibres of the central pad of the foot to nociceptive spinal flexor reflex pathways (FRA-type) and to nociceptive excitatory reflex pathways to foot extensors (non-FRA type) was investigated in high spinal cats. Persisting effects after complete blocking of A-fibres by tetrodotoxin (TTX) application were thus attributed to nociceptive C-fibres. The results revealed that both Aδ and C-fibre afferents contributed to nociceptive reflexes of a FRA-pattern and of a non-FRA pattern. Thereby, the effects of Aδ-fibres were evoked with a distinctly shorter delay than those of C-fibres. Furthermore, Aδ-fibres partly exerted a significant inhibitory influence on the C-fibre action in FRA and non-FRA pathways. Treatment with different opioid receptor agonists (DAMGO and DSLET) and subsequently with naloxone revealed that a distinct part of the opioid action on nociceptive reflex pathways of the FRA-type and of the non-FRA type was evidently exerted via Aδ-fibres.

Peripheral sensitisation of nociceptors via G‐proteindependent potentiation of mechanotransduction currents

Mechanical stimuli impinging on the skin are converted into electrical signals by mechanically gated ion channels located at the peripheral nerve endings of dorsal root ganglion (DRG) neurons. Under inflammatory conditions sensory neurons are commonly sensitised to mechanical stimuli; a putative mechanism that may contribute to such sensitisation of sensory neurons is enhanced responsiveness of mechanotransduction ion channels. Here we show that the algogens UTP and ATP potentiate mechanosensitive RA currents in peptidergic nociceptive DRG neurons and reduce thresholds for mechanically induced action potential firing in these neurones. Pharmacological characterisation suggests that this effect is mediated by the Gq-coupled P2Y2 nucleotide receptor. Moreover, using the in vitro skin nerve technique, we show that UTP also increases action potential firing rates in response to mechanical stimuli in a subpopulation of skin C-fibre nociceptors. Together our findings suggest that UTP sensitises a subpopulation of cutaneous C-fibre nociceptors via a previously undescribed G-protein-dependent potentiation of mechanically activated RA-type currents.

Long-term impact of neonatal intensive care and surgery on somatosensory perception in children born extremely preterm

Alterations in neural activity due to pain and injury in early development may produce long-term effects on sensory processing and future responses to pain. To investigate persistent alterations in sensory perception, we performed quantitative sensory testing (QST) in extremely preterm (EP) children (n=43) recruited from the UK EPICure cohort (born less than 26 weeks gestation in 1995) and in age and sex matched term-born controls (TC; n=44). EP children had a generalized decreased sensitivity to all thermal modalities, but no difference in mechanical sensitivity at the thenar eminence. EP children who also required neonatal surgery had more marked thermal hypoalgesia, but did not differ from non-surgical EP children in the measures of neonatal brain injury or current cognitive ability. Adjacent to neonatal thoracotomy scars there was a localized decrease in both thermal and mechanical sensitivity that differed from EP children with scars relating to less invasive procedural interventions or from those without scars. No relationship was found between sensory perception thresholds and current pain experience or pain coping styles in EP or TC children. Neonatal care and surgery in EP children are associated with persistent modality-specific changes in sensory processing. Decreases in mechanical and thermal sensitivity adjacent to scars may be related to localized tissue injury, whereas generalized decreases in thermal sensitivity but not in mechanical sensitivity suggest centrally mediated alterations in the modulation of C-fibre nociceptor pathways, which may impact on responses to future pain or surgery.

A Role For Wind-Up in Trigeminal Sensory Processing: Intensity Coding of Nociceptive Stimuli in the Rat

Wind-up is a progressive, frequency-dependent increase in the excitability of trigeminal and spinal dorsal horn wide dynamic range (WDR) nociceptive neurons evoked by repetitive stimulation of primary afferent nociceptive C-fibres. The correlate of wind-up in humans is temporal summation, which is an increase in pain perception to repetitive constant nociceptive stimulation. Although wind-up is widely used as a tool for studying the processing of nociceptive information, including central sensitization, its actual role is still unknown. Here, we recorded from trigeminal WDR neurons using in vivo electrophysiological techniques in rats and assessed the wind-up phenomenon in response to stimuli of different intensities and frequencies. First, we found that the amplitude of C-evoked responses of WDR neurons to repetitive stimulation increased progressively to reach a peak, then consistently showed a stable or slightly decreasing plateau phase. Only the first phase of this time course fitted in with the wind-up description. Therefore, to assess wind-up, we measured a limited number of initial responses. Second, we showed that wind-up, i.e. the slope of the frequency-dependent increase in the response to C-fibre stimulation, was linearly correlated to the stimulus intensity. Intensities of brief C-fibre inputs were thus coded into frequencies of action potentials by second-order neurons through frequency-dependent potentiation of the evoked responses. Third, wind-up also occurred at stimulation intensities below the threshold for C-evoked responses in WDR neurons, suggesting that wind-up can amplify subthreshold C-fibre inputs to WDR neurons. This might account for the observation that sparse, subliminal, neuronal activity in nociceptors can become painful via central integration of neural responses. Altogether, the present results show that wind-up can provide trigeminal WDR neurons with the capability to encode the intensity of short-duration orofacial nociceptive stimuli and to detect subthreshold nociceptive input. Thus, not only may wind-up play a physiological role in trigeminal sensory processing, but its enhancement may also underlie the pathophysiology of chronic orofacial pain conditions.

Disruption of axoplasmic transport induces mechanical sensitivity in intact rat C‐fibre nociceptor axons

Peripheral nerve inflammation can cause axons conducting through the inflamed site to become mechanically sensitive. Axonal mechanical sensitivity (AMS) of intact axons may explain symptoms in a diverse number of conditions characterized by radiating pain evoked by movements of the affected nerve. Because nerve inflammation also disrupts axoplasmic transport, we hypothesized that the disruption of axoplasmic transport by nerve inflammation could cause the cellular components responsible for mechanical transduction to accumulate and become inserted at the inflamed site, causing AMS. This was tested by examining AMS in C-fibre nociceptors following the application of axoplasmic transport blockers (colchicine and vinblastine) to the sciatic nerve. Both 10 mm colchicine and 0.1 mm vinblastine caused AMS to develop in 30.6% and 33.3% of intact axons, respectively (P < 0.05 compared to sham treatment). Since high doses of colchicine (> 50 mm) can damage axons, and inflammation is involved in the removal of axonal debris, experiments were performed to assess conduction across the treatment site as well as signs of inflammation. Results indicated minimal axonal loss (95% of A- and C-fibres conducting), consistent with the normal microscopic appearance of the colchicine treatment site and absence of ED1-positive (recruited) macrophages. In a separate series of experiments, the block of axoplasmic transport proximal to a localized neuritis significantly reduced inflammation-induced AMS (15.6% compared to 55.6%; P < 0.05), further supporting that the components necessary for AMS are moved by anterograde transport. In summary, nerve inflammation that causes the disruption of axoplasmic transport in patients with painful conditions may result in the accumulation and insertion of mechanosensitive elements at the inflamed site.

Selective inhibition from the anterior hypothalamus of C- versus A-fibre mediated spinal nociception

Modulation of spinal nociception from the anterior hypothalamus/preoptic area (AH/POA), and consequent alterations in the pain experience may contribute to integrated responses brought into play during fear or stress and as part of the sickness response. This study was designed to compare the effects of descending control from AH/POA on A- versus C-fibre-evoked spinal nociception, since any differential control is of behavioural and clinical importance given that A-fibre and C-fibre nociceptors convey different qualities of the pain signal (first and second pain, respectively), and play different roles in the development and maintenance of chronic pain states. In anaesthetised rats, electromyographic responses were recorded to monitor thresholds of withdrawal to slow (2.5 °C s −1) or fast (7.5 °C s −1) rates of skin heating of the hindpaw, to preferentially activate C- or A-nociceptors, respectively. Neuronal activation by microinjection of dl-homocysteic acid at sites within a specific region of AH/POA, lateral area of the anterior hypothalamus (LAAH), significantly increased response thresholds to slow heating rates ( p < 0.02, n = 11), but not those to fast rates of heating ( p = 0.48, n = 10). Injection of DLH adjacent to LAAH ( n = 9) had no significant effect on responses to slow ( n = 8) or fast ( n = 9) rates of skin heating. The functional significance of differential descending control of spinal processing of C- and A-nociceptive inputs is discussed with respect to roles both of the LAAH in pain processing, and of C- and A-nociceptive inputs in acute and chronic pain.

145 Spontaneous pain behaviour is related to spontaneous firing frequency in uninjured nociceptive c-fibre neurons after spinal nerve axotomy

145 Spontaneous pain behaviour is related to spontaneous firing frequency in uninjured nociceptive c-fibre neurons after spinal nerve axotomy

Acid-sensitive vagal sensory pathways and cough

Acid is an important mediator in the pathogenesis of cough. Inhalation of exogenous acid triggers cough and endogenous acid may contribute to cough in respiratory diseases. Acid directly stimulates vagal bronchopulmonary sensory nerves that regulate the cough reflex. Consistent with their putative role in defence against aspiration and inhaled irritants, A δ-fibre nociceptors in the large airways are most efficiently stimulated by rapid acidification. In contrast, acid-sensitive properties of the C-fibre nociceptors allow for continuous monitoring of pH which is likely important in inflammation. Acid is also the single most important mediator in the pathogenesis of cough due to gastro-oesophageal reflux (GOR). The cough pathways can be sensitized by the sensory inputs from the oesophagus. This sensitization is likely mediated by a subset of the vagal oesophageal sensory nerves distinguished by discriminative responsiveness to noxious stimuli (nociceptors). The receptors underlying acid sensitivity of vagal sensory nerves are incompletely understood. The role of TRPV1 has been established but the roles of acid-sensing ion channels (ASIC) and other receptors await more definitive investigation. Here, we provide a brief overview of the cough-related acid-sensitive sensory pathways and discuss the mechanisms of acid sensitivity.

Functional evaluation of regenerated and misrouted low threshold mechanoreceptors and polymodal nociceptors in the skin of Rat hindfeet after crush lesions to the sciatic nerve

Tracer studies on motor axons after nerve crush injuries have indicated that misrouting may occur even when the endoneurium is intact. Misrouting of regenerated polymodal nociceptive C-fibres and low threshold mechanoreceptive axons have been studied functionally in 50 rats three months after unilateral crush lesions to the sciatic nerve. Two weeks before evaluation the tibial fascicle (or the peroneal fascicle) above the lesion was cut and tied off. In this way only functional regeneration of misrouted axons was tested. Misrouted low threshold mechanoreceptive axons and polymodal nociceptor C-fibres were found after regeneration in both glabrous and hairy skin. We conclude that functional misdirection of both myelinated and unmyelinated sensory axons innervating either glabrous or hairy skin can occur after a crush lesion to a peripheral nerve in rats.

A reliable method for the preferential activation of C- or A-fibre heat nociceptors

There is strong evidence that A- and C-fibre nociceptors evoke significantly different sensory experiences, are differentially sensitive to pharmacological intervention, and play different roles in pain pathology. It is therefore of considerable interest to be able to selectively activate one fibre type or the other in studies of nociceptive processing. Here, we report significant modifications to a non-invasive technique, first described by Yeomans et al. [Pain 59 (1994) 85; Pain 68 (1996) 141; Pain 68 (1996) 133], which uses different rates of skin heating to preferentially activate A- or C-nociceptors. A copper disk (diameter: 4 mm) was used to transfer heat evenly across the dorsal surface of the rat hindpaw. Initial experiments established the relationship between the temperature at the skin surface and the sub-epidermal temperature. Subsequently, the vanilloid capsaicin, which sensitises unmyelinated C-mechanoheat nociceptors, was shown to decrease the thresholds of reflex responses evoked by slow rates of heating. In contrast thresholds of responses to fast rates of skin heating were unchanged, indicating that nociceptors activated by this stimulus were capsaicin-insensitive A-fibre heat nociceptors.

Non-phase locked electroencephalogram (EEG) responses to CO2 laser skin stimulations may reflect central interactions between A partial partial differential- and C-fibre afferent volleys.

By co-activating A partial partial differential- and C-fibre nociceptors, intense CO2 laser heat stimuli produce a dual sensation, composed of first and second pain, but induce only a single A partial partial differential-fibre related late laser evoked potential (LEP). However, when avoiding concomitant activation of A partial partial differential-fibres, C-fibre related ultra-late LEPs are recorded. This poorly understood phenomenon was re-investigated using a method which, unlike time-domain averaging, reveals electroencephalogram (EEG) changes whether or not phase-locked to stimulus onset. CO2 laser stimuli were applied to the dorsum of the hand. Reaction-time was used to discriminate between A partial partial differential- and C-fibre mediated detections. Analyses were performed using a method based on the time-frequency wavelet transform of EEG epochs. This study revealed: (1) a novel non-phase-locked component related to the activation of A partial partial differential-fibres occurring at similar latencies as the late LEP; and (2) a widespread post-stimulus event-related desynchronization (ERD) induced by both A partial partial differential- and C-fibres. A partial partial differential- and C-fibre related LEPs could be electrophysiological correlates of similar brain processes, which, when already engaged by A partial partial differential-fibres, cannot or do not need to be reactivated by the later arriving C-fibre afferent volley. A partial partial differential-fibre related ERD could reflect a transient change of state of brain structures generating these responses.

Transganglionic transport of choleragenoid by capsaicin-sensitive c-fibre afferents to the substantia gelatinosa of the spinal dorsal horn after peripheral nerve section

Choleratoxin B subunit-binding thick myelinated, A-fibre and unmyelinated, capsaicin-sensitive nociceptive C-fibre primary afferent fibres terminate in a strict topographic and somatotopic manner in the spinal cord dorsal horn. Injection of choleratoxin B subunit–horseradish peroxidase conjugate into injured but not intact peripheral nerves produced transganglionic labelling of primary afferents not only in the deeper layers (Rexed’s laminae III–IV), but also in the substantia gelatinosa (Rexed’s laminae II) of the spinal dorsal horn. This was interpreted in terms of a sprouting response of the Aβ-myelinated afferents and suggested a contribution to the pathogenesis of neuropathic pain [Nature 355 (1992) 75; J Comp Neurol 360 (1995) 121]. By utilising the selective neurotoxic effect of capsaicin, we examined the role of C-fibre sensory ganglion neurons in the mechanism of this phenomenon. Elimination of these particular, capsaicin-sensitive C-fibre afferents by prior intrathecal or systemic capsaicin treatment inhibited transganglionic labelling by the choleratoxin B subunit–horseradish peroxidase conjugate of the substantia gelatinosa evoked by chronic sciatic nerve section. More importantly, prior perineural capsaicin treatment of the transected nerve proximal to the anticipated site of injection of choleragenoid 12 hours later prevented the labelling of the substantia gelatinosa, but not that of the deeper layers. Electron microscopic examination of the dorsal roots revealed no significant difference in the proportion of labelled myelinated fibres relating to the intact (54.4±5.5%) and the transected (62.4±5.4%) sciatic nerves. In contrast, the proportion of labelled unmyelinated dorsal root axons relating to the transected, but not the intact nerves showed a significant, six-fold increase after sciatic nerve transection (intact: 4.9±1.3%; transected: 35±6.7%). These observations indicate that peripheral nerve lesion-induced transganglionic labelling of the substantia gelatinosa by choleratoxin B subunit–horseradish peroxidase may be primarily accounted for by the uptake and transganglionic transport of choleragenoid by injured capsaicin-sensitive C-fibre afferents rather than a sprouting response of A-fibre afferents. The present findings suggest an essential role of capsaicin-sensitive primary sensory neurons in lesion-induced spinal neuroplastic changes and provide further support for C-fibre nociceptor neurons being promising targets for the development of new strategies in pain management.

Quantitative responses of spinothalamic lamina I neurones to graded mechanical stimulation in the cat.

Nociceptive spinothalamic tract (STT) neurones in lamina I of the lumbosacral spinal cord of anaesthetized cats were characterized by recording their responses to graded mechanical stimulation with controlled forces of 10-120 g and probes of 5.0, 0.5 and 0.1 mm(2) contact area. Neurones were identified by antidromic activation from the contralateral thalamus, and cells that responded to noxious stimulation were categorized as either nociceptive specific (NS, n = 20) or as polymodal nociceptive (HPC, responsive to heat, pinch and cold, n = 19) based on their responses to quantitative thermal stimuli. The mean responses of the 39 units increased linearly as stimulus intensity increased, and the population stimulus-response curves evoked by each of the three probes were all significantly different from each other. Thresholds were 45 g for the 5.0 mm(2) probe, 30 g for the 0.5 mm(2) probe and 20 g for the 0.1 mm(2) probe. Further analysis showed that the NS neurones encoded both stimulus intensity and area (probe size) significantly better than HPC neurones in terms of their thresholds to individual probes, their peak discharge rates, their suprathreshold responsiveness and their ability to discriminate the three different probe sizes. These differences are consistent with the known differences between the mechanical encoding properties of A-fibre nociceptors, which provide the dominant inputs to NS neurones, and C-fibre nociceptors, which are the dominant inputs to HPC cells. Comparison of the stimulus-response curves of NS and HPC neurones indicated that the discharge of NS neurones better match the psychophysics of mechanical pain sensations in humans than the discharge of the HPC neurones do. Our findings support the view that NS neurones have a prominent role in mechanical pain and sharpness, and they corroborate the concept that the lamina I STT projection comprises several discrete channels that are integrated in the forebrain to generate qualitatively distinct sensations.

Roles of capsaicin-insensitive nociceptors in cutaneous pain and secondary hyperalgesia.

Polymodal nociceptors respond to mechanical, thermal and chemical stimuli. Whereas sensitivities to heat and to the irritant substance capsaicin have recently been linked via the properties of the vanilloid receptor type 1 receptor ion channel, sensitivity to noxious mechanical stimuli such as the pinpricks used in clinical neurology seems to be unrelated. We investigated the peripheral neural basis of pinprick pain using quantitative psychophysical techniques combined with selective conduction block by nerve compression and selective desensitization by topical capsaicin treatment. Complete A-fibre block by compression of the superficial radial nerve (criterion: loss of first pain sensation) lowered the stimulus-response function for pinprick pain (-82 +/- 6% versus baseline). Topical pretreatment of the skin with a 10% capsaicin cream also lowered the pinprick stimulus-response function (-32 +/- 10%), whereas laser-evoked heat pain was eliminated completely (-96 +/- 2%). Under combined capsaicin desensitization and A-fibre blockade, pinprick pain was eliminated completely (-98 +/- 1%). Intradermal injection of 40 microg capsaicin into normal skin between two skin areas that had been pretreated with either capsaicin cream or vehicle produced secondary hyperalgesia with a 260% enhancement of the stimulus-response function for pinprick pain in both areas. In contrast, axon reflexive flare spread only into the vehicle-treated area. These results suggest that capsaicin-sensitive afferents, including polymodal A-fibre and C-fibre nociceptors, make a small contribution to pinprick pain and that capsaicin-insensitive C-fibres do not contribute significantly to either mechanical or heat pain. Pinprick pain is mediated primarily by capsaicin-insensitive A-fibre nociceptors, which include high-threshold mechanoreceptors and type I mechano-heat nociceptors. In addition, central sensitization to input from these A-fibre nociceptors is the primary mechanism that accounts for the enhanced pain in response to punctate mechanical stimuli in the zone of secondary hyperalgesia. These capsaicin-insensitive A-fibre nociceptors may also mediate hyperalgesia in neuropathic pain.

Selective activation of nociceptors by P2X receptor agonists in normal and inflamed rat skin.

1. ATP can elicit pain in humans and, together with other P2X channel agonists, can produce nocifensive responses in rodents. We used the rat in vitro skin-nerve preparation to quantify primary afferent responses to ATP and its stable analogue alpha,beta-methylene ATP in normal and carrageenan-inflamed skin. 2. Both ATP and alpha,beta-methylene ATP were found to specifically activate the peripheral terminals of Adelta and C-fibre nociceptors in the skin. Thirty-nine per cent of the nociceptors tested responded to the maximal dose of alpha,beta-methylene ATP (5 mM). In contrast, non-nociceptive, low-threshold mechano-sensitive fibres were never activated by the same agonist concentrations. 3. Amongst the nociceptor population, C-mechanoheat fibres (C-MH or polymodal nociceptors) were markedly more responsive to P2X agonists than mechanonociceptors (C-M nociceptors) with Adelta- or C-fibre axons. Both C-mechanoheat and C-mechanonociceptors were activated by alpha,beta-methylene ATP doses as low as 50 microM. 4. In skin inflamed with carrageenan 3-4 h before recording both the number of responsive C-fibre nociceptors and their response magnitude increased. The increased neural response under inflammatory conditions was largely observed in C-mechanoheat or polymodal nociceptors. After low doses of P2X agonists C-MH fibres but not C-M fibres developed elevated ongoing activity and this effect was only seen after carrageenan inflammation. The time course of alpha,beta-methylene ATP-evoked discharges in nociceptors was found to correlate well with the time course of behavioural nocifensive responses in rats to the same agonist described in a previous study (Hamilton et al. 1999). 5. We conclude that the rapid increase in the number of alpha,beta-methylene ATP responsive nociceptors and the increased magnitude of the neural response following carrageenan inflammation explains why very low concentrations of such agonists can cause pain in inflammatory states.

Contribution of TTX-resistant C-fibres and Adelta-fibres to nociceptive flexor-reflex and non-flexor-reflex pathways in cats.

The contribution of Adelta-fibres and C-fibres activated by noxious heat stimulation of the central pad of the foot to nociceptive spinal flexor reflex pathways (FRA-type) and to nociceptive excitatory reflex pathways to foot extensors (non-FRA type) was investigated in high spinal cats. A-fibres were completely blocked by tetrodotoxin (TTX), leaving C-fibre conduction intact. Thus, effects persisting after TTX were attributed to nociceptive C-fibres while the contribution of nociceptive Adelta-fibres was defined by the difference between those effects and the control effects before TTX. The initial action of noxious stimulation on both types of reflex action was mediated predominantly by Adelta-fibres, while the later action was mainly mediated by C-fibres. In two (out of seven) experiments Adelta-fibres exerted a significant inhibitory influence on the C-fibre action in FRA pathways, but such an inhibitory interaction between the two fibre groups was absent in the non-FRA reflex pathways. The technique of TTX application at the peripheral nerve proved to be a reliable method for a long-lasting selective investigation of C-fibre effects. The results revealed that both Adelta- and C-fibres contributed to nociceptive FRA and non-FRA reflex pathways.

Secondary hyperalgesia to punctate mechanical stimuli. Central sensitization to A-fibre nociceptor input.

Tissue injury induces enhanced pain sensation to light touch and punctate stimuli in adjacent, uninjured skin (secondary hyperalgesia). Whereas hyperalgesia to light touch (allodynia) is mediated by A-fibre low-threshold mechanoreceptors, hyperalgesia to punctate stimuli may be mediated by A- or C-fibre nociceptors. To disclose the relative contributions of A- and C-fibres to the hyperalgesia to punctate stimuli, the superficial radial nerve was blocked by pressure at the wrist in nine healthy subjects. Secondary hyperalgesia was induced by intradermal injection of 40 microg capsaicin, and pain sensitivity in adjacent skin was tested with 200 micron diameter probes (35-407 mN). The progress of conduction blockade was monitored by touch, cold, warm and first pain detection and by compound sensory nerve action potential. When A-fibre conduction was blocked completely but C-fibre conduction was fully intact, pricking pain to punctate stimuli was reduced by 75%, but burning pain to capsaicin injection remained unchanged. In normal skin without A-fibre blockade, pain ratings to the punctate probes increased significantly by a factor of two after adjacent capsaicin injection. In contrast, pain ratings to the punctate probes were not increased after capsaicin injection when A-fibre conduction was selectively blocked. However, hyperalgesia to punctate stimuli was detectable immediately after block release, when A-fibre conduction returned to normal. In conclusion, the pricking pain to punctate stimuli is predominantly mediated by A-fibre nociceptors. In secondary hyperalgesia, this pathway is heterosynaptically facilitated by conditioning C-fibre input. Thus, secondary hyperalgesia to punctate stimuli is induced by nociceptive C-fibre discharge but mediated by nociceptive A-fibres.