Matthias Ringkamp

Thermoreceptors and thermosensitive afferents

Cutaneous thermosensation plays an important role in thermal regulation and detection of potentially harmful thermal stimuli. Multiple classes of primary afferents are responsive to thermal stimuli. Afferent nerve fibers mediating the sensation of non-painful warmth or cold seem adapted to convey thermal information over a particular temperature range. In contrast, nociceptive afferents are often activated by both, painful cold and heat stimuli. The transduction mechanisms engaged by thermal stimuli have only recently been discovered. Transient receptor potential (TRP) ion channels that can be activated by temperatures over specific ranges potentially provide the molecular basis for thermosensation. However, non-TRP mechanisms are also likely to contribute to the transduction of thermal stimuli. This review summarizes findings regarding the transduction proteins and the primary afferents activated by innocuous and noxious cold and heat.

Psychophysical and Physiological Evidence for Parallel Afferent Pathways Mediating the Sensation of Itch

The neuronal pathways for itch have been characterized mainly based on responses to histamine. Intracutaneous application of histamine produces intense itch and a large area of axon-reflexive vasodilation (“flare”) around the application site. Both phenomena are thought to be mediated through neuronal activity in itch-specific, mechanoinsensitive C-fiber afferents (CMi). However, mechanical and electrical stimuli that do not activate CMi fibers can cause the sensation of itch, and itch may occur without flare, suggesting that other neuronal itch pathways exist. Because cutaneous application of spicules from the plant Mucuna pruriens (cowhage) has been anecdotally reported to produce itch without flare, we performed psychophysical experiments to investigate whether the mechanisms underlying cowhage- and histamine-induced itch differ. Although histamine and cowhage produced itch of similar magnitude, the itch to cowhage was not correlated with the itch to histamine; some subjects had intense itch to cowhage and little itch to histamine and visa versa. Laser Doppler measurements of blood flow revealed that histamine led to a large area of vasodilation, whereas cowhage produced vasodilation restricted to the application site. Pretreatment of the skin with an antihistamine blocked the itch produced by histamine but did not prevent cowhage-induced itch. Desensitization of the skin with topical capsaicin abolished cowhage-induced itch but did not significantly alter histamine-induced itch. These findings indicate that cowhage itch is signaled through a population of capsaicin-sensitive afferent nerve fibers that is distinct from CMi fibers mediating histamine-induced itch. Cowhage may be useful to investigate the neural pathway mediating nonhistaminergic itch.

A Role for Polymodal C-Fiber Afferents in Nonhistaminergic Itch

Recent psychophysical and electrophysiological studies in humans suggest the existence of two peripheral pathways for itch, one that is responsive to histamine and a second pathway that can be activated by nonhistaminergic pruritogens (e.g., cowhage spicules). To explore the peripheral neuronal pathway for nonhistaminergic itch, behavioral responses and neuronal activity in unmyelinated afferent fibers were assessed in monkey after topical application of cowhage spicules or intradermal injection of histamine and capsaicin. Cowhage and histamine, but not capsaicin, evoked scratching behavior indicating the presence of itch. In single-fiber recordings, cowhage, histamine and/or capsaicin were applied to the cutaneous receptive field of 43 mechano-heat-sensitive C-fiber (CMH) nociceptors. The majority of CMHs exhibited a prolonged response to cowhage (39 of 43) or histamine (29 of 38), but not to capsaicin (3 of 34). Seven CMHs were activated by cowhage but not histamine. The average response to cowhage was more than twice the response to histamine, and responses were not correlated. The response of the CMHs to a stepped heat stimulus (49°C, 3 s) was either quickly adapting (QC) or slowly adapting (SC). In contrast, the cowhage response was characterized by bursts of two or more action potentials (at ∼1 Hz). The total cowhage response of the QC fibers (97 action potentials/5 min) was twice that of the SC fibers (49 action potentials/5 min). A subset of QC fibers exhibited high-frequency intraburst discharges (∼30 Hz). These results suggest multiple mechanisms by which CMHs may encode itch to cowhage as well as pain to mechanical and heat stimuli.

Sensory neurons and circuits mediating itch

Chemicals that are used experimentally to evoke itch elicit activity in diverse subpopulations of cutaneous pruriceptive neurons, all of which also respond to painful stimuli. However, itch is distinct from pain: it evokes different behaviours, such as scratching, and originates from the skin or certain mucosae but not from muscle, joints or viscera. New insights regarding the neurons that mediate the sensation of itch have been gained from experiments in which gene expression has been manipulated in different types of pruriceptive neurons as well as from comparisons between psychophysical measurements of itch and the neuronal discharges and other properties of peripheral and central pruriceptive neurons.

A Role for Nociceptive, Myelinated Nerve Fibers in Itch Sensation

Despite its clinical importance, the underlying neural mechanisms of itch sensation are poorly understood. In many diseases, pruritus is not effectively treated with antihistamines, indicating the involvement of nonhistaminergic mechanisms. To investigate the role of small myelinated afferents in nonhistaminergic itch, we tested, in psychophysical studies in humans, the effect of a differential nerve block on itch produced by intradermal insertion of spicules from the pods of a cowhage plant (Mucuna pruriens). Electrophysiological experiments in anesthetized monkey were used to investigate the responsiveness of cutaneous, nociceptive, myelinated afferents to different chemical stimuli (cowhage spicules, histamine, capsaicin). Our results provide several lines of evidence for an important role of myelinated fibers in cowhage-induced itch: (1) a selective conduction block in myelinated fibers substantially reduces itch in a subgroup of subjects with A-fiber-dominated itch, (2) the time course of itch sensation differs between subjects with A-fiber- versus C-fiber-dominated itch, (3) cowhage activates a subpopulation of myelinated and unmyelinated afferents in monkey, (4) the time course of the response to cowhage is different in myelinated and unmyelinated fibers, (5) the time of peak itch sensation for subjects with A-fiber-dominated itch matches the time for peak response in myelinated fibers, and (6) the time for peak itch sensation for subjects with C-fiber-dominated itch matches the time for the peak response in unmyelinated fibers. These findings demonstrate that activity in nociceptive, myelinated afferents contributes to cowhage-induced sensations, and that nonhistaminergic itch is mediated through activity in both unmyelinated and myelinated afferents.

Peripherally acting mu-opioid receptor agonist attenuates neuropathic pain in rats after L5 spinal nerve injury.

&NA; Studies in experimental models and controlled patient trials indicate that opioids are effective in managing neuropathic pain. However, side effects secondary to their central nervous system actions present barriers to their clinical use. Therefore, we examined whether activation of the peripheral mu‐opioid receptors (MORs) could effectively alleviate neuropathic pain in rats after L5 spinal nerve ligation (SNL). Systemic loperamide hydrochloride (0.3–10 mg/kg, s.c.), a peripherally acting MOR‐preferring agonist, dose‐dependently reversed the mechanical allodynia at day 7 post‐SNL. This anti‐allodynic effect produced by systemic loperamide (1.5 mg/kg, s.c.) was blocked by systemic pretreatment with either naloxone hydrochloride (10 mg/kg, i.p.) or methyl‐naltrexone (5 mg/kg, i.p.), a peripherally acting MOR‐preferring antagonist. It was also blocked by ipsilateral intraplantar pretreatment with methyl‐naltrexone (43.5 μg/50 μl) and the highly selective MOR antagonist CTAP (5.5 μg/50 μl). However, this anti‐allodynic effect of systemic loperamide was not blocked by intraplantar pretreatment with the delta‐opioid receptor antagonist naltrindole hydrochloride (45.1 μg/50 μl). The anti‐allodynic potency of systemic loperamide varied with time after nerve injury, with similar potency at days 7, 28, and 42 post‐SNL, but reduced potency at day 14 post‐SNL. Ipsilateral intraplantar injection of loperamide also dose‐dependently (10–100 μg/50 μl) reversed mechanical allodynia on day 7 post‐SNL. We suggest that loperamide can effectively attenuate neuropathic pain, primarily through activation of peripheral MORs in local tissue. Therefore, peripherally acting MOR agonists may represent a promising therapeutic approach for alleviating neuropathic pain.

Patterns of activity-dependent conduction velocity changes differentiate classes of unmyelinated mechano-insensitive afferents including cold nociceptors, in pig and in human

&NA; Activity‐dependent slowing of conduction velocity (ADS) differs between classes of human nociceptors. These differences likely reflect particular expression and use‐dependent slow inactivation of axonal ion channels and other mechanisms governing axonal excitability. In this study, we compared ADS of porcine and human cutaneous C‐fibers. Extracellular recordings were performed from peripheral nerves, using teased fiber technique in pigs and microneurography in humans. We assessed electrically‐induced conduction changes and responsiveness to natural stimuli. In both species, the group of mechano‐insensitive C‐fibers showed the largest conduction slowing (˜30%) upon electrical stimulation (2 Hz for 3 min). In addition, we found mechano‐insensitive cold nociceptors in pig that slowed only minimally (<10% at 2 Hz), and a similar slowing pattern was found in some human C‐fibers. Mechano‐sensitive afferents showed an intermediate conduction slowing upon 2 Hz stimulation (pig: 14%, human 23%), whereas sympathetic efferent fibers in pig and human slowed only minimally (5% and 9%, respectively). In fiber classes with more pronounced slowing, conduction latencies recovered slower; i.e. mechano‐insensitive afferents recovered the slowest, followed by mechano‐sensitive afferents whereas cold nociceptors and sympathetic efferents recovered the fastest. We conclude that mechano‐insensitive C‐fiber nociceptors can be differentiated by their characteristic pattern of ADS which are alike in pig and human. Notably, cold nociceptors with a distinct ADS pattern were first detected in pig. Our results therefore suggest that the pig is a suitable model to study nociceptor class‐specific changes of ADS.

Injured versus uninjured afferents : Who is to blame for neuropathic pain?

THE role of different classes of afferents in neuropathic pain is a controversial issue. The debate revolves around two questions: (1) What is the role of injured and uninjured afferents in neuropathic pain? (2) What is the role of myelinated and unmyelinated fibers? Although it is commonly accepted that sensitization of central painprocessing neurons is involved in neuropathic pain, it is unclear what afferents induce and maintain central sensitization under neuropathic conditions. A better understanding of the changes in injured and uninjured afferents after nerve injury is needed to improve strategies for the treatment of chronic pain. In this issue of ANESTHESIOLOGY, Sapunar et al. describe the electrophysiologic properties of isolated neurons from the L4 and L5 dorsal root ganglia after an L5 spinal nerve injury in rats. An advantage of the L5 spinal nerve ligation (SNL) model is that injured and uninjured neurons reside in different dorsal root ganglia, but their axons commingle in the sciatic nerve and target tissue (fig. 1). Like other animal models of neuropathic pain, SNL results in behavioral signs of spontaneous and stimulus evoked pain (i.e., mechanical and thermal hyperalgesia). What is the evidence that injured afferents are to blame for neuropathic pain? An obvious clinical example comes from patients with a traumatic nerve lesion that resulted in the development of a neuroma: Touching or tapping the neuroma produces paraesthesia and pain. Injection of local anesthetics at the site of the nerve injury may relieve not only ongoing pain but also mechanical hyperalgesia in the surrounding skin. Neuroma resection and relocation of the proximal nerve end may also produce pain relief in these patients. In animal neuroma models, ectopic mechanical sensitivity, thermosensitivity, and chemosensitivity as well as spontaneous activity are found in injured myelinated and unmyelinated afferents, and this is thought to be the neuronal basis of pain generated from neuromas in humans. Similar to other lesions of peripheral nerves, SNL results in neuroma formation. After SNL, however, development of spontaneous activity in injured afferents is restricted to myelinated nerve fibers, because it seems to be absent in injured unmyelinated afferents. The article by Sapunar et al. provides additional evidence that myelinated (but not unmyelinated) injured (but not uninjured) neurons develop enhanced excitability. Input from injured afferents seems to be important for the neuropathic pain behavior because application of tetrodotoxin to the L5 dorsal root ganglion reduces signs of mechanical hyperalgesia after SNL and because application of neomycin or gadolinium to the proximal, cut end of the L5 spinal nerve immediately after injury prevents or diminishes signs of mechanical hyperalgesia. Signs of mechanical hyperalgesia develop within hours after the lesion, similar to development of spontaneous activity in injured myelinated afferents, suggesting a causal link between the two. Studies that have used dorsal rhizotomies in the SNL model to directly investigate the role of injured afferents in neuropathic pain have unfortunately led to contradictory results: Some found reversal of neuropathic pain behavior, whereas others reported that an L5 dorsal rhizotomy did not prevent or reverse mechanical hyperalgesia. The interpretation of these data are complicated by the finding that dorsal rhizotomy by itself can produce signs of neuropathic pain. Is there a role for injured, unmyelinated afferents in neuropathic pain? Evidence for a role comes from the observation that artemin reversed the behavioral signs of neuropathic pain and normalized the SNL-induced changes in small L5 DRG neurons as well (e.g., IB4 binding; expression of P2X3, CGRP, galanin, and NPY). Artemin is a member of the glial-derived neurotrophic factor family, and the accessory protein GFR 3, through which it signals, is predominantly expressed in unmyelinated, nociceptive primary afferent neurons. The presence of mechanical hyperalgesia after an L5 ganglionectomy or an L5 ventral rhizotomy is direct evidence for a role of uninjured afferents in neuropathic pain. Both manipulations exclude injured primary afferents as contributors to neuropathic pain because either the soma of the injured neuron is removed (ganglionectomy) or only motor efferents are injured (ventral rhizotomy). Furthermore, there is accumulating indirect evidence for a role of uninjured afferents in neuropathic pain. The following evidence was mainly but not exclusively obtained in the SNL model. Uninjured afferents develop adrenergic sensitivity and an increased sensitivity to tumor necrosis factor . Uninjured afferents also up-regulate neuropeptides, neurotrophic factors, and signal transduction proteins (e.g., TRPV1). After L5 spinal nerve injury, the percentage of This Editorial View accompanies the following article: Sapunar D, Ljubkovic M, Lirk P, McCallum JB, Hogan QH: Distinct membrane effects of spinal nerve ligation on injured and adjacent dorsal root ganglion neurons in rats. ANESTHESIOLOGY 2005; 103:360–76.

Activity-dependent slowing of conduction velocity in uninjured L4 C fibers increases after an L5 spinal nerve injury in the rat

Abstract Growing evidence suggests that uninjured afferents may play an important role in neuropathic pain following nerve injury. The excitability of nociceptive neurons in the L4 spinal nerve appears to be enhanced following an injury to the adjacent L5 spinal nerve. In this study, we investigated whether the action‐potential conduction properties of unlesioned, unmyelinated fibers are also altered. A teased‐fiber technique was used to record from single C fibers from the L4 spinal nerve of the rat in vitro. Repeated electrical stimulation of the tibial nerve was used to investigate activity‐dependent slowing of conduction velocity. Twin pulse stimulation at a 50 ms interpulse interval allowed investigation of supranormal conduction velocity. Blinded experiments were performed 8–10 days after sham surgery and after an L5 spinal nerve ligation (L5 SNL). Activity‐dependent slowing revealed two populations of C fibers, a “nociceptor” population with a large degree of activity‐dependent slowing and a “non‐nociceptor” population with a smaller degree of activity‐dependent slowing. Both populations showed enhanced activity‐dependent slowing of conduction velocity and enhanced supranormal conduction velocities in lesioned animals compared to sham animals. Activity‐dependent slowing was also enhanced after an L5 SNL in the mouse. These alterations in conduction velocity may reflect changes in expression of ion channels responsible for the membrane excitability. These data provide additional evidence that a nerve injury leads to persistent alterations in the properties of adjacent uninjured, unmyelinated fibers.

Three functionally distinct classes of C-fibre nociceptors in primates

In primate C-fiber polymodal nociceptors are broadly classified into two groups based on mechanosensitivity. Here we demonstrate that mechanically-sensitive polymodal nociceptors that respond either quickly (QC) or slowly (SC) to a heat stimulus differ in responses to a mild burn, heat sensitization, conductive properties and chemosensitivity. Superficially applied capsaicin and intradermal injection of β-alanine, a MrgprD agonist, excite vigorously all QCs. Only 40% of SCs respond to β-alanine, and their response is only half that of QCs. Mechanically-insensitive C-fibers (C-MIAs) are β-alanine insensitive but vigorously respond to capsaicin and histamine with distinct discharge patterns. Calcium imaging reveals that β-alanine and histamine activate distinct populations of capsaicin responsive neurons in primate DRG. We suggest that histamine itch and capsaicin pain are peripherally encoded in C-MIAs and that primate polymodal nociceptive afferents form three functionally distinct subpopulations with β-alanine responsive QC fibers likely corresponding to murine MrgprD- expressing, non-peptidergic nociceptive afferents.