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Recent advances in research on sensory peptides and capsaicin mechanisms. A conference report
Neuroscience Letters, 122 (1991) 199-201
199
Elsevier Scientific Publishers Ireland Ltd. NSL 07496
Recent advances in research on sensory peptides and capsaicin mechanisms. A conference report C.A. Maggi and Fr.-K. Pierau Max-Planck-lnstitut ffir physiologische und klinische Forschung, W.G. Kerckhoff-lnstitut, Bad Nauheim ( F.R.G.) (Received 22 October 1990; Accepted 22 October 1990)
Key words: Sensory peptide; Capsaicin; Calcitonin gene-related peptide A mini-symposium (chaired by L. Chal, Australia, P. Lembeck, Austria, J. Szolcs~inyi, Hungary and M. Zimmermann, F.R.G.) was held in Bad Nauheim, F.R.G. on June 29, 1990, under the sponsorship of the European Neuropeptide Club, which focussed on recent advances of research on sensory peptides and mechanism of action of capsaicin. Attention was paid to both the sensory and 'efferent' functions of these primary afferents [1-3], to the detection of pathophysiologically relevant stimuli for their activation and to the development of new pharmacological tools for research in this field.
Anatomy. The connection between sensory peptides and the immunoresponse induced by inflammation was demonstrated by E. Weihe (Mainz, F.R.G.). In arthritic rats immunostaining for tachykinins and the coexisting calcitonin gene-related peptide (CGRP) was intensified in the inflamed tissue and sprouting of peptidergic nerve fibers was observed in areas infiltrated with mast cells and other inflammatory cells, which were partly stained for opioids. Staining for multiple opioid and the c-fos protooncogene protein was enhanced in spinal neurons which were the target of tachykinin/CGRP-reactive sensory fibers. In patients suffering from chronic pancreatitis, the number and diameter of tachykinin/CGRP stained fibers was strikingly increased, particularly in areas infiltrated by T-, B-lymphocytes and macrophages. Consequently, endogenous sensory peptides involved in nociception and inflammation appear to integrate the concerted response of the neuroimmune system to inflammatory pain. The induction of spinal opioid genes appears to be controlled by the c-fos protooncogenes suggesting a key-role in switching primary sensory immune signals into specifically coded neuronal responses. A detailed morphological analysis of changes in adult rat dorsal root ganglion (DRG) neurons following perineural application of capsaicin was presented by G. Jancs6 (Szeged, Hungary). The main finding was the demonstration of transganglionic degeneration of CCorrespondence: Fr.-K. Pierau, Max-Planck-Institut fiir physiologische und klinische Forschung, W.G. Kerckhoff-Institut, Parkstrasse 1, D-6350 Bad Nauheim, F.R.G. 0304-3940/91/$ 03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd.
fiber primary afferent terminals following a peripheral nerve lesion. These changes were associated with, and probably resulting from, the degeneration of small-sized D R G neurons. It was suggested that structural changes may significantly contribute to alterations in the functional properties of dorsal horn neurons which commence after peripheral nerve lesions. The experimental approach introduced and termed the 'capsaicin gap' technique, may be important for evaluating transganglionic degenerative changes of unmyelinated primary afferents following peripheral nerve injuries. The hypothesis was also advanced that not all classes of capsaicin-sensitive primary afferent neurons contain neuropeptides. The relationship between neuropeptide content and nociceptive afferents was investigated by U. Hanesch (Wiirzburg, F.R.G.) by combining an immunohistochemical and neurophysiological approach. The conclusion was reached that neither tachykinins nor CGRP or somatostatin can be considered as specific markers of nociceptive afferents. A detailed analysis of the distribution of primary afferents of both vagal and spinal origin to different targets in guinea-pig airways was presented by W. Kummer (Heidelberg, F.R.G.) who showed evidence for targetspecific differential chemical coding of neuropeptide content. Mechanisms of the action of capsaicin upon membrane properties and transmitter release. C.A. Maggi (Florence, Italy) presented an overview of the recent advances about the mechanism of the action of capsaicin, the ionic
200 basis for its excitatory and 'desensitizing' action on primary afferents and the action of Ruthenium red as a capsaicin antagonist. Of particular relevance has been the recent discovery of the capsaicin 'receptor' by using radiolabelled resiniferatoxin [4] and the demonstration that capsaicin activates a novel type of cation channel present on sensory neurons of mammals [5]. The development of these new techniques and the availability of ruthenium red as a selective capsaicin antagonist [6, 7] are expected to powerfully increase knowledge on the neurobiology of capsaicin-sensitive primary afferents and, particularly, to allow investigation about the possible existence of an endogenous ligand for the capsaicin receptor. Evidence for the existence of distinct modes for transmitter release from both peripheral and central endings of capsaicin-sensitive primary afferents was presented by C.A. Maggi (Florence, Italy) and J. Donnerer (Graz, Austria). Fr.-K. Pierau (Bad Nauheim, F.R.G.) presented further evidence for a selective antagonism between capsaicin and Ruthenium red using a pig skin axon reflex model. The question of unselective (e.g. not restricted to primary afferents) actions of capsaicin was addressed by J.R. Schwarz (Hamburg, F.R.G.) who presented evidence for a suppressive action of capsaicin on certain K current subtypes in myelinated nerves. As expected for the non-selective actions of capsaicin, this effect did not exhibit desensitization. Capsaicin-sensitive visceral afferents. P. Holzer (Graz, Austria) reviewed the gastric defence mechanisms underlying the role played by capsaicin-sensitive primary afferents in protecting the gastric mucosa against experimental ulcers (see below). H. Sann (Bad Nauheim, F.R.G.) showed that more than 90% of afferents in the guinea-pig ureter are capsaicin-sensitive (U2 units) and that their activation occurs more likely in pathophysiological conditions (ureteral obstruction). Capsaicin-insensitive (U 1) units were activated by ureteric contractions and might have a function under physiological conditions. The possible use of intravesicle capsaicin for treatment of pain arising from the urinary bladder was proposed on the basis of clinical data obtained in patients with hypersensitive bladder syndrome (C.A. Maggi). Protons as endogenous activators of primary afferents. A common topic of several presentations was the identification of protons (lowering of pH) as a key event for the activation of capsaicin-sensitive primary afferents during tissue injury/inflammation. P. Holzer (Graz, Austria) showed that acid back diffusion in the gastric mucosa during experimental ulcers activates a local axon reflex leading to mucosal vasodilation and proposed CGRP as the main mediator of this response. Removal
of this local defensive mechanism appears the most likely explanation for the aggravation of experimental ulcers in capsaicin-pretreated rats. Lowering of pH from 7.4 to 6 or 5 produced a prominent, calcium-dependent release of neuropeptides from capsaicin-sensitive primary afferents in the superfused guinea-pig urinary bladder or rat stomach, while increasing the pH up to 9 had no effect. Thus proton-dependent activation of the 'efferent' function of capsaicin-sensitive primary afferents was markedly reduced by Ruthenium red and partly attenuated by blockers of the voltage-dependent calcium channels (P. Geppetti, Florence, Italy). P.W. Reeh (Erlangen, F.R.G.) showed that exposure to acid produced mechanical hyperalgesia of polymodal nociceptors in the rat skin, an action not shared by chemical mediators of inflammation (bradykinin, prostaglandin etc.) applied simultaneously at concentrations similar to those recovered in inflammatory fluids (inflammatory 'soup'). Thus a local increase in proton concentration could be responsible for allodynia experienced in various inflammatory diseases. Peripheral opioid antinociception and pharmacological modulation of axon reflex. Recently, evidence has accumulated that opioids exert an action on the peripheral part of primary sensory neurons. A purely peripheral antinociceptive effect of systemically administered morphine is suggested from a rat model of unilateral paw inflammation. Both the lowered pain threshold in the inflamed paw and the antinociceptive effect of systemic morphine treatment was abolished by systemic capsaicin desensitization (L. Barth6, Prcs, Hungary). Using the same model, evidence was presented for a large increase in opioid content of the inflamed tissue indicated by the expression of opioid-like immunoreactivity in infiltrating immune cells. The local release of these opioids, particularly in response to stress, probably contributes to a local antinociceptive mechanism (C. Stein, Munich, F.R.G.). Systemic opioid agonists had divergent effects upon the axon reflex flare reaction in the pig's skin. While the flare reaction to antidromic electrical stimulation was inhibited (naloxone reversible) chemical, mechanical and heat-induced flare reactions were not affected, suggesting different molecular mechanisms or fiber populations responsible for the different flare responses. Evidence was presented that the frequency dependence of the electrical induced flare reaction is probably due to a peripheral release of endogenous opioids. Conditioning high frequency stimuli (I00 Hz) decreased the response to (I Hz) antidromic stimulation, an effect which was abolished by nalaxone (L. Barth6, P6cs, Hungary; Fr.-K. Pierau, Bad Nauheim, F.R.G.). Taehykinins and primary afferent transmission to the
201
spinal cord. H.G. Schaible (Wiirzburg, F.R.G.) pre-
sented data supporting an intraspinal release (particularly in the more superficial laminae of the dorsal horn) of both substance P and neurokinin A following the stimulation of peripheral nociceptors of joints, in which inflammation was induced by kaolin injection, as detected by the antibody microprobe technique. Different functions of both peptides in the transmission of noxious signals are indicated by the observations that neurokinin A but not substance P is spontaneously released after kaolin injection, and that flexion of the inflamed joints was necessary to increase the substance P content in the dorsal horn, while neurokinin A was already enhanced after noxious pinch. M. Wienrich (Frankfurt, F.R.G.) reported about electrical events produced by a receptor-selective (NK-1 and NK-3) tachykinin agonist on spinal neurones in culture and their blockade by an antagonist. The meeting was followed by a convivial 'open end' discussion which represented one of the most vivid and enjoyable moments. The mechanisms of capsaicin 'desensitization' was the main topic discussed. Although historically justified, this term should be abandoned because it has different meanings in the context of pharmacology (desensitization of receptor to which ligands bind to produce their effect) and physiology (desensitization of sensory receptors). J. Szolcs~inyi (Prcs, Hungary) briefly reiterated the time course of the action of capsaicin: stage 1 is represented by excitation and stages 2-4 correspond to what is collectively known as capsaicin 'desensitization' [8]. Stages 3 and 4 are accompanied by degenerative and neurotoxic events as well as by neuropeptide depletion. However, the sensory nerve endings become inexcitable by capsaicin and other agents within a few min from application of the drug, at a time when neither peptide depletion nor degeneration are evident (stage 2, corresponding to the sensory neuron blocking action of capsaicin). Ultimately, the four stages of capsaicin action on primary afferents can be viewed as consequential to the interaction of capsaicin with its receptor on sensory nerves and opening of a cation channel which leads to a massive entering of calcium ions. Thus in a short time after excitation (carried out by both sodium and calcium ions), the primary afferents are overloaded with harmful levels of intracellular calcium ions [9] leading to defunctionalization (stage 2), ultra-
structural changes/degeneration (stage 3), and, in some instances, to cell death (stage 4). Neuropeptide depletion is just a consequence and not a cause of defunctionalization of primary afferents by capsaicin. The possibility also exists that in low concentrations capsaicin produces a receptor-selective desensitization. The ability of low concentrations (< 1 pM) of Ruthenium red to protect primary afferents from stages 2-4 of the action of capsaicin may be linked to a partial blockade of the capsaicin receptor-coupled cation channel thereby limiting the influx of excess (toxic) calcium but still allowing enough cations to pass the membrane and produce excitation. At high concentrations (> 1 pM) of the dye, stage 1 is also blocked. In conclusion the Bad Nauheim meeting proved successful from both the scientific and social points of view and allowed a concentrated but yet exhaustive presentation and discussion of several topics related to the neurobiology of capsaicin-sensitive primary afferents together with mounting evidence for their pathophysiological roles. 1 B~lint, P., Capsaicin and the sensory system, Acta Physiol. Hung., 69 (1987). 2 Maggi, C.A. and Meli, A., The sensory-efferent function of capsaicin-sensitive sensory neurons, Gen. Pharmacol., 19 (1988) 1-43. 3 Holzer, P., Local effector function of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, CGRP and other neuropeptides, Neuroscience, 24 (1988) 739-768. 4 Szallasi, A. and Blumberg, P.M., Specific binding of resiniferatoxin, an ultrapotent capsaicin analog, by dorsal root ganglia membranes, Brain Res., 524 (1990) 106-I 11. 5 Forbers, C.A. and Bevan, S., Properties of single capsaicin-activated channels. Soc. Neurosci. Abstr., (1988) 642. 6 Maggi, C.A., Patacchini, R., Santicioli, P., Giuliani, S., Del Bianco, E., Geppett, P. and Meli, A., The 'efferent' function of capsaicinsensitive nerves: Ruthenium red discriminates between different mechanism of activation, Eur. J. Pharmacol., 170 (1989) 167-177. 7 Dray, A., Forbes, C.A. and Burgess, C.M., Ruthenium red blocks the capsaicin-induced increase in intracellular calcium and activation of membrane currents in sensory neurons, as well as the activation of peripheral nociceptors in vitro. Neurosei. Lett., 110 (1989) 52-59. 8 Szolcs~nyi, J., Sensory receptors and the antinociceptive effects of capsaicin. In R. Hakanson and F. Sundler (Eds.), Tachykinin Antagonists, Elsevier, Amsterdam, 1985, pp. 45-54. 9 Wood, J.N., Winter, J., James, I.F., Rang, H., Yeats, J. and Bevan, S., Capsaicin-induced ion fluxes in DRG cells in culture, J. Neurosci., (1988) 3208-3220.
199
Elsevier Scientific Publishers Ireland Ltd. NSL 07496
Recent advances in research on sensory peptides and capsaicin mechanisms. A conference report C.A. Maggi and Fr.-K. Pierau Max-Planck-lnstitut ffir physiologische und klinische Forschung, W.G. Kerckhoff-lnstitut, Bad Nauheim ( F.R.G.) (Received 22 October 1990; Accepted 22 October 1990)
Key words: Sensory peptide; Capsaicin; Calcitonin gene-related peptide A mini-symposium (chaired by L. Chal, Australia, P. Lembeck, Austria, J. Szolcs~inyi, Hungary and M. Zimmermann, F.R.G.) was held in Bad Nauheim, F.R.G. on June 29, 1990, under the sponsorship of the European Neuropeptide Club, which focussed on recent advances of research on sensory peptides and mechanism of action of capsaicin. Attention was paid to both the sensory and 'efferent' functions of these primary afferents [1-3], to the detection of pathophysiologically relevant stimuli for their activation and to the development of new pharmacological tools for research in this field.
Anatomy. The connection between sensory peptides and the immunoresponse induced by inflammation was demonstrated by E. Weihe (Mainz, F.R.G.). In arthritic rats immunostaining for tachykinins and the coexisting calcitonin gene-related peptide (CGRP) was intensified in the inflamed tissue and sprouting of peptidergic nerve fibers was observed in areas infiltrated with mast cells and other inflammatory cells, which were partly stained for opioids. Staining for multiple opioid and the c-fos protooncogene protein was enhanced in spinal neurons which were the target of tachykinin/CGRP-reactive sensory fibers. In patients suffering from chronic pancreatitis, the number and diameter of tachykinin/CGRP stained fibers was strikingly increased, particularly in areas infiltrated by T-, B-lymphocytes and macrophages. Consequently, endogenous sensory peptides involved in nociception and inflammation appear to integrate the concerted response of the neuroimmune system to inflammatory pain. The induction of spinal opioid genes appears to be controlled by the c-fos protooncogenes suggesting a key-role in switching primary sensory immune signals into specifically coded neuronal responses. A detailed morphological analysis of changes in adult rat dorsal root ganglion (DRG) neurons following perineural application of capsaicin was presented by G. Jancs6 (Szeged, Hungary). The main finding was the demonstration of transganglionic degeneration of CCorrespondence: Fr.-K. Pierau, Max-Planck-Institut fiir physiologische und klinische Forschung, W.G. Kerckhoff-Institut, Parkstrasse 1, D-6350 Bad Nauheim, F.R.G. 0304-3940/91/$ 03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd.
fiber primary afferent terminals following a peripheral nerve lesion. These changes were associated with, and probably resulting from, the degeneration of small-sized D R G neurons. It was suggested that structural changes may significantly contribute to alterations in the functional properties of dorsal horn neurons which commence after peripheral nerve lesions. The experimental approach introduced and termed the 'capsaicin gap' technique, may be important for evaluating transganglionic degenerative changes of unmyelinated primary afferents following peripheral nerve injuries. The hypothesis was also advanced that not all classes of capsaicin-sensitive primary afferent neurons contain neuropeptides. The relationship between neuropeptide content and nociceptive afferents was investigated by U. Hanesch (Wiirzburg, F.R.G.) by combining an immunohistochemical and neurophysiological approach. The conclusion was reached that neither tachykinins nor CGRP or somatostatin can be considered as specific markers of nociceptive afferents. A detailed analysis of the distribution of primary afferents of both vagal and spinal origin to different targets in guinea-pig airways was presented by W. Kummer (Heidelberg, F.R.G.) who showed evidence for targetspecific differential chemical coding of neuropeptide content. Mechanisms of the action of capsaicin upon membrane properties and transmitter release. C.A. Maggi (Florence, Italy) presented an overview of the recent advances about the mechanism of the action of capsaicin, the ionic
200 basis for its excitatory and 'desensitizing' action on primary afferents and the action of Ruthenium red as a capsaicin antagonist. Of particular relevance has been the recent discovery of the capsaicin 'receptor' by using radiolabelled resiniferatoxin [4] and the demonstration that capsaicin activates a novel type of cation channel present on sensory neurons of mammals [5]. The development of these new techniques and the availability of ruthenium red as a selective capsaicin antagonist [6, 7] are expected to powerfully increase knowledge on the neurobiology of capsaicin-sensitive primary afferents and, particularly, to allow investigation about the possible existence of an endogenous ligand for the capsaicin receptor. Evidence for the existence of distinct modes for transmitter release from both peripheral and central endings of capsaicin-sensitive primary afferents was presented by C.A. Maggi (Florence, Italy) and J. Donnerer (Graz, Austria). Fr.-K. Pierau (Bad Nauheim, F.R.G.) presented further evidence for a selective antagonism between capsaicin and Ruthenium red using a pig skin axon reflex model. The question of unselective (e.g. not restricted to primary afferents) actions of capsaicin was addressed by J.R. Schwarz (Hamburg, F.R.G.) who presented evidence for a suppressive action of capsaicin on certain K current subtypes in myelinated nerves. As expected for the non-selective actions of capsaicin, this effect did not exhibit desensitization. Capsaicin-sensitive visceral afferents. P. Holzer (Graz, Austria) reviewed the gastric defence mechanisms underlying the role played by capsaicin-sensitive primary afferents in protecting the gastric mucosa against experimental ulcers (see below). H. Sann (Bad Nauheim, F.R.G.) showed that more than 90% of afferents in the guinea-pig ureter are capsaicin-sensitive (U2 units) and that their activation occurs more likely in pathophysiological conditions (ureteral obstruction). Capsaicin-insensitive (U 1) units were activated by ureteric contractions and might have a function under physiological conditions. The possible use of intravesicle capsaicin for treatment of pain arising from the urinary bladder was proposed on the basis of clinical data obtained in patients with hypersensitive bladder syndrome (C.A. Maggi). Protons as endogenous activators of primary afferents. A common topic of several presentations was the identification of protons (lowering of pH) as a key event for the activation of capsaicin-sensitive primary afferents during tissue injury/inflammation. P. Holzer (Graz, Austria) showed that acid back diffusion in the gastric mucosa during experimental ulcers activates a local axon reflex leading to mucosal vasodilation and proposed CGRP as the main mediator of this response. Removal
of this local defensive mechanism appears the most likely explanation for the aggravation of experimental ulcers in capsaicin-pretreated rats. Lowering of pH from 7.4 to 6 or 5 produced a prominent, calcium-dependent release of neuropeptides from capsaicin-sensitive primary afferents in the superfused guinea-pig urinary bladder or rat stomach, while increasing the pH up to 9 had no effect. Thus proton-dependent activation of the 'efferent' function of capsaicin-sensitive primary afferents was markedly reduced by Ruthenium red and partly attenuated by blockers of the voltage-dependent calcium channels (P. Geppetti, Florence, Italy). P.W. Reeh (Erlangen, F.R.G.) showed that exposure to acid produced mechanical hyperalgesia of polymodal nociceptors in the rat skin, an action not shared by chemical mediators of inflammation (bradykinin, prostaglandin etc.) applied simultaneously at concentrations similar to those recovered in inflammatory fluids (inflammatory 'soup'). Thus a local increase in proton concentration could be responsible for allodynia experienced in various inflammatory diseases. Peripheral opioid antinociception and pharmacological modulation of axon reflex. Recently, evidence has accumulated that opioids exert an action on the peripheral part of primary sensory neurons. A purely peripheral antinociceptive effect of systemically administered morphine is suggested from a rat model of unilateral paw inflammation. Both the lowered pain threshold in the inflamed paw and the antinociceptive effect of systemic morphine treatment was abolished by systemic capsaicin desensitization (L. Barth6, Prcs, Hungary). Using the same model, evidence was presented for a large increase in opioid content of the inflamed tissue indicated by the expression of opioid-like immunoreactivity in infiltrating immune cells. The local release of these opioids, particularly in response to stress, probably contributes to a local antinociceptive mechanism (C. Stein, Munich, F.R.G.). Systemic opioid agonists had divergent effects upon the axon reflex flare reaction in the pig's skin. While the flare reaction to antidromic electrical stimulation was inhibited (naloxone reversible) chemical, mechanical and heat-induced flare reactions were not affected, suggesting different molecular mechanisms or fiber populations responsible for the different flare responses. Evidence was presented that the frequency dependence of the electrical induced flare reaction is probably due to a peripheral release of endogenous opioids. Conditioning high frequency stimuli (I00 Hz) decreased the response to (I Hz) antidromic stimulation, an effect which was abolished by nalaxone (L. Barth6, P6cs, Hungary; Fr.-K. Pierau, Bad Nauheim, F.R.G.). Taehykinins and primary afferent transmission to the
201
spinal cord. H.G. Schaible (Wiirzburg, F.R.G.) pre-
sented data supporting an intraspinal release (particularly in the more superficial laminae of the dorsal horn) of both substance P and neurokinin A following the stimulation of peripheral nociceptors of joints, in which inflammation was induced by kaolin injection, as detected by the antibody microprobe technique. Different functions of both peptides in the transmission of noxious signals are indicated by the observations that neurokinin A but not substance P is spontaneously released after kaolin injection, and that flexion of the inflamed joints was necessary to increase the substance P content in the dorsal horn, while neurokinin A was already enhanced after noxious pinch. M. Wienrich (Frankfurt, F.R.G.) reported about electrical events produced by a receptor-selective (NK-1 and NK-3) tachykinin agonist on spinal neurones in culture and their blockade by an antagonist. The meeting was followed by a convivial 'open end' discussion which represented one of the most vivid and enjoyable moments. The mechanisms of capsaicin 'desensitization' was the main topic discussed. Although historically justified, this term should be abandoned because it has different meanings in the context of pharmacology (desensitization of receptor to which ligands bind to produce their effect) and physiology (desensitization of sensory receptors). J. Szolcs~inyi (Prcs, Hungary) briefly reiterated the time course of the action of capsaicin: stage 1 is represented by excitation and stages 2-4 correspond to what is collectively known as capsaicin 'desensitization' [8]. Stages 3 and 4 are accompanied by degenerative and neurotoxic events as well as by neuropeptide depletion. However, the sensory nerve endings become inexcitable by capsaicin and other agents within a few min from application of the drug, at a time when neither peptide depletion nor degeneration are evident (stage 2, corresponding to the sensory neuron blocking action of capsaicin). Ultimately, the four stages of capsaicin action on primary afferents can be viewed as consequential to the interaction of capsaicin with its receptor on sensory nerves and opening of a cation channel which leads to a massive entering of calcium ions. Thus in a short time after excitation (carried out by both sodium and calcium ions), the primary afferents are overloaded with harmful levels of intracellular calcium ions [9] leading to defunctionalization (stage 2), ultra-
structural changes/degeneration (stage 3), and, in some instances, to cell death (stage 4). Neuropeptide depletion is just a consequence and not a cause of defunctionalization of primary afferents by capsaicin. The possibility also exists that in low concentrations capsaicin produces a receptor-selective desensitization. The ability of low concentrations (< 1 pM) of Ruthenium red to protect primary afferents from stages 2-4 of the action of capsaicin may be linked to a partial blockade of the capsaicin receptor-coupled cation channel thereby limiting the influx of excess (toxic) calcium but still allowing enough cations to pass the membrane and produce excitation. At high concentrations (> 1 pM) of the dye, stage 1 is also blocked. In conclusion the Bad Nauheim meeting proved successful from both the scientific and social points of view and allowed a concentrated but yet exhaustive presentation and discussion of several topics related to the neurobiology of capsaicin-sensitive primary afferents together with mounting evidence for their pathophysiological roles. 1 B~lint, P., Capsaicin and the sensory system, Acta Physiol. Hung., 69 (1987). 2 Maggi, C.A. and Meli, A., The sensory-efferent function of capsaicin-sensitive sensory neurons, Gen. Pharmacol., 19 (1988) 1-43. 3 Holzer, P., Local effector function of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, CGRP and other neuropeptides, Neuroscience, 24 (1988) 739-768. 4 Szallasi, A. and Blumberg, P.M., Specific binding of resiniferatoxin, an ultrapotent capsaicin analog, by dorsal root ganglia membranes, Brain Res., 524 (1990) 106-I 11. 5 Forbers, C.A. and Bevan, S., Properties of single capsaicin-activated channels. Soc. Neurosci. Abstr., (1988) 642. 6 Maggi, C.A., Patacchini, R., Santicioli, P., Giuliani, S., Del Bianco, E., Geppett, P. and Meli, A., The 'efferent' function of capsaicinsensitive nerves: Ruthenium red discriminates between different mechanism of activation, Eur. J. Pharmacol., 170 (1989) 167-177. 7 Dray, A., Forbes, C.A. and Burgess, C.M., Ruthenium red blocks the capsaicin-induced increase in intracellular calcium and activation of membrane currents in sensory neurons, as well as the activation of peripheral nociceptors in vitro. Neurosei. Lett., 110 (1989) 52-59. 8 Szolcs~nyi, J., Sensory receptors and the antinociceptive effects of capsaicin. In R. Hakanson and F. Sundler (Eds.), Tachykinin Antagonists, Elsevier, Amsterdam, 1985, pp. 45-54. 9 Wood, J.N., Winter, J., James, I.F., Rang, H., Yeats, J. and Bevan, S., Capsaicin-induced ion fluxes in DRG cells in culture, J. Neurosci., (1988) 3208-3220.