Visceral nociceptors: a new world order?

There has been a long-standing controversy as to whether or not internal organs are innervated by a special category of 'visceral nociceptor'. Recent experimental studies on the afferent supply of some viscera have thrown new light on this issue by demonstrating the presence of several categories of visceral sensory receptor, including high-threshold receptors, 'silent' nociceptors and intensity-encoding receptors. Advances in the understanding of how the CNS processes nociceptive signals have also helped to clarify the issue. The authors of this report, originally having different points of view, present here a common and closer approach to the visceral nociceptor controversy.

The RNA binding and transport proteins staufen and fragile X mental retardation protein are expressed by rat primary afferent neurons and localize to peripheral and central axons.

Neuronal proteins have been traditionally viewed as being derived solely from the soma; however, accumulating evidence indicates that dendritic and axonal sites are capable of a more autonomous role in terms of new protein synthesis. Such extra-somal translation allows for more rapid, on-demand regulation of neuronal structure and function than would otherwise be possible. While mechanisms of dendritic RNA transport have been elucidated, it remains unclear how RNA is trafficked into the axon for this purpose. Primary afferent neurons of the dorsal root (DRG) and trigeminal (TG) ganglia have among the longest axons in the neuraxis and such axonal protein synthesis would be advantageous, given the greater time involved for protein trafficking to occur via axonal transport. Therefore, we hypothesized that these primary sensory neurons might express proteins involved in RNA transport. Rat DRG and TG neurons expressed staufen (stau) 1 and 2 (detected at the mRNA level) and stau2 and fragile x mental retardation protein (FMRP; detected at the protein level). Stau2 mRNA was also detected in human TG neurons. Stau2 and FMRP protein were localized to the sciatic nerve and dorsal roots by immunohistochemistry and to dorsal roots by Western blot. Stau2 and FMRP immunoreactivities colocalized with transient receptor potential channel type 1 immunoreactivity in sensory axons of the sciatic nerve and dorsal root, suggesting that these proteins are being transported into the peripheral and central terminals of nociceptive sensory axons. Based on these findings, we propose that stau2 and FMRP proteins are attractive candidates to subserve RNA transport in sensory neurons, linking somal transcriptional events to axonal translation.

Painful stimuli induce in vivo phosphorylation and membrane mobilization of mouse spinal cord NKCC1 co-transporter.

The Na+ --Cl- --K+ isoform 1 (NKCC1) is a co-transporter that increases the intracellular concentration of chloride. NKCC1 plays a critical role in neuronal excitability and it has been recently suggested that it can contribute to hyperalgesic states by modulating the chloride concentration inside nociceptive neurons. In the spinal cord, trafficking of neurotransmitter receptors from the cytosol to the plasma membrane has been demonstrated to contribute to the development of hyperalgesia. However, it is unknown if trafficking of co-transporters can also occur in the nervous system or if it can be induced by painful stimulation. In this study, we have induced referred mechanical hyperalgesia in vivo by intracolonic instillation of capsaicin in mice. Using subcellular fractionation of proteins and cross-linking of membrane proteins we have observed that intracolonic capsaicin induced a 50% increase in NKCC1 in the plasma membrane of lumbosacral spinal cord 90 and 180 min after instillation, in parallel with a similar decrease in the cytosolic fraction. These effects returned to basal levels 6 h after capsaicin treatment. Intracolonic capsaicin also evoked a rapid (10 min) and transient phosphorylation of NKCC1, however, intracolonic saline did not produce significant changes in either NKCC1 trafficking or phosphorylation and none of the treatments induced any alterations of NKCC1 in the thoracic spinal cord. These results suggest that phosphorylation and recruitment of NKCC1 might play a role in referred mechanical hyperalgesia evoked by a painful visceral stimulus. The time course of the effects observed suggests that phosphorylation could contribute to the initial generation of hyperalgesia whereas trafficking could participate in the maintenance of hyperalgesic states observed at longer time points.

Distribution of somatic and visceral primary afferent fibres within the thoracic spinal cord of the cat.

Transport of horseradish peroxidase (HRP) through somatic and visceral nerve fibres was used to study the patterns of termination of somatic and visceral primary afferent fibres within the lower thoracic segments of the cat's spinal cord. A concentrated solution of HRP was applied for at least 5 hours to the central end of the righ greater splanchnic nerve and of the left T9 intercostal nerve of adult cats. Some animals remained under chloralose anaesthesia for the duration of the HRP transport times (up to 53 hours) whereas longer HRP application and transport times (4-5 days) were allowed in animals that recovered from barbiturate anaesthesia. Somatic afferent fibres and varicosities (presumed terminals) were found in laminae I, II, III, IV, and V of the ipsilateral dorsal horn and in the ipsilateral Clarke's column. The density of the somatic projection was particularly high in the superficial dorsal horn. In parasagittal sections of the cord, bundles of somatic fibres were seen joining the dorsal horn from the dorsal roots via the dorsal columns and Lissauer's tract. A medio-lateral somatotopic arrangement of somatic afferent terminations was observed, with afferent fibres from the ventral parts of the dermatome ending in the medial dorsal horn and afferent fibres from the dorsal parts of the dermatome ending in the lateral dorsal horn. The total rostro-caudal extent of the somatic projection through a single spinal nerve was found to be of 2 and 2/3 segments, including the segment of entry, the entire segment rostral to it and two-thirds of the segment caudal to it. A lateral to medial shift in the position of the somatic projection was observed in the rostro-caudal axis of the cord. Visceral afferent fibres and varicosities (presumed terminals) were seen in laminae I and V of the ipsilateral dorsal horn. The density of the visceral projection to the dorsal horn was substantially lower than that of the somatic projection. Visceral afferent fibres reached the dorsal horn via Lissauer's tract and joined a lateral bundle of fine fibres that run along the lateral edge of the dorsal horn. The substantia gelatinosa (lamina II) appeared free of visceral afferent fibres. These results are discussed in relation to the mechanisms of viscero-somatic convergence onto sensory pathways in the thoracic spinal cord.

Cutaneous receptive fields of somatic and viscerosomatic neurones in the thoracic spinal cord of the cat.

Extracellular single-unit recordings were made from 121 neurones in the thoracic spinal cord of the cat. All neurones could be driven by electrical stimulation of dorsal root afferent fibres. The neurones were classified, according to the absence or presence of inputs from the ipsilateral splanchnic nerve, as "somatic" or "viscerosomatic", respectively. Cutaneous receptive fields were identified for 75 of the neurones: 31 were somatic and 44 viscerosomatic. Only two of the somatic cells received cutaneous nociceptive inputs, compared with 33 of the viscerosomatic cells. Sixty-four percent of the whole sample of neurones had receptive fields which included three or more dermatomes. Viscerosomatic cells tended to have larger receptive fields than the somatic neurones, and six of them had fields which did not include the corresponding (T11) dermatome. Neurones with receptive fields in the dorsal one-third of the dermatome tended to be located in the lateral one-third of the dorsal horn, but those with receptive fields in the ventral two-thirds of the dermatome showed no differential distribution within the gray matter. This is discussed with respect to the results of anatomical studies on the dorsal horn projections of cutaneous afferent fibres from different regions of the dermatome. Preliminary results from intracellular staining with horseradish peroxidase reveal extensive branching of primary afferents in the dorsal horn, and large dendritic fields of dorsal horn neurones. Our physiological and morphological results indicate that the somatotopic organisation of the thoracic spinal cord is less well defined than that of the lumbosacral region.

Somatic and visceral primary afferents in the lower thoracic dorsal root ganglia of the cat.

Anterograde transport of horseradish peroxidase (HRP) through somatic and visceral nerves was used to estimate the proportions of somatic and visceral dorsal root ganglion (DRG) cells of the lower thoracic ganglia of the cat. A concentrated solution of HRP was applied for at least 5 hours to the central end of the right greater splanchnic nerve and of the left T9-intercostal nerve of adult cats. Some animals remained under chloralose anaesthesia for the duration of the HRP transport time (up to 53 hours) whereas longer HRP application and transport times (4-5 days) were allowed in animals that recovered from barbiturate anaesthesia. Visceral DRG cells were found in approximately equal numbers in all ganglia examined (T7-T11). Population estimates were obtained for the T8 and T9 ganglia where visceral DRG cells were found to be 6.2% (T8) and 5.2% (T9) of the total cell population. In contrast, somatic DRG cells were found in large numbers in the ganglia examined (T8 and T9) where they amounted to over 90% of the cell population. Measurement of cross-sectional areas and estimates of cell diameters of the DRG cells showed greater proportions of large somatic cells (diameter greater than 40 micron) than of large visceral cells. Similar distributions of cell size were found for both somatic and visceral DRG cells with diameters less than 40 micron. These results show that the proportion of visceral afferent fibres in the dorsal roots that mediate the spinal cord projection of the splanchnic nerve is very small. Since viscerosomatic convergence in the thoracic spinal cord is very extensive, the present results suggest considerable divergence of the visceral afferent input to the central nervous system.

Afferent activity evoked by natural stimulation of the biliary system in the ferret.

A technique for the natural stimulation of the biliary system which permits the distinction between noxious and innocuous intensities of stimulation has been developed in the ferret. Male ferrets anaesthetized with urethane have been used. Controlled distensions of the biliary system were produced and the nociceptive nature of the stimulus was ascertained by reflex increases in blood pressure. Levels of biliary pressure that did not evoke changes in blood pressure were considered innocuous. Using this approach electrical activity has been recorded from biliary afferents. Thirty-two recordings were obtained. Twenty-one were of afferents that could be excited by innocuous levels of biliary pressure (low threshold afferents) and 10 recordings were of afferents excited only by noxious stimulation of the biliary system (high threshold afferents). One fibre could not be activated by changes in biliary pressure. Twenty-seven receptive fields were located: 12 in the gallbladder and 15 in the biliary ducts. The relevance of high threshold biliary receptors to visceral nociception is discussed.

A psychophysical study of secondary hyperalgesia: evidence for increased pain to input from nociceptors.

Substantial evidence suggests that the hyperalgesia to mechanical stimuli that occurs in an area of uninjured skin surrounding a site of injury (area of secondary hyperalgesia) arises from activity in low-threshold mechanoreceptors (LTMs). In this study, we have investigated if activity in mechanically sensitive nociceptors also contributes to this secondary hyperalgesia. It is known that all woollen fabrics excite LTMs, but that only the prickly ones activate mechanically sensitive nociceptors. Therefore, we have conducted a psychophysical study using a range of prickly and non-prickly woollen fabrics applied to normal and hyperalgesic skin to assess the roles of LTMs and nociceptors in secondary hyperalgesia. We have studied in 10 normal volunteers the sensations of fabric-evoked prickle and pain in normal and hyperalgesic skin. Secondary hyperalgesia was produced by intradermal injection of capsaicin (25 micrograms) into the volar skin of the forearm. Five woollen fabrics (2 non-prickly, 2 prickly and 1 intermediate) were presented, in a blind manner, to the skin before and after the capsaicin injection. The sensation of fabric-evoked prickle was not changed in hyperalgesic skin. On the other hand, little if any pain was evoked by the fabrics when applied to normal skin, but substantial pain was produced by all fabrics when applied to hyperalgesic skin. The pain ratings were graded with the ratings of prickle so that fabrics that evoked the greatest prickle also evoked significantly more pain. The magnitude of pain increased linearly with prickle sensation; the slope of this regression function increased substantially in hyperalgesic skin.(ABSTRACT TRUNCATED AT 250 WORDS)

The representation of prolonged and intense, noxious somatic and visceral stimuli in the ventrolateral orbital cortex of the cat.

The responses of single neurones in the ventrolateral orbital (VLO) cortex to noxious pinch, heating of the skin, twisting of the joints and distension of the gall bladder were studied in cats anaesthetized with halothane. Of 60 neurones studied, 44 responded to prolonged (greater than 10 sec) stimuli that were well within the noxious range. Neurones were relatively unresponsive to innocuous stimuli or to the transient application of noxious stimuli. Many single neurones responded to a variety of modalities of noxious stimuli (e.g., skin heating and gall bladder distension). Many neurones studied showed a fluctuating level (5-15 Hz) of ongoing spontaneous activity. Neurones responded with either an increased frequency of spikes (excitation) or an inhibition of spontaneous discharge, irrespective of the source of noxious stimulation. Noxious stimuli delivered simultaneously to two different tissues (e.g., skin and visceral) sometimes produced excitation of the neurone under study, to levels above that produced by the application a noxious stimulus to only one of the tissues. Receptive fields were often large involving both contralateral and ipsilateral areas of the body, as well as both fore and hind limbs. No evidence of somatotopic organization was obtained. The responses of some neurones outlasted the application of the stimuli by many minutes. It is concluded that single neurones in the ventrolateral orbital cortex respond to the prolonged application of intensely noxious stimuli to a variety of body tissues, in a manner that is in keeping with the involvement of this cortical area in both the physiological, autonomic and experiential components of the affective-motivational aspect of pain. Furthermore, from the consequences of lesion studies in man and animals, it is proposed that the activation of cells in the orbital cortex by a variety of noxious stimuli reflects its more general role in the development and maintenance of behaviour in response to negative reinforcement of both social and physical origins.

Development of secondary hyperalgesia following non-painful thermal stimulation of the skin: a psychophysical study in man.

A psychophysical study has been carried out in 10 normal human subjects to examine whether conscious perception of pain is necessary for the development of secondary hyperalgesia. Prolonged thermal stimulation of the skin was applied to the subjects at intensities known to evoke discharges in polymodal nociceptors but insufficient to evoke pain sensations. During this stimulation the development of punctate and of stroking hyperalgesia was examined as was the presence of a skin flare indicative of nociceptor activation. All subjects developed a flare and an area of hyperalgesia following the application of the non-painful heat stimulus. The first change observed in the subjects was the appearance of an area of hyperalgesia to punctate stimuli, followed by flare and by stroking hyperalgesia. The onset of pain was always reported sometime after these events. Statistical analysis of these data for all subjects showed a highly significant difference between the time of onset of pain and the time of onset of any of the other 3 phenomena. Significant differences were also observed between the onset of punctate hyperalgesia and the onsets of flare and of stroking hyperalgesia. No difference was observed between the onset of flare and of stroking hyperalgesia. These results show that cutaneous hyperalgesia can be evoked in normal human subjects by prolonged thermal stimulation of the skin at temperatures that are not perceived as painful. The development of a flare in all subjects simultaneously with stroking hyperalgesia but before the perception of pain suggests that activation of nociceptors is necessary for the hyperalgesia to occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasodilatation in hyperalgesic rat skin evoked by stimulation of afferent A beta-fibers: further evidence for a role of dorsal root reflexes in allodynia.

In areas of secondary hyperalgesia, innocuous mechanical stimuli evoke pain (allodynia). We have proposed that this is produced by a central pre-synaptic interaction whereby A beta-fibers evoke spike activity (dorsal root reflexes) in nociceptive afferents (Pain, 68 (1996) 13). This activity should conduct centrally, evoking allodynia, and peripherally, evoking neurogenic vasodilatation. Here we tested this hypothesis by examining the effects of electrical stimulation of A beta-fibers on cutaneous blood flow before and after producing secondary hyperalgesia in anesthetized rats. Cutaneous blood flow was recorded in the hind paw skin innervated by the sural nerve using a laser Doppler flowmeter. The sural nerve was prepared for electrical stimulation, and the evoked activity was recorded from the sciatic nerve in continuity. Electrical stimulation (1 Hz, 4 x 0.2 ms pulses, 20 s) was applied to the sural nerve at 2T (A beta-fibers only) and 4T and 6T (A beta + A delta-fibers). Flux was recorded at baseline and after capsaicin or mustard oil application outside the sural nerve territory. The effects of intravenous administration of the calcitonin gene-related peptide (CGRP) receptor antagonist, alpha-CGRP(8-37), or of section of the sciatic nerve or of the L4-L6 dorsal roots were examined. Selective activation of the sural nerve A beta-fibers reliably evoked increases in cutaneous blood flow close to areas of chemical irritation or skin damage. A beta-fiber-evoked vasodilatation was abolished by sciatic nerve or dorsal root section and had a spatial arrangement and optimal stimulation pattern suggesting a central synaptic interaction similar to that responsible for dorsal root reflexes. The flux increases were dose-dependently and reversibly inhibited by alpha-CGRP(8-37), indicating that the A beta-fiber-evoked vasodilatation resulted from the antidromic activation of nociceptive cutaneous afferent fibers. These results support our hypothesis by showing activation of nociceptive primary afferents by A beta-fibers in areas of allodynia in a manner consistent with a pre-synaptic interaction evoking dorsal root reflexes.

Role of central and peripheral tachykinin NK1 receptors in capsaicin-induced pain and hyperalgesia in mice.

Substance P and its receptor (NK1) are thought to play an important role in pain and hyperalgesia. Here we have further examined this role by comparing the behavioural responses to intradermal capsaicin of mutant mice with a disruption of the NK1 receptor (NK1 KO) and wild-type (WT) mice. We have also evaluated the contribution of peripheral NK1 receptors to capsaicin-evoked behaviour by selective blockade of peripheral NK1 receptors in WT mice using a non-brain penetrant NK1 receptor antagonist. Injection of 6 microg capsaicin into the heel evoked paw licking with the same latency in WT and KO mice, but a significantly longer duration in WT mice. A higher dose (30 microg) evoked a similar duration of licking in both groups. There were no differences in mechanical sensitivity tested with von Frey hairs between WT and KO mice before capsaicin. Both capsaicin doses resulted in pronounced increases in responses to von Frey hairs (hyperalgesia) and novel responses to cotton wisps (allodynia) applied to the digits of the injected paw in WT mice, but no significant changes from baseline in KO mice. Selective blockade of peripheral NK1 receptors in WT mice resulted in a complete inhibition of capsaicin-evoked plasma extravasation, but the mechanical hyperalgesia induced by 30 microg capsaicin intraplantar was still significantly greater than that seen in KO mice. We conclude that the response to intradermal capsaicin is still present but abbreviated in mice lacking NK1 receptors, such that secondary hyperalgesia is not observed even after a high dose. Further, the lack of secondary hyperalgesia in NK1 KO mice is largely due to the loss of central rather than peripheral NK1 receptors. The phenotype of the NK1 KO mice is consistent with a loss of function of mechanically-insensitive nociceptors, and thus we propose that substance P may be expressed by this group of primary sensory neurones and required for their function.

A new model of visceral pain and referred hyperalgesia in the mouse.

The generation of transgenic mice that lack or overexpress genes relevant to pain is becoming increasing common. However, only one visceral pain model, the writhing test, is widely used in mice. Here we describe a novel model, chemical stimulation of the colon, which we have developed in mice. Mice of either sex were injected i.v. with 30 mg/kg Evan's Blue for subsequent determination of plasma extravasation. For behavioural testing, they were placed on a raised grid and 50 microl of saline, mustard oil (0.25-2.5%) or capsaicin (0.03-0.3%) was administered by inserting a fine cannula into the colon via the anus. Visceral pain-related behaviours (licking abdomen, stretching, contractions of abdomen etc) were counted for 20 min. Before intracolonic administration, and 20 min after, the frequency of withdrawal responses to the application of von Frey probes to the abdomen was tested. The colon was removed post-mortem and the Evan's Blue content measured. Mustard oil and capsaicin administration evoked dose-dependent visceral pain behaviours, referred hyperalgesia (significant increase in responses to von Frey hairs) and colon plasma extravasation. The peak behavioural responses were evoked by 0.1% capsaicin and by 1% mustard oil respectively. The nociceptive behavioural responses were dose-dependently reversed by morphine (ED50 = 1.9 +/- 1 mg/kg s.c.). We conclude that this model represents a useful tool both for phenotyping mutant mice and for classical pharmacology since information on visceral pain, referred hyperalgesia and colon inflammation can all obtained from the same animal.

Central and peripheral actions of the NSAID ketoprofen on spinal cord nociceptive reflexes.

Ketoprofen is a non-steroidal antiinflammatory drug (NSAID) which provides effective analgesia in situations of pain provoked by tissue inflammation. However, the location of its analgesic effects, (peripheral tissues versus central nervous system), have not been clearly identified and separated. In the present study the effectiveness of ketoprofen was examined in two different types of experiments: (i) Open field behavioural tests in conscious rats, and (ii) spinal cord nociceptive reflexes (single motor units) activated by electrical and thermal stimulation in chloralose anaesthetised rats. The experiments were performed in rats with carrageenan-induced inflammation of one hindpaw, or of one knee joint. The administration of ketoprofen significantly inhibited the reduction of exploratory movements caused by inflammation in open field experiments. Ketoprofen was also effective in depressing reflex activity evoked by electrical and noxious thermal stimulation of the skin, either in inflamed tissue or in normal tissue of monoarthritic animals. It was also effective in the reduction of reflex wind-up; a phenomenon in which the activity of spinal cord neurones increases progressively with high frequency electrical stimulation. We therefore conclude that ketoprofen has central as well as peripheral analgesic activity.

Responses of rat spinal neurons to distension of inflamed colon: role of tachykinin NK2 receptors.

Tachykinin NK2 receptors are implicated in nociception and the control of intestinal motility. Here we examined their involvement in responses of spinal lumbosacral neurons with colon input to distension of normal or inflamed colon in anesthetized rats. The responses of single neurons to colorectal distension (5-80 mmHg), to electrical stimulation of the pelvic nerve (bypassing sensory receptors) and to somatic stimulation were characterized. The effect of cumulative doses of an NK2 receptor antagonist, MEN 11420 (10-1000 microg kg(-1) IV), on responses to these stimuli was tested in control conditions (n=6), or 45 min after intracolonic instillation of acetic acid (n=6). After colonic inflammation, neuronal responses to colorectal distension and pelvic nerve stimulation were significantly greater. MEN 11420 dose-dependently inhibited the enhanced responses to colorectal distension after inflammation (ID50=402+/-14 microg kg(-1)), but had no significant effect on responses to pelvic nerve stimulation or distension of the normal colon, suggesting a peripheral action selective for the inflamed colon. We conclude that MEN 11420 possesses peripheral anti-hyperalgesic effects on neuronal responses to colorectal distension. These results provide a neurophysiological basis for a possible use of tachykinin NK2 receptor antagonists in treating abdominal pain in irritable bowel syndrome patients.

Afferent fibres from the guinea-pig ureter: size and peptide content of the dorsal root ganglion cells of origin.

A study was undertaken to determine the segmental organization of the dorsal root ganglion cells which give rise to ureteric primary afferent fibres in the guinea-pig. The size-distribution and peptide content of these dorsal root ganglion cells were examined and compared with a sample of all dorsal root ganglion cells from the same ganglia. Afferent fibres to the guinea-pig ureter were found to arise mainly from dorsal root ganglia L2-L3 and S1-S2. A large contralateral component of the afferent innervation of the ureter was found when either the right or the left ureter was injected with tracer. This amounted to approximately 40% of the total labelled cells. The cross-sectional areas of the dorsal root ganglion cells of ureteric afferents were found to be at the smaller end of the size-range for the whole ganglion. Most (90%) of the cells innervating the ureter were immunoreactive for one of the peptides studies, substance P or calcitonin gene-related peptide, and a large proportion (65%) were immunoreactive for both. This was very different for the ganglia as a whole, where only about 50% of the cells were immunoreactive for either of the peptides and only 14% were immunoreactive for both peptides. These results show a bilateral afferent innervation of the ureter by nerve fibres which, in the vast majority, contain substance P and/or calcitonin gene-related peptide.

Evidence for a visceral afferent origin of substance P-like immunoreactivity in lamina V of the rat thoracic spinal cord.

A visceral afferent origin of substance P-like immunoreactivity in lamina V of the lower thoracic spinal cord of the rat was investigated. In transverse sections from normal animals there was a moderately dense substance P-immunoreactive innervation of lamina V. In some sections there was a dorsoventrally orientated fibre bundle from the superficial dorsal horn entering lamina V. In parasagittal sections, substance P-immunoreactivity in lamina V was found arranged in clusters, with a periodicity in the rostrocaudal axis of 200-600 microns. In some cases these were seen to be continuous with a dorsoventrally orientated fibre bundle from the superficial dorsal horn. After section of the splanchnic nerve there was a consistent reduction in the density of the substance P-like immunoreactivity in lamina V, with fewer clusters on the operated side. Adult rats treated neonatally with capsaicin showed a substantial reduction of substance P-immunoreactivity in laminae I and II and the virtual abolition of staining in lamina V. These results provide evidence of a visceral origin for some of the substance P-like immunoreactivity in lamina V of the rat thoracic spinal cord. In addition, they confirm that most of the substance P-immunoreactivity in the dorsal horn is of primary afferent origin.

Visceral and somatic afferent origin of calcitonin gene-related peptide immunoreactivity in the lower thoracic spinal cord of the rat.

The origin of calcitonin gene-related peptide in the thoracic spinal cord of the rat was investigated by radioimmunoassay and immunohistochemistry. In transverse sections from normal animals there was a dense staining of calcitonin gene-related peptide-immunoreactivity in laminae I, II and V of the dorsal horn. In parasagittal sections this was found to consist of rostrocaudally orientated fibres in laminae I and II and longitudinal bundles of fibres interspersed with a plexus of immunoreactivity in lamina V. After sectioning the thoracic spinal nerves there was a significant reduction in immunoreactivity in the dorsal horn of the spinal cord which was seen as a marked reduction of staining in lamina II and in the bundles of fibres in lamina V. Section of the splanchnic nerve slightly reduced staining in lamina I and virtually abolished the plexuses of immunoreactivity in lamina V. However, measurement of calcitonin gene-related peptide in samples from coeliac-ganglionized rats revealed an increase in immunoreactivity in regions of the spinal cord containing lamina V. These results provide evidence of a visceral and somatic afferent origin of calcitonin gene-related peptide in the thoracic spinal cord of the rat.

Deficits in visceral pain and hyperalgesia of mice with a disruption of the tachykinin NK1 receptor gene.

Studies in mice lacking genes encoding for substance P or its receptor (NK1), or with NK1 antagonists, have shown that this system contributes to nociception, but the data are complex. Here, we have further examined the role of NK1 receptors in pain and hyperalgesia by comparing nociceptive responses to mechanical and chemical stimulation of viscera and the resulting hyperalgesia and inflammation in NK1 knockout (-/-) and wild-type (+/+) mice. We concentrated on visceral nociception because substance P is expressed by a much greater proportion of visceral than cutaneous afferents. NK1 -/- mice showed normal responses to visceral mechanical stimuli, measured as behavioural responses to intraperitoneal acetylcholine or hypertonic saline or reflex responses to colon distension in anaesthetized mice, although -/- mice failed to encode the intensity of noxious colon distensions. In contrast, NK1 -/- mice showed profound deficits in spontaneous behavioural reactions to an acute visceral chemical stimulus (intracolonic capsaicin) and failed to develop referred hyperalgesia or tissue oedema. However, in an identical procedure, intracolonic mustard oil evoked normal spontaneous behaviour, referred hyperalgesia and oedema in -/- mice. The inflammatory effects of capsaicin were abolished by denervation of the extrinsic innervation of the colon in rats, whereas those of mustard oil were unchanged, showing that intracolonic capsaicin evokes neurogenic inflammation, but mustard oil does not. Tests of other neurogenic inflammatory stimuli in NK1 -/- mice revealed impaired behavioural responses to cyclophosphamide cystitis and no acute reflex responses or primary hyperalgesia to intracolonic acetic acid. We conclude that NK1 receptors have an essential role mediating central nociceptive and peripheral inflammatory responses to noxious stimuli that evoke neurogenic inflammation, and modulating responses to noxious mechanical stimuli. We propose that two separate hyperalgesia pathways exist, one of which is NK1 receptor dependent, whereas the other does not require intact substance P/NK1 signalling.