Salt stress differentially regulates mobilisation of carbon and nitrogen reserves during seedling establishment of Pityrocarpa moniliformis.

Seedling establishment is a critical step in environment colonisation by higher plants that frequently occurs under adverse conditions. Thus, we carried out an integrated analysis of seedling growth, water status, ion accumulation, reserve mobilisation, metabolite partitioning and hydrolase activity during seedling establishment of the native Caatinga species Piptadenia moniliformis (Benth.) Luckow & R.W. Jobson under salinity. Two-day-old seedlings were cultivated in vitro for 4 days in water agar (control) or supplemented with 50 or 100 mm NaCl. Biochemical determinations were performed according to standard spectrophotometric protocols. We found that 100 mm NaCl stimulated starch degradation, amylase activity and soluble sugar accumulation, but limited storage protein hydrolysis in the cotyledons of P. moniliformis seedlings. Although Na+ accumulation in the seedling affected K+ partitioning between different organs, it was not possible to associate the salt-induced changes in reserve mobilisation with Na+ toxicity, or water status, in the cotyledons. Remarkably, we found that starch content increased in the roots of P. moniliformis seedlings under 100 mm NaCl, probably in response to the toxic effects of Na+ . The mobilisation of carbon and nitrogen reserves is independently regulated in P. moniliformis seedlings under salt stress. The salt-induced delay in seedling establishment and the resulting changes in the source-sink relationship may lead to storage protein retention in the cotyledons. Possibly, the intensification of starch mobilisation in the cotyledons supported starch accumulation in the root as a potential mechanism to mitigate Na+ toxicity.

Regulation of reserve mobilisation in sunflower during late seedling establishment in continuous darkness.

Reserve mobilisation, metabolite partitioning and reserve-degrading enzyme activity were studied in sunflower seedlings cultivated in vitro under a 12-h photoperiod or in the dark to investigate the involvement of source-sink relation and carbon starvation in the regulation of reserve mobilisation under continuous darkness. Reserves, metabolites and enzyme activity were determined with standard spectrophotometric methods. At the first 24 h of treatment (acclimation phase), darkness did not affect growth, but restricted carbon and nitrogen use, as indicated by sugar and amino acid accumulation in the different seedling parts. After 5 days of treatment (survival phase), extended darkness limited growth and retarded storage lipid mobilisation due to carbon starvation, as evidenced by the depletion of carbohydrates in cotyledons and hypocotyl, as well as the consumption of amino acids in hypocotyls and roots. Alterations in the source-sink relationship might have been a response to prolonged darkness, instead of a mechanism used to regulate reserve mobilisation, as these alterations cannot be associated with negative feedback mediated by metabolite accumulation. Storage lipid degradation depends, at least in part, on mechanisms that co-ordinately regulate the activities of lipases and isocitrate lyase. Taking these results together, it is possible that reserve mobilisation in sunflower seedlings cultivated in the dark might be regulated by mechanisms that perceive the absence of light and predict carbon starvation, adjusting reserve use according to future energy demands to allow, at least in the short term, seedling survival.

Urinary bladder inflammation induces changes in urothelial nerve growth factor and TRPV1 channels.

BACKGROUND AND PURPOSE: The urinary bladder urothelium expresses various receptors and in response to chemical and mechanical stimuli releases mediators, thereby modulating bladder sensory pathways. Transient receptor potential vanilloid 1 (TRPV1) ion channels and nerve growth factor (NGF) in those cells are implicated in this modulatory effect and play a role in sensitizing pain-related afferent pathways during inflammation. In this study, we investigated the interaction between NGF and TRPV1 channels in urothelial cells. EXPERIMENTAL APPROACH: Urothelial cells from female Sprague-Dawley rat bladders were cultured to quantify membrane expression of TRPV1 channels and capsaicin-induced ATP release in the presence of NGF alone or with TrKA or PI3K inhibitors. Pain scores from rats with cyclophosphamide (CYP)-induced bladder inflammation were assessed after treatment with a TrkA antagonist. Bladders (from control and CYP rats) were collected and analysed for NGF content and TRPV1 channel expression. KEY RESULTS: Cultured cells responded to NGF with increased TRPV1 channel expression in the cell membrane and increased release of ATP. Both responses were blocked by either a TrkA antagonist or a PI3K inhibitor. Treatment in vivo with the TrkA antagonist alleviated pain symptoms and reduced CYP-induced NGF overexpression in the mucosa. Furthermore, in urothelial cells from animals with bladder inflammation, expression of TRPV1 channels in the membrane was significantly increased. CONCLUSIONS AND IMPLICATIONS: During bladder inflammation, increased production of NGF in urothelial cells induced increased expression and activity of TRPV1 channels in the cell membrane. This effect was primarily mediated by the PI3K pathway.

Identification of the ghrelin-GHSR 1 system and its influence in the modulation of induced ocular hypertension in rabbit and rat eyes.

Recent studies evidenced a decrease in ghrelin's aqueous humor levels in patients with glaucoma. The goal of our investigation was to study the effect of the ghrelin-GHSR-1a system in the modulation of intraocular pressure in acute ocular hypertension models and its expression and distribution in ocular tissues. Two animal models of acute ocular hypertension were used to study the effect of the ghrelin-GHSR-1a system in the modulation of intraocular pressure: the rabbit and the rat. Ocular hypertension was induced by an intravitreal injection of 20% NaCl. Ghrelin or des-acyl ghrelin were delivered subconjunctivally and the intraocular pressure was assessed by a rebound tonometer that was calibrated for each species. In addition, we have studied the influence of nitric oxide and prostaglandins on ghrelin's effect in the rabbit animal model. Finally, we determined by immunofluorescence the expression of ghrelin and GHSR-1 in the rat's ocular tissue. Ghrelin decreased the intraocular pressure in both animal models (maximum decrease: 43.8±12.0% in the rabbit and 29.0±7.46% in the rat). In the rabbit, this effect was blunted in the presence of l-NAME and ketorolac. Des-acyl ghrelin only decreased the intraocular pressure in the rat (maximum decrease: 34.9±8.15%). Ghrelin expression was detected in the ciliary processes and GHSR-1 expression was detected in the trabecular meshwork and ciliary body. The ghrelin-GHSR-1 system is expressed in the anterior segment of the eye. Ghrelin and des-acyl ghrelin are responsible for a hypotensive effect in acute ocular hypertension animal models.

Intrathecal administration of botulinum toxin type A improves urinary bladder function and reduces pain in rats with cystitis.

BACKGROUND: Botulinum toxin A (Onabot/A) has been shown to have an antinociceptive effect. This might be due to an impairment of sensory nerves not only in the peripheral but also in the central nervous system. In this work, we analysed both systems by studying the effect of intrathecal (i.t.) administration of botulinum toxin A in an animal model of bladder pain and hyperactivity induced by cyclophosphamide (CYP). METHODS: Rats were implanted with an i.t. catheter at the L6 segment. Bladder pain was induced by intraperitoneal (i.p.) injection of CYP. Five experimental groups were created: (1) Saline i.p. + i.t.; (2) Onabot/A i.t.; (3) CYP i.p. + saline i.t.; (4) CYP i.p. + Onabot/A i.t. 48 h after CYP; and (5) Onabot/A i.t. 30 days. Mechanical sensitivity was assessed in the abdomen and hindpaws. Motor activity was observed in an open-field arena. Bladder reflex activity was evaluated by cystometry. At the end, bladders and spinal cord were immunoreacted (IR) against cleaved SNAP-25 (cSNAP-25), c-Fos, p-ERK, calcitonin gene-related peptide (CGRP) and GAP43. RESULTS: The toxin reduced pain symptoms, bladder hyperactivity, expression of neuronal activation markers and CGRP, typically up-regulated in this inflammatory model. The presence of cSNAP-25 was detected in the spinal cord and bladder fibres from animals treated with Onabot/A. No somatic or visceral motor impairments were observed. CONCLUSIONS: Our findings suggest that i.t. Onabot/A has a strong analgesic effect in a model of severe bladder pain. This route of administration can be further explored to treat intractable forms of pain.

Transient receptor potential channels in bladder function.

The transient receptor potential (TRP) superfamily of cationic ion channels includes proteins involved in the transduction of several physical and chemical stimuli to finely tune physiological functions. In the urinary bladder, they are highly expressed in, but not restricted to, primary afferent neurons. The urothelium and some interstitial cells also express several TRP channels. In this review, we describe the expression and the known roles of some members of TRP subfamilies, namely TRPV, TRPM and TRPA, in the urinary bladder. The therapeutic interest of modulating the activity of TRP channels to treat bladder dysfunctions is also discussed.

Peptidergic sensory and parasympathetic fiber sprouting in the mucosa of the rat urinary bladder in a chronic model of cyclophosphamide-induced cystitis.

In this study, we used a well-established animal model to investigate changes in the peptidergic and parasympathetic innervation of the bladder following chronic bladder inflammation. Adult female Sprague-Dawley rats were injected with either 70 mg/kg cyclophosphamide diluted in saline, i.p., once every 3 days or saline. After 10 days, all animals were tested for urinary frequency and number of low volume voids, as well as symptoms of spontaneous pain. At the end of 12 days, all animals were perfused with histological fixatives and the urinary bladders processed for immunofluorescence using antibodies against calcitonin gene-related peptide and the vesicular acetylcholine transporter as markers, respectively, of peptidergic primary afferent fibers and parasympathetic efferent fibers. We show that animals treated with cyclophosphamide had inflamed bladders and displayed high urinary frequency as well as some indicators of spontaneous pain, such as piloerection and a rounded-back posture. Furthermore, they had a significant increase in the density of both parasympathetic and peptidergic sensory fibers in the bladder mucosa and an increase in peptidergic sensory fibers in the detrusor muscle. Based on these results, we suggest that peripheral sprouting of parasympathetic and peptidergic fibers could be a mechanism responsible for sensitization of the bladder, leading to urinary symptoms. Since we observed that the parasympathetic and peptidergic fibers often wrapped around one another and that their varicosities were very close, these two fiber populations may be interacting with each other to lead to and maintain sensitization. Future studies are required to establish the role of this fiber sprouting in bladder symptoms.

Peptidergic sensory and parasympathetic fiber sprouting in the mucosa of the rat urinary bladder in a chronic model of cyclophosphamide-induced cystitis.

In this study, we used a well-established animal model to investigate changes in the peptidergic and parasympathetic innervation of the bladder following chronic bladder inflammation. Adult female Sprague-Dawley rats were injected with either 70 mg/kg cyclophosphamide diluted in saline, i.p., once every 3 days or saline. After 10 days, all animals were tested for urinary frequency and number of low volume voids, as well as symptoms of spontaneous pain. At the end of 12 days, all animals were perfused with histological fixatives and the urinary bladders processed for immunofluorescence using antibodies against calcitonin gene-related peptide and the vesicular acetylcholine transporter as markers, respectively, of peptidergic primary afferent fibers and parasympathetic efferent fibers. We show that animals treated with cyclophosphamide had inflamed bladders and displayed high urinary frequency as well as some indicators of spontaneous pain, such as piloerection and a rounded-back posture. Furthermore, they had a significant increase in the density of both parasympathetic and peptidergic sensory fibers in the bladder mucosa and an increase in peptidergic sensory fibers in the detrusor muscle. Based on these results, we suggest that peripheral sprouting of parasympathetic and peptidergic fibers could be a mechanism responsible for sensitization of the bladder, leading to urinary symptoms. Since we observed that the parasympathetic and peptidergic fibers often wrapped around one another and that their varicosities were very close, these two fiber populations may be interacting with each other to lead to and maintain sensitization. Future studies are required to establish the role of this fiber sprouting in bladder symptoms.

Vanilloid receptor 1 expression in the rat urinary tract.

Previous findings have shown that the capsaicin sensitivity of sensory fibres is due to the expression of a specific membrane protein, the vanilloid receptor type 1 (VR1). In the present work we studied the distribution, morphology and the neurochemical content of nerve fibres expressing this receptor in the rat urinary tract. Immunolabelling was performed against the VR1 and the positive fibres were examined by light and electron microscopy. Colocalisation of VR1 and substance P or calcitonin gene-related peptide immunoreactivities, and isolectin B4 binding, was evaluated under the confocal microscope. In addition, the effect of intravesical administration of resiniferatoxin, an ultra-potent vanilloid receptor agonist, in the receptor expression in the bladder was also studied. Numerous VR1-immunoreactive fibres were found in the mucosa and muscular layer of the entire urinary tract except the kidney. In the bladder, most fibres were also substance P- or calcitonin gene-related peptide-immunoreactive but did not bind isolectin B4. Under the electron microscope VR1 immunoreactivity was confined to unmyelinated axons and varicosities containing small clear and large dense-core synaptic vesicles. They occurred beneath or among epithelial cells or closely apposed to smooth muscle cells. Intravesical resiniferatoxin decreased VR1 immunoreactivity transiently. These data indicate that primary sensory fibres expressing VR1 are extremely abundant in the rat urinary tract and that, in contrast to the skin, they belong almost exclusively to the peptide-containing sub-population of primary afferents. As capsaicin-sensitive bladder afferents are involved in nociception and reflex micturition control, the numerous free terminal nerve endings expressing VR1 in the mucosa seem more adequate to accomplish the former function. However, the close apposition between VR1-expressing fibres and smooth muscle cells suggests that they may also encode the tonus of the muscular layer.

A light- and electron-microscopic histopathological study of human bladder mucosa after intravesical resiniferatoxin application.

OBJECTIVE: To determine the morphology of bladder mucosa and the integrity of its mucin coat in patients with detrusor hyper-reflexia treated with intravesical resiniferatoxin. PATIENTS AND METHODS: Seven patients with detrusor hyper-reflexia were treated intravesically with resiniferatoxin dissolved in 10% ethanol in saline (50 nmol/L solution in two and 100 nmol/L in five). Patients were clinically evaluated by a voiding chart and filling cystometry before and 3 months after each resiniferatoxin application. In addition, they underwent cystoscopy and bladder biopsies at 22-33 months after the first instillation and at 7-23 months after the last one. Tissue samples for light microscopy were fixed in 4% paraformaldehyde, embedded in paraffin and stained with haematoxylin-eosin or periodic acid-Schiff reagent (PAS). Those for electron microscopy were fixed in 5% glutaraldehyde and embedded in resin. RESULTS: The resiniferatoxin instillation was not painful. Three months after treatment the mean voiding frequency decreased and five incontinent patients became continent. The maximum cystometric capacity increased in all patients; at cystoscopy the bladders appeared normal. On light microscopy the urothelium was of normal morphology and stained with PAS in the luminal cells and in the basement membrane. Mononuclear inflammatory cells were occasionally apparent in the lamina propria. On electron microscopy epithelial cells were visible in a thick basal lamina. Superficial cells had the usual irregular contour and contained numerous membrane-coated vesicles. In the lamina propria, unmyelinated axonal profiles with occasional varicosities could be identified. CONCLUSIONS: Intravesical resiniferatoxin improved urinary frequency, incontinence and bladder capacity in patients with detrusor hyper-reflexia, causing no morphological change in the bladder mucosa. The PAS reactivity of the carbohydrate moieties present in the mucin coat and the basement membrane was unchanged by resiniferatoxin.

Peptide immunoreactivity and ultrastructure of rat urinary bladder nerve fibers after topical desensitization by capsaicin or resiniferatoxin.

In the present study the decrease of neuropeptide containing nerve fibers and the increase in the volume threshold to reflex micturition occurring in the rat bladder after intravesical application of capsaicin or resiniferatoxin were compared. The ultrastructure of bladder terminal axons was evaluated at the moment of maximal peptide depletion and compared to that of nerve fibers after systemic capsaicin application. Adult Wistar rats were treated intravesically for 30 min with 0.5 ml of 100 nM RTX, 1 mM capsaicin or 30% ethanol in saline, the vehicle solution. Twenty-four hours and 1, 2, 3, 4 and 8 weeks later the bladders were immunostained for CGRP, SP, VIP and NPY. Cystomanometric studies were performed 24 h and 1, 8, and 12 weeks after vanilloid instillation. Twenty-four hours after systemic capsaicin or intravesical capsaicin or RTX, bladders were prepared for electron microscopic (EM) observation. Intravesical capsaicin or RTX decreased, in a similar way, the number of CGRP and SP-IR (immunoreactive) fibers coursing in the muscular layer and the mucosa. IR fibers amounted to less than 20% of controls at 24 h and returned to normal levels in the eighth week. At the EM level, bladders treated with topical vanilloids did not show morphological changes in terminal axons coursing in the mucosa. In contrast, bladders from animals treated systemically with capsaicin contained numerous grossly degenerated nerve fibers. VIP and NPY-IR fibers were not affected by the treatment. Cystometrograms showed an increase of the volume threshold to reflex micturition that started at 24 h and disappeared at 12 weeks. We conclude that intravesical capsaicin or RTX were equally effective in terms of reducing the number of SP and CGRP-IR fibers and increasing the volume threshold for reflex micturition. Both changes were transient and were not associated with ultrastructural changes of the bladder nerve fibers, excluding terminal axon degeneration as the main mechanism of action of intravesical vanilloids.

Intravesical resiniferatoxin desensitizes rat bladder sensory fibres without causing intense noxious excitation. A c-fos study.

In this study the desensitizing power of increasing concentrations of resiniferatoxin applied topically to the bladder mucosa, and the irritating properties of the most effective desensitizing dose, were determined with the aid of the spinal expression of the proto-oncogene c-fos. Desensitization was assessed by the decrease in the number of Fos-immunoreactive spinal neurons induced by the intravesical instillation of 1% acetic acid, when the latter was preceded by resiniferatoxin in concentrations between 1 and 1000 nM. Irritation, as shown by the noxious excitation of vesical sensory innervation, was measured by the c-fos response evoked by a single application of resiniferatoxin. As to the desensitizing power, resiniferatoxin produced a dose-dependent effect with a maximum at 100 nM, which decreased Fos-immunoreactive cell numbers to less than 10% of controls. No further decrease of c-fos activation occurred at 1000 nM. As to the irritating power, the saturation dose of resiniferatoxin (100 nM) produced a very weak c-fos activation in lumbosacral spinal cord segments. These data show that in an effective desensitizing concentration, resiniferatoxin is virtually devoid of nociceptive effects, in agreement with current clinical observations.

Lidocaine prevents noxious excitation of bladder afferents induced by intravesical capsaicin without interfering with the ensuing sensory desensitization: an experimental study in the rat.

PURPOSE: The effects of the local anesthetic lidocaine on the noxious excitation and subsequent desensitization of bladder sensory fibers, produced by intravesical capsaicin, were evaluated through c-fos activation in the spinal cord. MATERIALS AND METHODS: Noxious excitation was demonstrated by counting Fos-immunoreactive (IR) cells occurring in the rat spinal cord 2 hours after intravesical administration of 1 mM. capsaicin, preceded or not by 2% lidocaine. Desensitization was studied by comparing the number of Fos-IR cells induced by 1% acetic acid in rats treated 24 hours before with 1 mM. intravesical capsaicin preceded or not by 2% lidocaine. RESULTS: Lidocaine instilled previously markedly reduced the number of Fos-IR spinal cells responding to capsaicin-induced bladder afferent excitation. Numbers of Fos-IR cells induced by acetic acid instillation in bladders desensitized by capsaicin administrated 24 hours before were not changed by lidocaine application prior to capsaicin. CONCLUSIONS: These findings suggest that local anesthetic pretreatment of the bladder with lidocaine reduces the capsaicin-induced noxious excitation of the sensory fibers without decreasing their subsequent desensitization.

Modulated expression of c-Fos in the spinal cord following noxious thermal stimulation of monoarthritic rats.

Peripheral noxious stimulation evokes functional and biochemical changes in the spinal cord which results in central sensitization and hyperalgesia, but at the same time also induces the activation of inhibitory control systems. The purpose of the present study was to investigate whether the adaptive changes induced by ongoing peripheral inflammation influence the spinal cord expression of c-Fos (a commonly used marker of neuronal activity) following an additional acute noxious stimulus. Therefore, the spinal expression of c-Fos was immunohistochemically investigated following noxious thermal stimulation of a rat monoarthritic hindpaw at various time points (1, 4, 8, 21 days) after induction of monoarthritis. Compared to normal rats, c-Fos expression following ipsilateral noxious thermal stimulation of monoarthritic rats was strongly modified in the deep laminae of the dorsal horn depending on the time course of inflammation. At 1 day of monoarthritis, an enhanced ipsilateral expression (135% and 208% of normal rats in laminae III-VI and VII, respectively) and at 3 weeks a reduced expression (38% and 23% of normal rats in laminae III-VI and VII, respectively) was detected. The amount of c-Fos-positive neurons in the ipsilateral superficial laminae I and II was unchanged at all time points investigated. To assess excitability changes on the contralateral side at an early stage of inflammation, a group of monoarthritic rats received a contralateral noxious stimulus at day 1 of monoarthritis. This resulted in a potentiated expression of c-Fos ipsilateral to the acute noxious stimulus (i.e., contralateral to the monoarthritic hindpaw) restricted to lamina II (137% of normal rats) of the dorsal horn. The data showed that changes in c-Fos expression depended on the time point of noxious heat stimulation (NHS) of monoarthritic rats, and differed in the ipsi- and contralateral side of the spinal cord. In addition to a possible habituation of c-Fos expression, it may be speculated that the time course-dependent changes reflect laminae-specific modulations of excitatory and inhibitory mechanisms during monoarthritis. Further studies are necessary in order to provide more insights into the contribution of these mechanisms on noxious stimulus-evoked c-Fos expression.

Sites of renal pain processing in the rat spinal cord. A c-fos study using a percutaneous method to perform ureteral obstruction.

The sites of renal pain processing in the rat spinal cord were studied by mapping the spinal cord neurons expressing c-fos after acute ureteral distension due to obstruction. A new experimental model is presented. A nylon knot was loosely placed around the ureter and the ends of the thread exteriorized through the retroperitoneal wall. Eight days later, when all c-fos expression due to nociceptive input from the abdominal wound and the manipulation of the intestines had disappeared, the nylon ends were pulled to produce ureteral occlusion. C-fos activation occurred at spinal segments T10-L4 with a peak at L1-L2. The activated neurons were concentrated in laminae I, lateral IV-V, medial VII and X. While in lamina I nearly all Fos-immunoreactive cells were ipsilateral, in the deeper laminae taken together 60% cells were ipsilateral and 40% contralateral to the distended ureter. It is suggested that renal nociceptive input giving rise to conscious pain perception is transmitted through ipsilateral lamina I, whereas input triggering autonomic reflexes may be mainly processed, ipsi- and contralaterally, in the deep laminae.

Spinal c-fos expression is differentially induced by brief or persistent noxious stimulation.

The influence of stimulus duration on spinal induction of the c-fos proto-oncogene was analysed in the rat by pinching or heating the skin for periods varying from 20 s to 2 h. At stimulation periods shorter than 20 min, c-fos activation occurred in laminae I-IIi following mechanical stimulation and I-IIo following thermal stimulation. Mechanical stimulation produced delayed activation in laminae III-IV, V and VII at 30 min, 60 min and 2 h, respectively, and thermal stimulation in lamina IIi at 50 min. It is suggested that late c-fos activation signals inflammatory pain and is due to sensitization of primary afferent neurones.

Activation of the c-fos proto-oncogene in the spinal cord following noxious stimulation of the urinary bladder.

Activation of the c-fos proto-oncogene following mechanical or chemical noxious stimulation of the urinary bladder was studied at T12-L2 and L5-S1, the spinal cord segments of projection of the hypogastric nerve (HGN) and pelvic nerve (PN) fibers, respectively. In intact adult rats, c-fos expression was found at T12-L2 only in lamina I. At L5-S1, Fos cells occurred in lamina I, the intermediolateral gray matter (ILG), and the dorsal commissure (DCM). These two areas contained the highest number of immunoreactive cells. Although more Fos cells were induced by mechanical than by chemical stimulation, the distribution of the reactive neurons was similar after both types of stimuli. In adult rats that had been treated neonatally with capsaicin, there was a marked fall in c-fos activation by mechanical or chemical noxious stimuli in all immunoreactive areas. The loss of Fos cells was more pronounced in ILG and DCM at L5-S1 (95%) than in lamina I at the two spinal domains (70%). The confinement of c-fos activation to lamina I at T12-L2, the spinal cord domain of the HGN, suggests that the input carried from the bladder by this nerve is preferentially used for pain perception. The same function is expected for noxious input reaching lamina I at L5-S1, the spinal cord territory of termination of the PN. However, the striking number of Fos cells in ILG and DCM supports the important role played by this nerve in the control of the micturition reflex.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential activation of c-fos in spinal neurones by distinct classes of noxious stimuli.

The laminar distribution of spinal cord neurones expressing immunoreactivity to the Fos protein was evaluated in the rat following chemical, thermal or mechanical noxious stimulation of the skin for 2 h. After stimulation by 20% or 5% formalin, Fos-immunoreactive neurones prevailed in lamina I where they accounted for 64% and 59%, respectively, of the entire population of Fos-immunoreactive spinal cells. Values in the remaining laminae were low (2-10%). Following thermal stimulation by radiant heat at 65 degrees C or 58 degrees C, Fos cells were concentrated in laminae I and IIo, amounting to 57% and 62%, respectively, in lamina I, and to 26% and 29% in lamina IIo. Values were lower than 10% in the remaining laminae. Following mechanical stimulation by pinching or needle prick, Fos-positive cells were regularly distributed throughout laminae I-V amounting to 25-26% in lamina I, and 10-20% in each of the remaining laminae. These findings suggest that the spinal neuronal groups upon by prolonged noxious stimulation differ according to the nature of the stimulus.