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This article is the fifth of a series aimed at mapping brain activities as they result from the development of cyclophosphamide (CP) cystitis in behaving rats using c-fos and Krox-24 expression. The inactive hepatic metabolites of CP are metabolized in the kidney to produce acrolein, which generates cystitis. Data come from animals which were injected once i.p. with either 1 ml saline (sham) or 100 mg/kg CP in 1 ml saline under transient volatile anesthesia and which behaved freely for 1-4 h postinjection, 4 h being the minimum time for cystitis to completely develop. Survival times longer than 4 h were not studied owing to ethical considerations. The first 2 h postinjection cover a period of time over which inputs of multifactorial origin (stress and pain due to the intraperitoneal injection process, possible effects due to the presence of hepatic CP metabolites in blood, cystitis onset) interact in an indistinguishable way; the last 2 h are more cystitis specific as the other effects have vanished. Complete screening of telencephalic levels has been performed. These data complete previously published data at both spinal and subtelencephalic levels. Of all the telencephalic structures, only the bed nucleus of the stria terminalis in the dorsal part of its lateral division (BSTLd) and, to a lesser degree, the nucleus centralis of the amygdala, mostly in its caudal portion (cCeA), appeared to be significantly driven over the most specific cystitis period. Both of these structures had related, but not identical patterns of expression. They both reacted shortly after CP injection, but, while cCeA maintained its activity throughout cystitis development, BSTLd showed a rebound, reaching a peak value when cystitis was fully developed. Both of these areas are the only telencephalic areas to contain high PACAP38 immunoreactivity. This is evidence that, (1) both the BSTLd and cCeA could be the most rostral areas that visceronociceptive inflow would reach when cystitis genesis is under way, and (2) PACAP38 could be one of the neurochemical agents involved in telencephalic visceronociceptive processing. From our complete mapping of brain activities under a fully developed cystitis situation (4 h postinjection), it appears that the activities in BSTLd and cCeA are concomitant with those of both the dorsal vagal complex (DVC), paratrigeminal nucleus (PaT), and the ventrocaudal bulbar reticular formation (vcBRF) at brainstem levels, suggesting they all form the main part of the neural network that subserves the central processing of cystitis-related inputs, comprising pain and associated pseudoaffective responses. Both the DVC and BSTLd, which are the most powerfully driven areas, would be particularly important in such a way. The origin of these activities should be found in both vagal (as sensed through PaT activity) and spinal (pelvic) influences. This network profoundly differs from those reported for painful situations, either somatic or visceral, which controversally accompany positive cardiac inotropism.

The evoked expression of the immediate-early gene-encoded proteins c-Fos and Krox-24 was used to study activation of mesodiencephalic structures as a function of the development of cyclophosphamide (CP) cystitis in behaving rats. This article is the third of a series and completes previously published data obtained at both spinal and hindbrain levels. CP-injected animals received a single dose of 100 mg/kg i.p. under transient volatile anesthesia and survived for 1-4 h in order to cover the entire postinjection period during which the disease develops. Survival times longer than 4 h were not used owing to ethical considerations. Results from CP-injected groups are compared with those from either noninjected controls or saline-injected animals having survived for the same times as CP-injected ones. Quantitative results come from c-fos expression. At mesodiencephalic levels a high and widespread basal c-fos expression was observed in control animals; maximum staining was observed at the midthalamic level. Four groups of nuclei were identified with regard to the density of staining. The first group included nuclei showing clustered, intensely labeled cells; these areas were restricted in extent and related to the maintenance of circadian rythms (intergeniculate leaf, suprachiasmatic nucleus, dorsal parts of either paraventricular thalamic nuclei or central gray), sleep-arousal cycle (supramamillary nucleus), or changes in arterial pressure (laterodorsal tegmental nucleus). The second group included nuclei showing scattered, moderately labeled cells; these areas were widespread at all rostrocaudal levels and related to either autonomic/neuroendocrine regulations (central gray, lateral habenula, hypothalamus) or motor behavior, orienting reflex and oculomotor coordination (unspecific subdivisions of both colliculi and their adjoining mesencephalic regions, zona incerta dorsal). The third group included nuclei with evenly distributed, faintly labeled cells; these areas, which, with few exceptions, covered almost the entire diencephalon, mainly concerned nuclei of multisensory convergence having functions in either discriminative tasks (laterodorsal and lateroposterior thalamic nuclei) or emotional responses (intralaminar and midline thalamic nuclei). The fourth group included nuclei free of labeling; these were areas that received the bulk of unimodal sensory/motor inputs (central inferior colliculus, pretectal optic nuclei, ventral medial geniculate nucleus, ventral anterior pretectal nucleus, dorsal lateral geniculate nucleus, ventrobasal complex; zona incerta ventral, parafascicular thalamic nucleus) and are thus the most discriminative regarding specific modalities. Variations in staining were of the same magnitude in both saline- and CP-injected animals. A sequential study spanning every postinjection hour revealed maximum staining at 1 h postinjection, which was followed by a progressive, time-related decrease. Increases in the number of labeled cells 1 h postinjection were significant in only a restricted number of nuclei showing low basal expression (Edinger-Westphal nucleus and paraventricular, supraoptic, and lateral hypothalamic nuclei); time-related reductions in staining that were correlated to sleep or quiescence behaviors finally resulted in staining equal to or below that seen in control animals. No structures showed significantly increased staining in relation to the full development of cystitis, i.e., with the increase of visceronociceptive inputs. Comparing the present results with those previously obtained at more caudal levels, it appears that subtelencephalic levels primarily driven by visceronociceptive inputs, i.e., those that increase and/or maintain their activity in parallel with the degree of nociception, are confined to brainstem-spinal cord junction levels and only comprise certain subdivisions of the nucleus of the solitary tract (nucleus medialis, nucleus commissuralis, and ventralmost part of area po

The evoked expression of the immediate early gene-encoded proteins c-Fos and Krox-24 was used to study activation of hindbrain neurons as a function of the development of cyclophosphamide (CP) cystitis in behaving rats. CP-injected animals received a single dose of 100 mg/kg i.p. under transient volatile anesthesia and survived for 1 to 4 h in order to cover the whole postinjection period during which the disease develops. CP-injected groups included: (1) animals with minor simple chorionic edema, an early characteristic of inflammation (1 h postinjection); (2) animals with well-developed simple chorionic edema (2 h postinjection); (3) animals with mild inflammation (chorionic edema accompanied by epithelial cleavage; 3 h postinjection); and (4) animals with complete inflammation (4 h postinjection). In addition to onset of chorionic edema, the earliest postinjection period also included the general aspects of the nervous reaction consecutive to the injection process (handling, transient volatile anesthesia and postanesthesia awakening, abdominal pinprick, CP-blood circulating effects). Controls included both noninjected animals and saline-injected animals surviving for the same times as CP-injected ones. Quantitative results come from c-Fos expression. It has been shown that: (1) saline injection is a significant stimulus for only nucleus O and central gray pars alpha and nucleus medialis of the dorsal vagal complex; (2) all structures driven by CP injection (nucleus O and central gray pars alpha, locus coeruleus, Barrington's nucleus and parabrachial area mostly in its ventral and lateral subdivisions, dorsal vagal complex, ventrocaudal portion of lateral bulbar reticular formation) responded vigorously shortly after injection, but only two (dorsal vagal complex, ventrocaudal portion of lateral bulbar reticular formation) showed increased or renewed activity when cystitis completely developed, i.e., when noxious visceral inputs reached highest levels. Regarding the sequential activation of these structures in relation to postinjection time, evidence is given that: (1) a large variety of hindbrain structures are differentially involved in either the general reaction consecutive to the injection process or to various degrees of cystitis; (2) these structures extend from the brain-spinal cord to the pons-mesencephalon transitional junction levels; (3) the two structures most powerfully driven by visceronociceptive inputs are also the most caudal ones, being located at the brain-spinal cord junction level; and (4) the dorsal vagal complex could be the main hindbrain visceral pain center, with three particular subdivisions, the nucleus medialis, nucleus commissuralis, and ventralmost part of area postrema, being involved.

The evoked expression of the immediate early gene (IEG)-encoded proteins c-Fos and Krox-24 was used to monitor spinal visceronociceptive processing that results from cyclophosphamide cystitis in behaving rats. Animals received a single dose of 100 mg/kg i.p. of cyclophosphamide and survived for 30 min to 5 h. Longer survival times were not considered because of ethical considerations. Cyclophosphamide-injected animals developed characteristic behavioral signs in parallel with development of bladder lesions and spinal evoked expression of IEG-encoded proteins. Histological examination of the urinary bladder was used to evaluate the degree of cystitis and as a criterion for selection of groups of animals to be quantitatively analyzed. Controls consisted of freely behaving animals including control (un-injected), sham (saline-injected) or diuretic (furosemide-injected) animals. Behavioral modifications consisted of lacrimation, piloerection, assumption of a peculiar "rounded-back" posture, which was accompanied by head immobility and various brief "crises" (tail hyperextension, abdominal retractions, licking of the lower abdomen, backward withdrawal movements). Abnormal behaviors, which first appeared (lacrimation, piloerection) at the end of postinjection hour 1, progressively increased in severity (rounded-back posture) over the following 90 min to reach a plateau at about postinjection hour 2; the rounded-back posture was maintained up to time of death. Histological modifications of bladder tissue were assessed using a 4-grade scale in a blind setting. The 1st grade consisted of control or sham animals with no bladder lesion; 2nd grade, animals with simple chorionic edema; 3rd grade, animals with chorionic edema associated with mucosal abrasion, fibrin deposit, and onset of polymorphonuclear leukocyte infiltration; 4th grade, animals with complete cystitis corresponding to an increase in severity and spread of all the signs of cystitis described above plus petechial hemorrhage. Simple chorionic edema was observed from 30 min to 3 h postinjection, but with a progressive increase in severity over time. Edema accompanied by epithelial abrasion was observed for animals that survived 3-4 h postinjection; complete inflammation was observed in animals that survived 4-5 h postinjection. The study of c-Fos- and Krox-24-encoded protein expression demonstrated that few lumbosacral spinal areas were specifically involved in the processing of visceral inputs in response to bladder stimulation. These areas were the parasympathetic column (SPN), the dorsal gray commissure (DGC as the caudal extent of lamina X), and superficial layers of the dorsal horn.(ABSTRACT TRUNCATED AT 400 WORDS)

We have used the evoked expression of the immediate early gene-encoded proteins (c-Fos, Fos B, Jun B, Jun D, c-Jun and Krox-24) to monitor sensory processing in the hindbrain structures of rats undergoing somatic inflammation. Experiments were performed on freely moving animals that did not experience constraints other than those imposed by the disease itself. Local injections of chemicals were used to cause subcutaneous inflammation of the plantar foot or monoarthritis by intracapsular injection. Labelling was studied at survival times that corresponded either to the time points of maximum labelling in the spinal cord (4 h for the subcutaneous model, 24 h and two weeks for the monoarthritis model) or at survival times that corresponded to the chronic phase of monoarthritis evolution (six, nine and 15 weeks). Controls consisted of freely moving, unstimulated animals. Basal expression was observed for all immediate early genes and in a variety of structures, but always remained moderate. All immediate early gene-encoded protein expressions except c-Jun were evoked, but except for c-Fos, and to a lesser extent Jun D, intensities of staining always remained faint. The following results will be mainly based on c-Fos expression, as this protein proved to be the most effective marker for all the survival times studied. Somatic pain evoked c-Fos expression in a subset of discrete subregions of both the caudal medulla oblongata and transitional areas of the pontomesencephalic junction. In the caudal medulla oblongata, structures involved were the caudal intermediate reticular nucleus, the subnucleus reticularis dorsalis, the ventrolateral reticular formation and the lateral paragigantocellular nucleus. Structures involved at the pontomesencephalic junction level mostly included the superior and dorsal lateral subnuclei of the parabrachial area, the nucleus cuneiformis and the most caudal portions of the lateral central gray, also including the laterodorsal tegmental nucleus; labelling in other lateral subnuclei of the parabrachial area always remained moderate. Staining in the caudal reticular areas was evident only at short survival times (4 and 24 h survival times in subcutaneous and monoarthritis models, respectively). Staining in nuclei of the pontomesencephalic junction was evident in all cases except for the very long survival periods (six to 15 weeks) of monoarthritis. In all cases staining was bilateral with contralateral predominance with regard to the stimulated limb. The present work demonstrates that hindbrain structures involved in somatic pain processing can be effectively identified in behaving animals and that c-Fos is the most reliable activity marker in this case.(ABSTRACT TRUNCATED AT 400 WORDS)

We have used the evoked expression of the immediate early gene-encoded proteins (Krox-24, c-Fos, Fos B, Jun D, Jun B, c-Jun) to monitor visceral processing in both the spinal cord and hindbrain structures of rats undergoing either mechanical colorectal or chemical intraperitoneal stimulation. Experiments were conducted under controlled volatile anaesthesia to suppress affective reactions that visceral stimulations may induce. The results refer to the effects of anaesthesia alone, and of both innocuous and noxious stimulations. Non-nociceptive and nociceptive stimulation but not anaesthesia were effective in evoking c-Fos, c-Jun, Jun B and Krox-24 expressions in the spinal cord. Intraperitoneal injections labelled cells mostly at the thoracolumbar junction levels, while colorectal distension labelled cells mostly at the lumbrosacral junction levels. Labelling was widely distributed throughout the gray matter including superficial layers, deep dorsal horn, lamina X and sacral parasympathetic columns. Krox-24- and, to a lesser degree, c-Jun-labelled cells were quite numerous in the superficial layers of the dorsal horn; Jun B, and especially c-Fos, were very effective in demonstrating inputs to all parts of the spinal cord. Both anaesthesia and noxious visceral stimulation were effective in evoking c-Fos, Krox-24 and Jun B expressions in discrete hindbrain subregions. The structures which are primarily labelled under anaesthesia are the rostral ventrolateral medulla, the external medial and lateral nuclei of the parabrachial area, the medial and dorsal subnuclei of the nucleus of the solitary tract, the area postrema, the central gray including pars alpha and nucleus O, the nucleus beta of the inferior olive, the locus coeruleus, and the inferior colliculi and adjacent parts of central gray. The structures which are primarily labelled following noxious visceral stimulation are the caudal intermediate reticular nucleus as part of the caudalmost ventrolateral medulla and the superior lateral nucleus of the rostrolateral parabrachial area. Labelling in the caudal intermediate reticular nucleus was maximal for colorectal distension. Labelling in the superior lateral nucleus was specific to peritoneal inflammation. The Edinger-Westphal nucleus is a structure in which noxious-evoked labelling was superposed onto the anaesthesia-evoked labelling. Nociception-evoked overexpression in this nucleus was maximal for intraperitoneal inflammation. The present work demonstrates that the central effects induced by either anaesthesia or visceroception including pain can be effectively monitored through the induction of an array of immediate early genes.(ABSTRACT TRUNCATED AT 400 WORDS)

We have used the evoked expression of both immediate early gene (IEG)-encoded proteins (Krox-24, c-Fos, Fos B, Jun D, Jun B, c-Jun), and dynorphin to monitor sensory processing in the spinal cords of rats undergoing subacute or chronic somatic inflammation (i.e., subcutaneous inflammation of the plantar foot and monoarthritis, respectively). Behavioral and immunocytochemical approaches were conducted in parallel up to 15 weeks postinjection in order to detect possible relationships between clinical evolution and spatiotemporal pattern of IEG-encoded protein expression. Each disease had specific characteristics both in terms of their clinical evolution and pattern of evoked protein expression. All IEG proteins were expressed in both cases. Most of the staining was observed in both the superficial layers of the dorsal horn and deep dorsal horn (laminae V-VII and X). Monoarthritis was distinguished by a high level of total protein expression. Staining was especially dense in the deep dorsal horn. More labelled cells were observed at 1-2 days and at 2 weeks postinjection, corresponding to the initiation and progressive phases of the disease, respectively. Subcutaneous inflammation was characterized by a moderate level of total IEG expression. More labelled cells were observed in the first day following injection. It is the relative degree of expression of each IEG-encoded protein with regard to the others that characterized the progression of the diseases. Early stages of the diseases coincided with the expression of all Fos and Jun proteins, while late stages showed an increase in Jun D and Fos B involvement; Krox-24 was induced mostly during the early phases and/or periods of paroxysm of the diseases. Persistent stimulation was characterized by a predominant expression in deep versus superficial layers of the dorsal horn. Evoked expression of c-Jun in motoneurons was only observed in monoarthritis. The peak of dynorphin expression was late in regard to both the induction of inflammation and period of maximal IEG-encoded protein expression. The present work indicates that the neural processing that takes place during progression of these diseases can be monitored well at the spinal cord level by using the expression of an array of IEG-encoded proteins. Study of long term evolutive diseases and especially those that evolve into chronicity can largely benefit from such an approach.