The effects of viscero-somatic interactions on thalamic mast cell recruitment in cystitic rats.

Mast cells accessing the brain parenchyma through the blood-brain barrier in healthy animals are limited to pre-cortical sensory relays - the olfactory bulb and the thalamus. We have demonstrated that unilateral repetitive stimulation of the abdominal wall generates asymmetry in midline thalamic mast cell (TMC) distribution in cyclophosphamide-injected rats, consisting of contralateral side-prevalence with respect to the abdominal wall stimulation. TMC asymmetry 1) was generated in strict relation with cystitis, and was absent in disease-free and mesna-treated animals, 2) was restricted to the anterior portion of the paraventricular pars anterior and reuniens nuclei subregion, i.e., the rostralmost part of the paraventricular thalamic nucleus, the only thalamic area associated with viscero-vagal and somatic inputs, via the nucleus of the solitary tract, and via the medial contingent of the spinothalamic tract, respectively, and 3) originated from somatic tissues, i.e., the abdominal wall where bladder inflammation generates secondary somatic hyperesthesia leading to referred pain in humans. Present data suggest that TMCs may be involved in thalamic sensory processes, including some aspects of visceral pain and abnormal visceral/somatic interactions.

Evidence for serotonin influencing the thalamic infiltration of mast cells in rat.

Serotonin (5-HT) is involved in neuroimmunomodulation. We analyzed the effects of sumatriptan, a 5-HT(1B/1D) receptor agonist, and ondansetron, a 5-HT(3) receptor antagonist, on thalamic mast cell (TMC) population, the only immunocytes known to infiltrate the brain in physiological conditions. Only sumatriptan was effective, significantly increasing TMC numbers versus controls, and especially those containing 5-HT. 5-HT(1B) receptors are concentrated in the median eminence on non-serotonergic axonal endings, probably hypothalamic terminal fibers, involved in hypothalamic-pituitary neuroendocrine modulating processes. TMC variations could reflect serotonergic actions on these fibers. TMCs would thus be cellular interfaces mediating immune action in the nervous system in relation with the hormonal status of the organism.

Differential effects of two analgesic drugs, morphine chlorhydrate and acetylsalicylic acid, on thalamic mast cell numbers in rat.

Thalamic mast cells (TMCs), the only immunocytes known to infiltrate the brain in physiological conditions, respond to pharmacological agents including sumatriptan - a serotonergic anti-migraine agent - that increases their number. We analysed the effects of two other main analgesics: morphine chlorhydrate, a micro opioid agonist, and acetylsalicylic acid (ASA), a non-steroidal anti-inflammatory drug. All three drugs have specific modes of action, and morphine and ASA, unlike sumatriptan, are also known to interact with peripheral mast cells. Only ASA was effective in promoting TMC number decrease. TMCs, unlike other mast cells, do not express cyclooxygenase (COX) - the key enzyme in the production of prostanoids and the main site of action of ASA - thus dismissing a direct local cellular COX-mediated action. Direct TMC COX-independent mechanisms or effects mediated via distant populations of COX-positive cells such as platelets, leptomeningeal, endothelial and peripheral mast cells are thus probable. ASA, morphine and sumatriptan have distinct TMC effects, suggesting that the TMC number variations they induce are more likely to derive from systemic vasoactive actions than from pharmacological mechanisms devoted to pain relief.

Origins of Spinal Ascending Pathways that Reach Central Areas Involved in Visceroception and Visceronociception in the Rat.

The location of spinal cells projecting rostrally to central areas that process visceroception and visceronociception were studied in rat using the retrograde transport of a protein - gold complex. Origins of afferents to the nucleus tractus solitarius (the spinosolitary tract), the parabrachial area (the spinoparabrachial tract), the hypothalamus (the spinohypothalamic tract) and the amygdala (the spinoamygdalar tract) were studied at thoracic, lumbar and sacral levels, where spinal visceroceptive areas are concentrated. All of the afore-mentioned pathways have common origins in the lateral spinal nucleus and in the reticular formation of the neck of the dorsal horn at all the levels studied, and also in the dorsal grey commissure and adjacent areas at sacral levels. The spinosolitary and the spinoparabrachial tracts are dense pathways, both of which are also characterized by afferents from the superficial layers of the dorsal horn at all the levels studied and from cells lying in close proximity to some autonomic spinal areas. These autonomic areas are the central autonomic nucleus (dorsal commissural nucleus) of lamina X at thoracolumbar levels and the parasympathetic column at sacral levels; some projections from the intermediolateral cell column at thoracic levels were also noted. Projections from all these autonomic structures to the parabrachial area have not yet been recognized. Thus, the origin of the spinoparabrachial tract closely resembles that of the spinomesencephalic tract that reaches the periaquaductal grey and adjacent areas. The spinohypothalamic and the spinoamygdalar tracts are smaller pathways. Direct spinal connections to the amygdala have not been reported previously. Both the hypothalamus and amygdala receive projections from lamina VII cells at low thoracic and upper lumbar levels in a pattern that resembles that of the preganglionic cells of the intercalated nucleus. Hypothalamic projections from the sacral parasympathetic area were also noted. The use of c-fos as a functional marker to identify spinal neurons that are activated by noxious visceral stimulation suggests that both the spinoparabrachial and the spinosolitary tracts contribute significantly to the central transmission of visceronoceptive messages. Most of the visceronociceptive ascending projections in these pathways issued from lamina I cells. The results presented here confirm previous observations regarding the spinosolitary and the spinohypothalamic tracts and also demonstrate, for the first time, the complex origin of the spinoparabrachial tract and the existence of direct spinal afferents to the amygdala. These findings suggest that rostral transmission and central integration of visceral inputs require several parallel routes. The spinosolitary and spinoparabrachial tracts clearly play a role in conveying information regarding visceronociception.

Calbindin-D28K (CaBP28k)-like Immunoreactivity in Ascending Projections.

This study concerns the involvement of calbindin-D28K (CaBP28k)-containing neurons in the efferent projections of both the trigeminal nucleus caudalis and the dorsal vagal complex (nucleus of the solitary tract and area postrema) in rats. Recent evidence has shown that these projections are particularly important for the processing of visceroception and/or nociception at central levels. The trigeminal nucleus caudalis has dense projections to both the nucleus of the solitary tract and the parabrachial area; the dorsal vagal complex is intimately connected to the parabrachial area. CaBP28k is a calcium-binding protein the function of which could be a determining factor in controlling the excitability of cells by acting on intrinsic calcium metabolism. CaBP28k content of projections was ascertained using a double labelling approach that combined the retrograde transport of a protein - gold complex to identify projection cells and immunocytochemistry to identify CaBP28k-positive cells. The trigeminal nucleus caudalis is rich in both CaBP28k-immunoreactive cells and cells projecting to the parabrachial area or the nucleus of the solitary tract. Cells containing both the protein and the retrograde tracer, however, were mostly restricted to the superficial layers (laminae I and outer II) and to their rostral extensions, the dorsal paramarginal and paratrigeminal nuclei. These trigeminal subdivisions are targets for nociceptive, visceroceptive and thermal inputs of peripheral origins. The dorsal vagal complex is rich in CaBP28k. Dense populations of immunoreactive cells are observed in the ventrolateral part of the area postrema and all of the three main subdivisions of the nucleus of the solitary tract (rostral gustatory, ventrolateral respiratory and medial cardiovascular subregions). The subnucleus commissuralis, subnucleus centralis and dorsal subnuclei are particularly densely stained. The subnucleus centralis, which is involved in regulating food and water intake, does not project to the parabrachial area. The area postrema, subnucleus commissuralis and dorsal subnuclei, which are implicated in cardiovascular and/or ingestive behaviours, have dense projections to the parabrachial area, many of which contain CaBP28k. The present results demonstrate that CaBP28k-containing cells form a major part of the solitary and trigeminal projection systems, including subregions that are involved in visceroception and/or nociception processing. The location of solitary nucleus projection cells overlaps those of some neuropeptidergic projecting populations, suggesting colocalization. Consequently, certain neuropeptidergic actions may be CaBP28k-dependent.

Calbindin-D28K (CaBP28k)-like Immunoreactivity in Ascending Projections.

This study concerns the involvement of calbindin-D28K (CaBP28k)-containing neurons in ascending spinal projections to the brainstem (nucleus of the solitary tract, lateral reticular nucleus area), pontine (parabrachial area) and mesencephalic (periaqueductal grey) structures. All these central structures are important in the processing of visceroception and visceronociception and all are targets for spinal efferents from similar areas. CaBP28k controls the excitability of cells by acting on intrinsic calcium metabolism. Results refer to the caudal spinal areas where the visceroceptive regions are concentrated. Experiments were performed through a double labelling approach that combined the retrograde transport of a protein - gold complex to identify the projection cells and immunohistochemistry to identify the CaBP28k-positive cells. The caudal spinal cord is rich in both CaBP28k-containing and projection cells. Cells colocalizing the protein and the retrograde tracer were quite numerous, with a particularly high concentration in the superficial layers of the dorsal horn (laminae I and outer II) and the lateral spinal nucleus. The other spinal areas containing immunoreactive projection cells were the reticular part of the neck of the dorsal horn, the medial laminae VII and VIII, lamina X and the sacral parasympathetic nucleus. The superficial layers and the neck of the dorsal horn are targets for nociceptive, visceroceptive and thermal inputs; the sacral parasympathetic column and lamina X are involved in visceroceptive integration. A functional role for the lateral spinal nucleus has not yet been established. Quite similar results were obtained for each of the ascending pathways under study. The high incidence of CaBP28k in spinal pathways suggests that calbindin has a major role in controlling the excitability of spinal cells subserving the processing of visceroception and/or visceronociception information to supraspinal levels. The participation of CaBP28k-immunoreactive cells in spinal ascending tract cells largely outnumbers those previously reported for various neuropeptides (Leah et al., Neuroscience, 24, 195 - 207, 1988)

Spinal and trigeminal projections to the nucleus of the solitary tract: a possible substrate for somatovisceral and viscerovisceral reflex activation.

This study used the retrograde transport of a protein-gold complex to examine the distribution of spinal cord and trigeminal nucleus caudalis neurons that project to the nucleus of the solitary tract (NST) in the rat. In the spinal grey matter, retrogradely labeled cells were common in the marginal zone (lamina I), in the lateral spinal nucleus of the dorsolateral funiculus, in the reticular part of the neck of the dorsal horn (lamina V), around the central canal (lamina X), and in the region of the thoracic and sacral autonomic cell columns. The pattern of labeling closely resembled that seen for the cells at the origin of the spinomesencephalic tract and shared some features with that of the spinoreticular and spinothalamic tracts. Labeled cells in lamina IV of the dorsal horn were only observed when injections spread dorsally, into the dorsal column nuclei, and are thus not considered to be at the origin of the spinosolitary tract. They are probably neurons of the postsynaptic fibers of the dorsal column. Retrogradely labeled cells were also numerous in the superficial laminae of the trigeminal nucleus caudalis, through its rostrocaudal extent. The pattern of marginal cell labeling appeared to be continuous with that of labeled neurons in the paratrigeminal nucleus, located in the descending tract of trigeminal nerve. Since the NST is an important relay for visceral afferents from both the glossopharyngeal and vagus nerves, we suggest that the spinal and trigeminal neurons that project to the NST may be part of a larger system that integrates somatic and visceral afferent inputs from wide areas of the body. The projections may underlie somatovisceral and/or viscerovisceral reflexes, perhaps with a significant afferent nociceptive component.

Wheat germ agglutinin-apoHRP gold: a new retrograde tracer for light- and electron-microscopic single- and double-label studies.

In this report, we describe a new colloidal-gold-labelled retrograde tracer, wheat germ agglutinin (WGA) conjugated to enzymatically inactive horseradish peroxidase (apoHRP). This protein gold complex (WGAapoHRP-Au) is a sensitive marker for retrograde tracing of the projections of CNS neurons at the light-microscopic (LM) level when a silver-enhancement procedure is used to detect the gold in the tracer. For electron-microscopic (EM) analysis, the silver-enhanced sections undergo a further gold-toning step. This protects against rapid oxidation and dissolution of the silver precipitate during the osmication procedure. A major advantage of WGAapoHRP-Au is that it can be used in a variety of multiple-labelling studies. When the retrograde transport of the new tracer is combined with that of the fluorescent dye, True Blue, neurons that have bifurcating axons can be readily demonstrated. Simultaneous immunofluorescent detection of the cytochemistry of the double-retrogradely labelled neurons is also possible. In contrast to a WGA-HRP gold complex, the new complex has no enzymatic activity. Thus HRP-based techniques (e.g., anterograde transport of WGA-HRP or peroxidase-antiperoxidase immunocytochemistry) can be performed on tissue that contains retrogradely labelled neurons marked with WGAapoHRP-Au without having to pretreat tissue so as to destroy endogenous HRP enzyme activity. At the EM level, the gold is readily distinguished from DAB immunoreaction product. This makes both LM and EM double-labelling studies possible. The great sensitivity of the new tracer, its compatibility with a variety of aldehyde fixatives, its ease of detection, and the fact that it can be simultaneously used with several fluorescent and HRP-based immunocytochemical and tracing techniques make WGAapoHRP-Au a valuable tool for LM and EM characterization of CNS cytochemistry and connectivity.

Neuropeptides and catecholamines in efferent projections of the nuclei of the solitary tract in the rat.

This study focuses on the involvement of catecholamines and nine different peptides in efferents of the nucleus of the solitary tract to the central nucleus of the amygdala, the bed nucleus of the stria terminalis, and different parabrachial and hypothalamic nuclei in the rat. A double-labeling technique was used that combines a protein-gold complex as the retrograde tracer with immunohistochemistry. Catecholaminergic projection neurons were the most numerous type observed and projected mainly ipsilaterally to all targets studied. Most projections arose from areas overlying the dorsal motor nucleus, mainly the medial nucleus. Neurons synthesizing somatostatin, met-enkephalin-Arg-Gly-Leu, dynorphin B, neuropeptide Y, and neurotensin projected to all structures examined. Somatostatin and enkephalin immunoreactive projection cells were the most numerous. They were located in close proximity to each other, including all subnuclei immediately surrounding the solitary tract, bilaterally. Most dynorphin and neuropeptide Y immunoreactive projection cells were found rostral to that of enkephalinergic and somatostatinergic projections, and mainly in the ipsilateral medial nucleus. Neurotensinergic projections were sparse and from dorsal and dorsolateral nuclei. Substance P and cholecystokinin contribute to parabrachial afferents. The location of substance P immunoreactive projection cells closely resembled that of enkephalinergic and somatostatinergic projections. Projecting cholecystokinin immunoreactive cells were observed in dorsolateral nucleus. Bombesin immunoreactive cells in dorsal nucleus projected to either the parabrachial or hypothalamic nuclei. No vasoactive intestinal polypeptide-containing cells were detected. Thus, most catecholaminergic and neuropeptidergic efferents originated from different populations of cells. It is proposed that catecholaminergic neurons constitute the bulk of solitary efferents and that they may contribute to autonomic neurotransmission. Peptidergic neurons mainly form other subgroups of projections and may play a role in modulating the physiological state of the target nuclei.

Spinal neurons reaching the lateral reticular nucleus as studied in the rat by retrograde transport of horseradish peroxidase.

An anatomical technique based on the retrograde transport of horseradish peroxidase (HRP) was used to investigate the projections of spinal cord neurons to the lateral reticular nucleus (LRN). Labeled cells were found at all spinal levels and in particular large numbers in cervical and lumbar segments. Various spinal areas gave rise to cells of origin of this tract, which appears to be more prominent than any other tract previously studied with a similar approach. Labeling common to all spinal segments was observed in (1) ventromedial parts of both intermediate zone and ventral horn (laminae VII, VIII and X), mainly contralaterally; (2) the reticular extension of the neck of the dorsal horn, partly bilateral; and (3) superficial layers of the dorsal horn and nucleus of the dorsolateral funiculus (NDLF), mainly contralateral and projecting essentially to the lateral zone of the LRN. Additional labeling was observed at cervical and lumbar levels, each with specific qualities: (1) the cervical enlargement, which displayed labeling in the central part of the ipsilateral intermediate zone (lamina VII); (2) the rostral lumbar levels, which had projections from the contralateral median portion of the neck of the dorsal horn. These latter projections appear to be specific to pathways reaching the lateral reticular nucleus and the inferior olive. Control injections in neighboring structures demonstrated the similarity between the afferents to the lateral reticular nucleus and the inferior olive. Control injections in neighboring structures demonstrated the similarity between the afferents to the lateral reticular nucleus and the inferior olive (except lamina I and NDLF projections) and the differences between these afferents and those projecting to the dorsal reticular formation, i.e., the nucleus reticularis ventralis.

Expression of c-fos protein in interneurons and projection neurons of the rat spinal cord in response to noxious somatic, articular, and visceral stimulation.

This study used immunocytochemistry to examine the pattern of noxious-stimulus evoked expression of the proto-oncogene c-fos in the spinal cord of the rat. Both noxious somatic and joint stimulation in awake rats evoked the expression of c-fos protein in similar areas of the lumbar spinal cord. C-fos-immunoreactive neurons were found in laminae I and outer II, in the lateral part of the neck of the dorsal horn, and in laminae VII, VIII, and X. All of the labelled neurons were located ipsilateral to the injured hindpaw, except for lamina VIII where bilateral labelling was recorded. The c-fos-immunoreactive neurons in lamina I extended from the L3 segment to the rostral sacral cord; staining in outer lamina II was only found at the L4 segment. The more deeply located cells, of the dorsal and medioventral horns, had the most extensive rostrocaudal spread; they were found from L1 through the rostral sacral segments. The pattern of c-fos-immunoreactivity produced by visceral stimulation, in anesthetized rats, differed in several ways from that produced by somatic stimulation. First, there was considerable bilateral, symmetrical labelling of cells. Second, there was a much more extensive rostrocaudal spread of the labelling, from cervical through sacral cord. Third, the greatest rostrocaudal spread was found for neurons in the superficial dorsal horn; labelled cells in the neck of the dorsal horn and in lamina X were restricted to segments at the thoracolumbar junction, which is also where the superficial dorsal horn cells were most concentrated. Fourth, there were very few labelled neurons in the outer part of the substantia gelatinosa. To determine whether any neurons that express the c-fos protein in response to noxious stimulation project to supraspinal sites, we combined the immunocytochemical localization of c-fos with the localization of a retrogradely transported protein-gold complex that was injected into the thalamic and brainstem targets of the major ascending spinal pathways. In rats that received the somatic noxious stimulus, 90% of all of the c-fos projection neurons were recorded in four major areas of the cord: lamina I (37%), the lateral part of the neck of the dorsal horn (24%), laminae VIII (9%), and X (29%). The remainder were scattered throughout the spinal gray. With the exception of lamina VIII, which contained c-fos projection neurons contralateral to the inflamed paw, all of the c-fos projection neurons were located ipsilateral to the injured paw.(ABSTRACT TRUNCATED AT 400 WORDS)

Neurons at the origin of the medial component of the bulbopontine spinoreticular tract in the rat: an anatomical study using horseradish peroxidase retrograde transport.

An anatomical technique based on the retrograde transport of horseradish peroxidase (HRP) was used to investigate the projections of spinal cord neurons to the reticular formations in the rat. Both large and restricted injections were staggered all along the bulbar and pontine levels, involving the nucleus gigantocellularis, the nuclei reticularis pontis, pars oralis and caudalis and in some cases the nucleus raphé magnus. Labeled cells were constantly encountered in the reticular part of the neck of the dorsal horn throughout the whole length of the cord, mainly contralateral to the central core of the injection site. This area was taken as the equivalent of lamina V in the cat. Other labeled cells were observed in the medial parts of the intermediate and ventral horns, in areas considered similar to laminae VII and VIII in the cat. The two most rostral cervical segments were characterized by an additional bilateral projection originating from the dorsolateral part of ventral horns. Thus, this study is a clear confirmation that the bulbopontine reticular formations constitute a target for various somatosensory inputs originating in spinal cord. It demonstrates that the medial spinoreticular tract (mSRT) differs from the other main ascending tracts by the absence of projections from (1) superficial layers and nucleus of the dorsolateral funiculus contrary to the spinomesencephalic tract; (2) ventromedial zone of the lumbar dorsal horn unlike the spinothalamic tract; (3) the neck of the dorsal horn in its medial portion contrary to the spinoreticular component reaching the lateral reticular nucleus; and (4) central cervical nucleus and Clarke's columns, unlike the spinocerebellar tracts. The difficulty in demonstrating retrograde labeling from discrete injections could result from the fact that mSRT neurons have sparsely ramified collaterals on their terminal zones.

The origin of the spinomesencephalic tract in the rat: an anatomical study using the retrograde transport of horseradish peroxidase.

An anatomical technique based on the retrograde transport of horseradish peroxidase (HRP) was used to investigate the projections of spinal cord neurons to the mesencephalic tegmentum in the rat. Restricted unilateral injections were confined to central grey, cuneiformis areas, and superior colliculus. Injections into all these loci produced labeling in similar spinal areas. Only quantitative differences were noted. In the spinal grey matter, numerous labeled cells were regularly encountered in the marginal zone, the lateral part of the neck of the dorsal horn, and the dorsal grey commissure. Projections from the marginal zone and neck of the dorsal horn were predominantly contralateral. In the white matter, a pronounced bilateral labeling was observed in the nucleus of the dorsolateral funiculus, thus confirming our previous electrophysiological findings (Menétrey et al., '80). This distribution of labeled cells was commonly observed throughout the whole length of the cord. Additional sites of projecting cells have also been identified at the most rostral levels (obex, C1, C2). They mostly derived from spinal extensions of the dorsal column nuclei and lateral cervical nucleus contralaterally; from the lateral ventral horns bilaterally and from the nucleus commissuralis ipsilaterally. This study is thus a clear confirmation that the mesencephalic tegmentum constitutes a target for various somatosensory inputs originating from spinal cord, dorsal column nuclei, and lateral cervical nucleus. Moreover, from these results together with those obtained for the spinothalamic tract in the rat, it appears that marginal and dorsolateral funiculus neurons preferentially project to the mesencephalic tegmentum. The importance of marginal zone projections underlines the involvement of the spinomesencephalic tract in pain mechanisms.

Differential time course and spatial expression of Fos, Jun, and Krox-24 proteins in spinal cord of rats undergoing subacute or chronic somatic inflammation.

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.

Responses of spinal cord dorsal horn neurones to non-noxious and noxious cutaneous temperature changes in the spinal rat.

(1) Lumbar dorsal horn units characterized by their mechanical cutaneous sensitivities were tested for their responses to temperature changes of the skin in the decerebrate spinal rats. (a) Class 1 units (i.e. driven by non-noxious mechanical stimuli) were rarely thermally sensitive. (b) Nearly all class 2 units (i.e. driven by both non-noxious and noxious mechanical stimuli) and 4 of the 5 class 3 units (i.e. driven by noxious mechanical stimuli) were sensitive to temperature changes. (2) According to their thermal response threshold and their response range, these units were divided into 3 groups. (a) Warming units whose response threshold and maximum response were below 42.5 degrees C. Such units were rarely encountered. (b) Warming/noxious heat units whose response threshold was below 42.5 degrees C but with a maximum response above this temperature. They represented approximatively one-third of the radiant heat-sensitive units. (c) Noxious heat units whose response threshold was above 42.5 degrees C and maximum discharge several degrees above it. Approximately 50% of units activated by radiant heat belonged to this group. (3) Responses to radiant heat stimulation were frequently affected by a first noxious heat application. It consisted: --in a threshold decrease and/or an increase of their cellular discharge for a same temperature range. Only observed for warming/noxious heat units and noxious heat units, this sensitization phenomenon predminantly affected noxious heat units. --in a decrease of cellular discharge for a same temperature range. This desensitization phenomenon was observed for the 3 groups of units driven by radiant heat but mainly for warming units. (4) Supramaximal transcutaneous electrical stimulation revealed that nearly all the thermally sensitive units received A delta and/or C inputs. These units were largely distributed throughout the dorsal horn (laminae I, IV and V). Ten of the 12 lamina I units responded to noxious thermal stimuli. (5) These data indicate that an increase in skin temperature is coded at the level of the rat spinal cord dorsal horn by both an increase in discharge of low threshold temperature sensitive units and a progressive recruitment of high threshold units.

Sex, time-of-day and estrous variations in behavioral and bladder histological consequences of cyclophosphamide-induced cystitis in rats.

This study examined how cyclophosphamide (CP)-induced cystitis related manifestations (bladder inflammation and behavioral impairment) differed in female and male Sprague-Dawley rats. Under transient halothane-O2-N2O gas anesthesia, a single dose of CP was injected (100 mg/kg i.p. in 1 ml saline) and the animal's behaviors analyzed for a period of 4 h using a protocol that permits quantitative analysis of behavioral impairment. The rats were then sacrificed and their bladders removed for histological quantification of inflammation. All CP-injected, but not control rats, exhibited a range of impairment behaviors that increased rapidly over a period of 2 h, gradually reaching plateau levels over the next 2 h. Female rats initially developed behavioral responses faster than male rats, but reached the same mean peak values overall as males. No sex differences were observed in CP-induced bladder inflammation. Influences of time-of-day and estrous stage were further examined in females. Time-of-day had no effect on the degree of bladder inflammation. Although there were also no significant time-of-day differences in behavioral impairments, impairment scores from 90 min after the injection consistently tended to be lower for rats injected 5 h versus 9 h after lights on. Overall, the effects of estrous stage were also insignificant. However, a subset of rats who were in the estrous stage of their cycle early in the morning of the experimental day developed the most severe degree of bladder inflammation, but failed to develop the severe behavioral impairments shown by all the other rats. These results show that there are seemingly only minor sex differences in the overall behavioral and inflammatory consequences of CP injections, as evidenced by similar final degrees of behavioral impairment and inflammation. These results also suggest, however, that there are sex differences in the etiology of the disease process. These differences are evidenced by the more rapid development of behavioral symptoms in females and the susceptibility of some of those having shown morning estrous smears to develop very severe bladder inflammation in absence of corresponding behavioral impairment. The multiple influences of sex and estrous condition on CP-induced cystitis related manifestations observed here underline the complexity of the etiological factors associated with the cystitis disease process.

The distribution of substance P-, enkephalin- and dynorphin-immunoreactive neurons in the medulla of the rat and their contribution to bulbospinal pathways.

This study examined the medullary distribution of peptide-containing neurons at the origin of bulbospinal pathways in the rat. Antisera directed against substance P, methionine-enkephalin-arg-gly-leu and dynorphin B were used on sections in which spinally projecting brainstem neurons had been identified by the retrograde transport of a protein-gold complex that was injected into the spinal cord. Both the relative numbers and distribution of the different peptide-immunoreactive spinally projecting neurons differed. Methionine-enkephalin-immunoreactive neurons were twice as numerous as the substance P-immunoreactive cells and seven times more numerous than the dynorphin B-positive neurons. The methionine-enkephalin cells were found in all medullary raphé nuclei, and in the ventromedial and ventrolateral medullary reticular formation. Caudally, the methionine-enkephalin cells were concentrated laterally; more rostrally they were located more medially. Three major loci of methionine-enkephalin-immunoreactive cells were found: (1) the nucleus reticularis paragigantocellularis lateralis, at levels caudal to the facial nucleus, (2) the B3 cell group (nucleus raphé magnus and the nucleus reticularis magnocellularis, pars alpha) and the most rostral part of the B1 and B2 cell groups (nuclei raphé pallidus and obscurus), (3) a dense cluster of cells that flanks the dorsal surface of the dorsal accessory olive (referred to as the nucleus interfascicularis hypoglossi, pars dorsalis). Substance P-like cells were seen in all raphé nuclei except for the most anterior portion of the B3 cell group. Substance P-immunoreactive cells were also seen in both the ventromedial (nuclei reticularis ventralis and magnocellularis) and ventrolateral medulla (nucleus reticularis paragigantocellularis lateralis). Finally there was a dense concentration of substance P neurons in the nucleus interfascicularis hypoglossi, pars ventralis. The distribution of dynorphin-immunoreactive neurons differed significantly from that of methionine-enkephalin and substance P. Dynorphin cells were almost exclusively found in the ventrolateral medulla (nucleus reticularis paragigantocellularis lateralis), at all levels between the lateral reticular nucleus and the caudal pole of the facial nucleus. The proportion of each of these peptidergic-immunoreactive cells at the origin of bulbospinal pathways differed considerably. Substance P spinally projecting neurons were more numerous than methionine-enkephalin spinally projecting neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

neuropeptides in long ascending spinal tract cells in the rat: evidence for parallel processing of ascending information.

A study has been made of the involvement of spinal peptidergic neurons in ascending tracts at lumbar-sacral levels in rats, by combining the retrograde transport of a protein-gold complex with immunocytochemistry. Ten neuropeptides have been considered for their presence in the cells of origin of the following six ascending tracts, including some involved in pain transmission: the spinosolitary tract, the medial and lateral spinoreticular tracts, the spinomesencephalic tract, the spinothalamic tract and the postsynaptic dorsal column tract. Although there was overlap in the distribution of several of the types of peptidergic cells and some ascending tract cells only a very small percentage of long ascending tract cells were found to contain neuropeptides. Most (90%) of those peptidergic ascending tract cells, however, were clearly congregated in two distinct spinal regions: the lateral spinal nucleus and the region surrounding the central canal (including lamina X). Ascending tract cells in both of these regions contained a wide variety of neuropeptides. Immunoreactivities for a total of seven different peptides were seen. The lateral spinal nucleus had the highest percentage of neuropeptide containing ascending tract cells; cells of all the four populations of peptidergic neurons lying in this region were involved in supraspinal projections; they stained for vasoactive intestinal polypeptide, bombesin, substance P or dynorphin and their axons projected in the spinomesencephalic, spinoreticular and spinosolitary tracts. The region surrounding the central canal contained bombesin-, enkephalin-, cholecystokinin- and somatostatin-immunoreactive ascending tract cells; these cells were found at the origin of the spinothalamic, spinomesencephalic, spinoreticular and spinosolitary tracts. In this region only the cells staining for substance P were not involved in supraspinal projections. The peptidergic ascending tract cells in other spinal regions were few; they were found in either lamina I or lateral part of lamina V. Ascending tract lamina I cells reacted for dynorphin or vasoactive intestinal polypeptide and their axons projected in the spinosolitary and spinomesencephalic tracts. Ascending tract lamina V cells reacted for somatostatin and were found at the origin of the medial component of the spinoreticular tract. It is proposed that peptidergic ascending tract cells form minor but distinct subgroups within each ascending tract. Each of the ascending tracts are divisible into peptide- and nonpeptide-containing groups of cells which convey information in a parallel fashion.(ABSTRACT TRUNCATED AT 400 WORDS)

Cholinergic and peptidergic projections from the medial septum and the nucleus of the diagonal band of Broca to dorsal hippocampus, cingulate cortex and olfactory bulb: a combined wheatgerm agglutinin-apohorseradish peroxidase-gold immunohistochemical study.

We have examined the distribution pattern and the density of various neuropeptide, neurotransmitter and enzyme containing neurons in the rat medial septum and the nucleus of the diagonal band of Broca to assess their possible involvement in the septohippocampal, septocortical and septobulbar pathways. Immunohistochemical methods were combined with the retrograde transport of a protein-gold complex injected in the hippocampus, the cingulate cortex or the olfactory bulb. Cholinergic neurons were the most numerous. Galanin-positive neurons were about two or three times less numerous than cholinergic cells. Both these cell types had a similar location though the choline acetyl transferase-like immunoreactive cells extended more caudally in the horizontal limb of the nucleus of the diagonal band of Broca. Immunoreactive cells for other neuroactive substances were few (calcitonin gene-related peptide, luteinizing hormone releasing hormone. [Met]enkephalin-arg-gly-leu) or occasional (dynorphin B, vasoactive intestinal polypeptide, somatostatin, neurotensin, cholecystokinin, neuropeptide Y and substance P). No immunoreactive cells for bombesin, alpha atrial natriuretic factor, corticotropin releasing factor, 5-hydroxytryptamine, melanocyte stimulating hormone, oxytocin, prolactin, tyrosine hydroxylase or arg-vasopressin were present. Choline acetyltransferase- and galanin-like immunoreactive cells densely participate to septal efferents. Cholinergic neurons constituted the bulk of septal efferent neurons. Galanin-positive cells were 22% of septohippocampal, 8% of septocortical, and 9% of septobulbar neurons. Galanin containing septohippocampal neurons were found in the medial septum and the nucleus of the diagonal band of Broca; galanin-positive septobulbar and septocortical cells were limited to the nucleus of the diagonal band of Broca. Occasional double-labellings were noticed with some peptides other than galanin. Luteinizing hormone-releasing hormone, calcitonin gene-related peptide and enkephalin were the most often observed; some other projecting cells stained for vasoactive intestinal polypeptide or dynorphin B. Luteinizing hormone-releasing hormone, calcitonin gene-related peptide and enkephalin were observed in septohippocampal neurons; luteinizing hormone-releasing hormone and vasoactive intestinal peptide were observed in septocortical neurons and calcitonin gene-related peptide, luteinizing hormone-releasing hormone and dynorphin B were observed in septo-bulbar cells. These results show that, in addition to acetylcholine, galanin is a major cellular neuroactive substance in septal projections to the hippocampus, the cingulate cortex and the olfactory bulb. The presence of septal projecting neurons immunoreactive for other peptides shows that a variety of distinct peptides may also participate, but in a smaller number, to septal efferent pathways.

Hindbrain structures involved in pain processing as revealed by the expression of c-Fos and other immediate early gene proteins.

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)