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Visceral disorders are always accompanied by pain and/or a sense of ill-being that entails people to isolate themselves both physically and socially. By analogy with what happens in human beings, we have transferred to the rat the question of whether a protective, dark and quiet environment would influence the brain activities induced by visceral chemically-induced (cyclophosphamide [CP], 100 mg/kg/ip) adverse conditions of life. CP is an antitumoral drug that induces severe side effects (cystitis, headache, nausea, photophobia, phonophobia) and produces a strong state of ill-being in human beings. Brain activities were quantified using the expression of the Fos protein, a molecular marker of neuronal activity. The results compare data from groups of paired animals having been offered a shelter or not. Data were collected 4 h after the injection of CP, i. e., when cystitis was fully developed. Sheltered and unsheltered groups did not differ in bladder pathology. Intentional sheltering was shown to attenuate the expression of the CP-related Fos-Li activity within the locus coeruleus (LC) without affecting that of the structures known preferentially to process nociceptive inputs of bladder origin (dorsal vagal complex, ventrocaudal bulbar reticular formation, nucleus centralis of amygdala, dorsolateral portion of bed nucleus of stria terminalis). The LC levels of tyrosine hydroxylase and galanin neuronal contents were not affected. The LC belongs to the emotional activation system and can respond to a wide range of somatosensory and viscerosensory stimuli. Our hypothesis is that the LC would be processing the nervous activities that accompany the sense of ill-being coming from adverse conditions of life, including visceral disorders, and that voluntary isolation, by reducing its activity, would enable animals to minimize their level of distress.

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.

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. © 1993 Wiley-Liss, Inc.

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.

Small amounts of the retrograde tracer WGA-apoHRP-Au complex were injected in the caudal medulla to study the spinal afferents to the subnucleus reticularis dorsalis (SRD). Labelled neurones were found at all levels of the spinal cord: the highest numbers were in the ipsilateral cervical spinal cord (mainly laminae I, V, VI, VII, VIII and X), the lowest were at the thoracic and lumbar levels bilaterally, while an intermediate density was found bilaterally at the sacral level. When injection sites were located in the underlying subnucleus reticularis ventralis (SRV), labelling was bilateral and mainly in the deep layers of the cervical spinal cord. Together with our previous electrophysiological and anatomical data, this study suggests that the SRD provides a link in spino-reticulo-spinal loops implicated in the processing of pain.

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.

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.

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.

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.

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.

Cyclophosphamide administration generates systemic toxicity having immune and nervous consequences. After focusing on nervous consequences by studying neuronal activity, we now consider cyclophosphamide impact on diencephalic mast cells as part of the brain immune system. Diencephalon, the ultimate sensory relay before neocortical processing, is the only brain structure containing mast cells. Single cyclophosphamide administration (100 mg/(kg 1 ml ip)) was performed in naturally behaving rats and diencephalic mast cell numbers were analyzed once all drug effects had developed (4 h postinjection). Significant increases were observed only in the medial habenular nucleus--bilaterally and especially in its caudal portion. Mast cell increase is temporally related to behavioral impairment and evoked neuronal activity in a restricted number of visceral/limbic extrathalamic structures. The medial habenular nucleus belongs to the limbic system involved in processing emotional reactions and regulation of the autonomic nervous system. Its involvement during toxic challenge is highly compatible with its presumed function in the maintenance of vital functions.

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.

My commentary on mcmahon addresses the fact that only peripheral data have been considered for explaining differential sensibility in somato- and viscerosensory systems. This fails to take it into account that central processing for visceral and somatic inputs is now known to depend on different functional pathways. My commentary on berkley points out that the hypothalamus-pituitary axis is more responsive to stress in females than in males.

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.

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-glγ-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 raphenuclei, 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 raphemagnus and the nucleus reticularis magnocellularis, pars alpha) and the most rostral part of the B1 and B2 cell groups (nuclei raphepallidus 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 raphenuclei 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 Ievels 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. The majority of the substance P projection neurons were found in the Bl and B2 cell groups, in the adjacent ventromedial medulla and in the nucleus interfascicularis hypoglossi, pars ventralis. A few substance P projection neurons were also found in the caudal reaches of the B3 cell group. Methionine-enkephalin projection neurons were located in the rostral B1/B2 cell groups, B3 cell group and in the nucleus interfascicularis hypoglossi, pars dorsalis. None were found in caudal B1/B2 cell groups. Methionine-enkephalin projecting neurons were thus located rostral to the majority of the double-labelled substance P cells. Only scattered projection neurons were found among the methionine-enkephalin- and substance P-immunoreactive cells of the nucleus reticularis paragigantocellularis lateralis. In contrast to the large numbers of double-labelled methionine-enkephalin and substance P cells, no spinally projecting dynorphin neurons were found. We suggest that there are differential methionine-enkephalin 5-hydroxytryptamine inhibitory and substance P/5 hydroxytryptamine excitatory influences that issue from different medullary areas and are, respectively, exerted upon dorsal horn nociceptive neurons, and visceral/somatic motoneurons and intermediolateral preganglionic neurons.

Postsynaptic fibers reaching the dorsal column nuclei were investigated in rat by means of retrograde transport of wheat germ agglutinin-horseradish peroxidase conjugate. Each nucleus received only ipsilateral afferents with most of the labeled cells forming a band which covered the mediolateral extent of the dorsal horn in an area that resembled lamina IV in the cat. The labeling excluded the reticular extension of the neck of the dorsal horn. Lumbosacral afferents were restricted to the gracilis nucleus and cervicothoracic afferents to the cuneatus nucleus. Cervical and anterior lumbar levels showed additional projections coming from their most medial parts. The organization of this second-order pathway in rat is similar to that in cat and monkey.

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.

Electrophysiological properties of neurones in the spinal cord dorsal horn were studied in decerebrated, immobilized spinal rats. Extracellular recordings were performed at the thoraco-lumbar junction level. Each track was systematically located by extracellular injection of pontamine sky blue. According to their responses to mechanical peripheral stimuli, cells were classified in four classes: Class 1 cells: Cells activated only by nonnoxious stimuli. They were divided into - 1A: hair movement and/or touch and 1B: hair movement and/or touch and pressure or pressure only. Class 2 cells: Cells driven by both nonnoxious and noxious stimuli, divided into - 2A: hair movement and/or touch, pressure, pinch and/or pin-prick, and 2B: pressure, pinch and/or pin-prick. Class 3 cells: Cells only activated by noxious stimuli (pinch and/or pin-prick). Class 4 cells: Cells responding to joint movement or pressure on deep tissues. Peripheral transcutaneous or sural nerve stimulation clearly showed that class 1 cells were activated only by A fiber input while 68% of classes 2 and 3 cells received A and C input. Histological examination indicated that cells driven only by noxious input were located either in the deepest part or in the marginal zone (lamina I) of the dorsal horn. Nevertheless, some lamina I cells were also driven by both nonnoxious and noxious stimuli. In addition, there is a great deal of overlap between class 1 and class 2 cells. This fact was confirmed by considering the wide distribution in the dorsal horn of cells receiving A and C input. However, spinal organization of the different classes of cells consists of a preferential distribution rather than a strict lamination. This study indicates that properties of dorsal horn inter-neurones in the rat have a high degree of similarity with those previously described in other species (cat and monkey).

In this study, we have tested the possible use of protein-gold complexes as neuronal markers for long-term tracing studies in rat. The tracer we have used consisted of colloidal gold particles coupled to wheat-germ agglutinin apohorseradish peroxidase conjugate (WGA-apoHRP). The neuronal labeling was studied for survival periods of up to nineteen months following injection in the central nervous system. Maximal visualization of the gold particles was achieved through gold silver intensification. The tracer could be detected throughout the entire range of periods considered. The injection site consisted of a dense black core and retrogradely labeled cells were characterized by round black granules over the cell body. The retrogradely labeled cells were cytochemically characterized by demonstrating their transmitter content. Thus protein-gold complexes may be used as long-term neuronal markers compatible with the persistance of the vital functions of the labeled cells.

The effects of diazepam (DZ), 0.5–1 mg/kg, i.v., on inhibitory presynaptic mechanisms induced at the spinal level by the appearance of cortical seizures produced by pentetrazol have been studied in intact cats anaesthetized with pentobarbital. Spontaneous dorsal root potentials (DRP), induced by isolated discharges and by paroxysmal cortical seizures, were abolished by DZ. After the paroxysmal activity, heterosensory and heterosegmental DRP's, due to cortical hyperexcitability, were suppressed by DZ. In contrast, the segmental DRP which was reduced by pentetrazol, was increased by DZ. These data suggest that: (1) DZ exerts a direct action at the spinal level by increasing segmental presynaptic inhibition as previously described by others. (2) DZ decreases or suppresses presynaptic controls of supraspinal origin by its depressive action on supraspinal structures involved in these phenomena.

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)

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)

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, α 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.

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.

This paper describes a sensitive method for tracing neural connections at the electron microscopic (EM) level using a new compound produced through the coupling of colloidal gold particles to a wheat germ agglutinin horseradish peroxidase conjugate (the WGA*HRP-gold complex). Visualization of retrogradely labeled cells at the EM level was achieved either directly by gold particles scanning or after silver enhancement. By using different sizes of gold particles individually coupled to WGA*HRP and injected in different brain areas EM detection of multiple retrograde labeling was possible. Thus retrogradely labeled cells were first identified at the light microscopic level through HRP histochemistry with tetramethylbenzidine as a chromogen and then examined under the electron microscope after osmication and embedding. Gold particles were readily identified as electron dense, round dots in spherical grey vesicles. Identification of different sizes of gold particles often localized in the same vesicle established that the protein-gold complex can be used to study collateralisation of parental axons.

In this study I have used a tracer complex made of wheat germ agglutinin horseradish peroxidase conjugate (WGA*HRP) coupled to colloidal gold for retrograde tracing of neuronal pathways at the light microscopic level. Visualization of the gold was achieved by silver precipitation (the gold silver intensification method) with gold particles acting as specific cores of nucleation. The presence of horseradish peroxidase in the protein conjugate allowed this method to be compared with classical histochemistry using tetramethylbenzidine as a chromogen. The gold silver intensification method proved to be reliable, specific and sensitive. It has been demonstrated to be useful with fixatives containing a high percentage of paraformaldehyde and compatible with histochemical procedures to show projections of transmitter specific pathways.

The efferent projections of the paratrigeminal nucleus in the rat were investigated by means of retrograde transport techniques. Injections were made in most of the supraspinal structures known to receive afferents from the spinal cord or the trigeminal nucleus caudalis. Spinal injections were also performed. Dense paratrigeminal efferents were seen to be directed to the nucleus of the solitary tract and to the peribrachial area, the latter including the cuneiformis and parabrachial nuclei. Projections were mostly ipsilateral. These results are discussed in relation to a possible role of the paratrigeminal nucleus in thermoreception and/or vegetative regulation processing.