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A detailed account of the distribution of immunoreactive substance P-containing structures in the rat central nervous system is presented, from results obtained by applying an indirect immunofluorescent technique. High densities of substance P-containing nerve terminals were present in sensory nuclei and other non-sensory structures such as thalamus, hypothalamus and extrapyramidal system. Substance P-reactive neuron cell bodies were present in spinal root ganglia, nucleus habenulae medialis, nucleus interpeduncularis, caudoputamen and globus pallidus. Most of the neocortex and the cerebellar cortices had no substance P-positive elements. The results support the hypothesis that substance P may be a widespread neurotransmitter in the central nervous system.

The immunocytochemical distribution of [Leu]enkephalin and an adrenal enkephalin precursor fragment (BAM-22P) immunoreactivity was investigated in the diencephalon and brainstem of rats pretreated with relatively high doses of colchicine (300-400 micrograms/10 microliters intracerebroventricularly). The higher ranges of colchicine pretreatment allowed the visualization of extensive enkephalin-containing systems in these brain regions, some of which are reported for the first time. Immunoreactive perikarya were found in many hypothalamic and thalamic nuclei, interpeduncular nucleus, substantia nigra, the colliculi, periaqueductal gray, parabrachial nuclei, trigeminal motor and spinal nuclei, nucleus raphe magnus and other raphe nuclei, nucleus reticularis paragigantocellularis, vestibular nuclei, several noradrenergic cell groups, nucleus tractus solitarius, as well as in the spinal cord dorsal horn. In addition to the above regions, immunoreactive fibers were also noted in the habenular nuclei, trigeminal sensory nuclei, locus coeruleus, motor facial nucleus, cochlear nuclei, dorsal motor nucleus of the vagus, and hypoglossal nucleus. When adjacent sections of those stained for [Leu]enkephalin were processed for BAM-22P immunoreactivity, it was found that these two immunoreactivities were distributed identically at almost all anatomical locations. BAM-22P immunoreactivity was generally less pronounced and ws preferentially localized to neuronal perikarya. The results of the present as well as the preceding studies (Khachaturian et al., '83) strongly suggest substantial structural similarity between the adrenal proenkephalin precursor and that which occurs in the brain. Also discussed are some differences and parallels between the distribution of [Leu]enkephalin and dynorphin immunoreactivities.

Olfactory marker protein (OMP), previously thought to be expressed only by olfactory receptor neurons and their processes, was localized anatomically with immunocytochemical techniques to a number of brain regions in three rodent species, the mouse, rat, and hamster. In addition, the amount of antigen was quantified by radioimmunoassay (RIA) and characterized by an immunoblot procedure. In all three species the antigen could be detected immunocytochemically in the preoptic region and hypothalamus. The rat did not exhibit immunostaining in any other brain region. However, in the mouse neuronal labelling was observed throughout the neural axis, including cellular labelling in the bed nucleus of the anterior commissure, the median preoptic nucleus, the bed nucleus of the stria terminalis, the periventricular region, the anterior parvicellular subnucleus of the paraventricular nucleus, around the dorsomedial hypothalamic nucleus (pars compacta), the subincertal region, the arcuate nucleus, the anterior cortical nucleus of the amygdala, the suprageniculate nucleus, the lateral lemniscal nuclei, the lateraldorsal and lateralventral central gray, the posterior aspects of the commissural and marginal nuclei of the inferior colliculus, the paragenule nucleus, the A-5 region, the area postrema, the ventromedial nucleus of the solitary tract, area X, the spinal trigeminal nucleus (pars zonale), and superficial laminae of the spinal cord. The hamster displayed a different pattern of labelling including cells in the periventricular gray, the pontine reticular tegmental nucleus, the A-5 region, the medial vestibular complex, the prepositus hypoglossal nucleus, the parvicellular reticular nucleus, the lateral paragigantocellular nucleus, the raphe obscuras, the lateral reticular nucleus, and the lateral nucleus of the cerebellum. Immunostaining was seen in fibers within the red nucleus and within mossy fibers of the cerebellum. OMP levels could only be quantified by radioimmunoassay in the olfactory bulb of the three species and in the hamster cerebellum where they were 1/1,000 of those determined in the olfactory bulb. The authenticity of OMP measured in the RIA and detected immunocytochemically was verified by a double-antibody immunoisolation/immunodetection procedure, which confirmed that the antigen being visualized had the molecular properties expected for OMP. In summary, these experiments demonstrate that authentic OMP exists in small groups of neurons in many areas of the central nervous system.

The atrial natriuretic peptide, atriopeptin, is a circulating hormone that plays an important role in the regulation of fluid and electrolyte homeostasis. Several recent studies have shown that atriopeptin-like immunoreactivity is present within the central nervous system as well as peripheral tissues. In the present report, we describe in detail the organization of atriopeptin-like immunoreactive (APir) perikarya and fibers in the central nervous system of the rat. The most prominent collection of APir perikarya was found in the hypothalamus, adjacent to the anteroventral tip of the third ventricle. Additional groups of APir perikarya were observed along the wall of the third ventricle and in the paraventricular and arcuate nuclei. Separate, smaller groups with distinctive morphology were seen in the lateral hypothalamic area, in the supra-mammillary, medial, and lateral mammillary nuclei, medial habenular nucleus, bed nucleus of the stria terminalis, and the central nucleus of the amygdala. In the pons and brain-stem, APir neurons were observed in the pedunculopontine and laterodorsal tegmental nuclei, as well as in the ventral tegmental area, Barrington's nucleus, the parabrachial nucleus, and the nucleus of the solitary tract. The densest terminal fields of APir fibers were found in the paraventricular nucleus of the hypothalamus, the bed nucleus of the stria terminalis, the median eminence, and the interpeduncular nucleus. The presence of atriopeptin immunoreactivity within the central nervous system suggests that atriopeptin may function as a central neuromediator. Potential functions of this candidate neuromediator deduced from its anatomical distribution are discussed, including the possibility that atriopeptin may function as both a central neuromediator and a systemic hormone in the regulation of the cardiovascular system.

The distribution of CGRP immunoreactivity in the cervical, thoracic, lumbar, and sacral levels of the human spinal cord was mapped at the light microscopic level with the aid of a rabbit-generated antiserum against human calcitonin gene-related peptide (CGRP). CGRP-positive fibers formed a dense plexus in lamina I, II, the reticulated region of lamina V, and the tract of Lissauer at all spinal cord levels. The distribution of fibers showed some variations dependent on the cord level analyzed. At the light microscopic level, intervaricose fiber diameters consistently measured 1.0 micron or less, and two types of CGRP varicosities were observed: a small (1 to 2 microns in diameter), relatively round profile and a larger, (3 to 4 microns in diameter) oval or oblong profile. At the electron microscopic level, immunostained varicosities contained a mixture of round clear vesicles and vesicles that contained dense cores. The CGRP immunoreaction product was often associated with vesicles containing dense cores. The reaction product was also seen associated with clear round vesicles or in the cytoplasmic matrix. Postsynaptic elements included dendritic spines, small and large diameter dendritic shafts and vesicle containing profiles. The presence of CGRP in the superficial dorsal horn of human spinal cord is highly suggestive of a role in primary afferent transmission as postulated in lower vertebrates. This study establishes the distribution of CGRP at four different spinal levels in human cord and will serve as a basis for future studies related to the pathologic conditions affecting sensory systems.

Using the indirect antibody peroxidase-antiperoxidase method of Sternberger, we localized substance P (SP), somatostatin (SOM), enkephalin (ENK), and serotonin (5HT, 5-hydroxytryptamine) in the spinal cord of Rana pipiens. This is the first study to demonstrate all four substances in adjacent sections of frog spinal cord. The distribution patterns of ENK, SP, SOM, and 5HT in our study differ from that described for laminae I and II in amniotes. A high density of ENK, SP, and SOM fibers is present in a band ventral to the dorsal terminal field of cutaneous primary afferent fibers and slightly overlapping the ventral terminal field of muscle primary afferent fibers. However, a high density of 5HT fibers is present in the dorsal terminal field.

The distribution of cholecystokinin (CCK)-like immunoreactivity in the brainstem and spinal cord of lampreys was studied by using CCK antisera with different properties. In the spinal cord, three separate systems reacted with CCK antisera: (1) A ventral and lateral fiber system descending from a group of neurons in the posterior reticular nucleus of the rhombencephalon was labeled by both a C-terminal-directed CCK antiserum and a monoclonal CCK antibody. (2) A dorsal root-dorsal column system of fibers originating from cell bodies in the dorsal root ganglia was labeled only by the C-terminal CCK antiserum. This CCK immunoreactivity could be abolished by preabsorption with calcitonin-gene-related peptide (CGRP), suggesting that it was due to cross-reactivity with a CGRP-like peptide. This system also contained 5-hydroxytryptamine (5-HT)-, bombesin-, and CGRP-like immunoreactivities. (3) An intraspinal system of 5-HT neurons was labeled with an antiserum to the midportion of CCK-33 but not by the other CCK antisera. The CCK labeling of this system was difficult to reduce by preabsorption with CCK peptide and thus appeared to be nonspecific. Groups of cell bodies in the middle reticular nucleus of the rhombencephalon, the reticular nucleus of the mesencephalon, and the hypothalamus were labeled by both the C-terminal and the monoclonal CCK antisera. The gut contained two types of CCK-like immunoreactivity, one of which appeared to be due to cross-reactivity with CGRP. A biochemical analysis showed that the content of CCK was low in the spinal cord compared to the brain, and these results agreed with the immunohistochemical findings.

The developmental patterns of neurofilament triplet proteins, peptide and amine immunoreactivities were compared in motor (ventral spinal cord), sensory (dorsal spinal cord, dorsal root ganglia, epidermis), and autonomic (intermediolateral cell columns, dermis) regions in the rat and human. In the rat, neurofilament triplet proteins first appeared in motoneurones (embryonic day 13). In the youngest human fetuses studied (6 weeks), immunoreactivity was present throughout the spinal cord. Peptides and amines occurred later. Calcitonin gene-related peptide, galanin, somatostatin, neuropeptide Y and its C-flanking peptide (CPON) were the first to appear localized to motoneurones (embryonic days 15-17 rat; fetal weeks 6-14 human). Numbers of immunoreactive motoneurones decreased toward birth, but immunoreactive fibers increased in the ventral horn with enkephalin, thyrotrophin-releasing hormone, and the monoaminergic markers 5-hydroxytryptamine and tyrosine hydroxylase (all presumably of supraspinal origin) the last to appear perinatally. In the dorsal horn, particularly in the rat, a transient expression of substance P-, somatostatin-, and neuropeptide Y/CPON-immunoreactive cells was detected (embryonic days 15-17). A pronounced increase of calcitonin gene-related peptide-, galanin-, somatostatin- and substance P- immunoreactive fibers was found perinatally in both species. This coincided with an increased detection of cells in the dorsal root ganglia containing these peptides and the earliest appearance of calcitonin gene-related peptide-, somatostatin-, and substance P-immunoreactive fibers in the rat epidermis. Few antigens were localized to the intermediolateral cell columns before embryonic day 20 (rat), fetal week 20 (human), with thyrotrophin-releasing hormone-, 5-hydroxytryptamine-, tyrosine hydroxylase-, and vasoactive intestinal polypeptide-immunoreactive nerves appearing perinatally. In the rat dermis, tyrosine hydroxylase-immunoreactive fibers (sympathetic fibers) and fibers immunoreactive for neuropeptide Y/CPON and vasoactive intestinal polypeptide were detected from postnatal day 1. In conclusion, 1) peptide and amine immunoreactivity develops in motor before sensory or autonomic regions, 2) many peptide-containing cells are transient in fetal life, and 3) central terminals of dorsal root ganglion cells express peptides before terminals in the skin.

Previous findings have indicated the presence of local circuit neurons in the lateral cervical nucleus (LCN). An immunohistochemical study with gamma-aminobutyric acid (GABA) antiserum was therefore performed both to investigate whether GABA-immunoreactive neurons are present in the LCN, and if so, to compare their characteristics with those previously assigned to probable internuncial neurons in the nucleus. The fine structure and synaptology of GABA-positive boutons in the LCN were also studied. Transversely cut sections from the upper cervical spinal cord of three cats were processed for GABA immunohistochemistry with the free-floating PAP technique. On light microscopic examination immunoreactive neurons were observed within the ventromedial half of the LCN. Their total number was estimated to be 42.5 +/- 11.7 in the entire LCN on one side of the cervical spinal cord, but this may have been an underestimation, as the penetration by the antisera was limited. The labeled neurons were small and had a relatively large nucleus and a low bouton covering ratio. In their number, localization, and ultrastructural appearance the GABA-positive neurons closely resembled the population of neurons previously suggested to be local circuit neurons. Immunoreactive bouton-sized puncta were scattered throughout the LCN. Ultrastructural examination showed labeled terminals with a mean sectional area of 0.85 micron 2 and a relatively high density of synaptic vesicles. The vast majority of GABA-positive terminals were in contact with dendrites and only a minority had synaptic contact with cell bodies. No axoaxonal synapses were observed. The GABA-positive boutons probably derive at least partly from the observed GABA-positive neurons, but there is also a possibility of extrinsic GABAergic input.

Rat dorsal root ganglia and spinal cord were stained with 12 monoclonal antibodies reacting with phosphorylated epitopes of two neurofilament proteins (NF 150K and NF 200K). Three monoclonal antibodies were axon-specific in both locations; neuronal perikarya were not stained. Nine monoclonal antibodies stained a subpopulation of neurofilament-positive sensory neurons, as indicated by double labeling experiments with polyclonal antibodies reacting with phosphorylated and dephosphorylated forms of the neurofilament protein triplet. Of these nine antibodies, two stained motor neuron perikarya in the spinal cord, while the remaining seven antibodies were axon-specific in this location. Subpopulations of stained and unstained motor neurons were not observed. With all 12 antibodies, the staining pattern in the lumbar dorsal root ganglia and spinal cord remained unchanged following sciatic nerve crush and ligature. The findings suggest that, in the neurofilament, some phosphorylated epitopes are axon specific, while other phosphorylated epitopes are present in both axons and perikarya. Furthermore, they suggest that differences exist between neuronal populations as to the presence of phosphorylated epitopes in perikaryal neurofilaments. It remains to be seen whether phosphorylation events in perikarya and axons have similar or different effects on neurofilament structure and function.

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)

The distribution of central neurons displaying somatostatin immunoreactivity was studied using three monoclonal antibodies to cyclic somatostatin. The sensitive ABC immunoperoxidase technique was employed. A large number of positive cell groups including many previously undescribed populations were detected throughout the brain and spinal cord. Telencephalic somatostatin neurons included periglomerular cells in the olfactory bulb, mitral cells in the accessory olfactory bulb, and multipolar cells in the anterior olfactory nuclei, neocortex, amygdala, hippocampus, lateral septum, striatum, and nucleus accumbens. Within the hypothalamus, positive neurons were found in the periventricular, suprachiasmatic, and arcuate nuclei, and throughout the anterior and lateral hypothalamus. The entopeduncular nucleus and zona incerta contained many positive neurons, and the lateral habenula had a dense terminal field suggesting a pallidohabenula somatostatin pathway. Somatostatin neurons were also found in association with many sensory systems. Positive cells were present in the superior and inferior colliculi, the ventral cochlear nuclei, the ventral nucleus of the lateral lemniscus, nucleus cuneatus, nucleus gracilus, and the substantia gelatinosa. Various cerebellar circuits also displayed somatostatin immunoreactivity. Golgi cells throughout the cerebellar cortex were intensely stained, and some Purkinje cells in the paraflocculus also showed a positive reaction. Positive fibers were present in the granular layer and large varicose fibers were present in the inferior cerebellar peduncle. Many nuclei known to project to the cerebellum, including the nucleus reticularis tegmenti pontis, the medial accessory inferior olive, the nucleus prepositus hypoglossi, and many areas of the reticular formation contained positive neurons. These studies demonstrate that these new monoclonal antibodies are of great value for the study of central somatostatin systems. Previously described somatostatin systems are readily detected with these antibodies, and in addition, many otherwise unrecognized somatostatin cell groups have been discovered.

By means of the aluminum-formaldehyde (ALFA) fluorescence technique for monoamine visualization the distribution of catecholamines was studied in the caudal spinal cord, particularly in relation to motoneurons innervating pelvic structures. In the lumbosacral cord all parts of the spinal gray matter were found to contain catecholamines. In the dorsal horn the most intense fluorescence was seen in the superficial layers. The motoneuron neuropil exhibited the most prominent catecholamine-fluorescence of the ventral horn layers. In the sixth lumbar segment, which contains the motor nuclei that innervate the pelvic striated muscles as well as one innervating muscles in the lower limb, a differential distribution of the density of catecholamine fluorescence was presented by the individual nuclei. The catecholamine fibers in the motoneuron neuropil were seen closely surrounding the motoneuron somata, suggesting the existence of axosomatic contacts, and by utilizing the fluorescent retrograde tracer True Blue in combination with the ALFA method tentative axosomatic noradrenergic synapses on identified neurons innervating small striated pelvic muscles could be visualized in the light microscope. In the intermediate gray the intermediolateral nucleus in thoracic and upper lumbar segments was the most heavily innervated area, followed by the medial lumbar sympathetic group, which contains the majority of the sympathetic preganglionic neurons innervating the pelvic organs. The parasympathetic intermediolateral nucleus in the upper sacral segments received a catecholamine innervation of moderate density. The catecholamine innervation pattern is discussed in relation to the patterns of other putative transmitters. The distribution of catecholamine fluorescence in relation to nuclei that control the pelvic organs differs from the arrangement of other transmitters in this region. The complexity of the innervation of the pelvic organs and their related striated muscles is thus further stressed.

The cells in the gracile nucleus that project to the dorsal accessory olive were identified in cats with retrograde tracing techniques. In the same animals, the retrograde labeling patterns in the lateral cervical nucleus and the lumbosacral spinal cord were also examined. Small injections of wheat germ agglutinin complexed to horseradish peroxidase were made in the ventrolateral portion of the dorsal accessory olive without involving the medial accessory olive and without damaging the medial lemniscus. The tissue was processed with tetramethyl benzidine. In each of the three relay nuclei, the neurons that project to the ventrolateral portion of the contralateral dorsal accessory olive are highly concentrated in small regions. In the gracile nucleus these cells are found almost exclusively in the transitional zone, just caudal to the obex and rostral to the clusters. In the lateral cervical nucleus they are concentrated in the dorsolateral tip. In the lumbosacral spinal cord in segments L5 and L6 the cells are found primarily in lamina V, while in segments L7 and S1 they are found along the ventromedial edge of the ventral horn. In the gracile and lateral cervical nuclei there is no segregation of neurons that project to the rostral and caudal portions of the dorsal accessory olive. Comparison of these results with physiological data suggests that each of the three sources of ascending somatic input conveys some distinct aspect of sensory information from the hindlimb to the dorsal accessory olive.

Developmental changes of serotonin (5-hydroxytryptamine) neurons and fibers in the spinal cord of the embryo and posthatching chick were studied with immunohistochemical techniques with the aid of an antibody against serotonin. The first serotonin-immunoreactive fibers were found in the marginal layer of the cervical and lumbar spinal cord on embryonic days 6 and 8, respectively. There was a time lag of a few days between the first appearance of serotonin fibers in the marginal layer (embryonic days 6-8) and the time of penetration of serotonin fibers into the mantle layer (embryonic day 8 or older). The developments of serotonin innervation in the rostral parts of the spinal cord precedes that of caudal regions. Serotonin fibers penetrating into the mantle layer of the lumbar spinal cord were first found in lamina VII on embryonic day 8, whereas there were no serotonin-immunoreactive fibers in lamina IX by embryonic day 10. Large differences were found between embryonic day 16 and posthatching day 5 with regard to the density of serotonin varicosities and fibers in lamina IX, where profiles of soma and large-sized dendrites were heavily covered with varicosities. Laminae I and II first received serotonin fibers on embryonic day 16 and had a much denser innervation by posthatching day 5. There were no traces of serotonin fibers in lamina III in the stages examined up to posthatching day 5. Serotonin fibers were located in the lateral and ventral marginal layers in all specimens examined; only a few fibers were found in the dorsal marginal layer. Although few, serotonin-immunoreactive cell bodies were found in an area around the central canal of all animals from embryonic day 8 to adult. Some of these were located in the ependymal layer and sent processes toward the central canal; there were a small number of cells with long, fine processes. Serotonin-immunoreactive fibers in the spinal cord were not altered in regions rostral to the spinal transection, whereas all the serotoninergic fibers of the supraspinal origin were eliminated in the spinal cord caudal to the gap.

This paper describes the vasopressin (VP) and oxytocin (OXT) immunoreactive structures in the brain and upper spinal cord of the adult male and female Macaca fascicularis. Immunocytochemistry following intraventricular application of colchicine displayed VP neurons in the diagonal band of Broca (DBB), bed nucleus of the stria terminalis (BST), medial amygdaloid nucleus, dorsomedial hypothalamic nucleus, area of the locus coeruleus (LC), solitary tract nuclei (NTS), and the dorsal horn of the cervical spinal cord in addition to those known to exist in the paraventricular, supraoptic, and suprachiasmatic hypothalamic nuclei. Furthermore, a dense accumulation of VP fibers was observed in areas such as the DBB, medial septum, BST, amygdala, hippocampus, ventral tegmental area, periaquaductal gray, dorsal and ventral raphe, area of Forel, LC region, parabrachial nuclei, and NTS. The lateral septum and lateral habenula displayed no and very few VP fibers, respectively. No extrahypothalamic OXT neurons were found in the brain of this macaque monkey. Dense concentrations of OXT fibers were demonstrated in the amygdala, NTS, and marginal layer of the cervical spinal cord. No sexual dimorphism was found in this primate VP or OXT system. The results show a distribution of the central VP and OXT systems in this primate which is quite different from that in the rat. However, in various aspects it agrees with current data on the VP and OXT systems of the human brain. The present results suggest, therefore, that this monkey might serve as a better model for the human VP system than the rat.

Monoclonal antibodies were obtained upon immunization of mice with chicken brain antigen and with the two high molecular weight neurofilament proteins (NF 150K and NF 200K) isolated from bovine spinal cord by anion exchange chromatography. By the immunoblotting procedure, the antibodies selected for this study reacted with bovine NF 150K and NF 200K. By the same procedure the antibodies reacted with sea raven, goldfish, sea bass, shark, and trout spinal cord extracts. In goldfish and sea raven the antibodies stained a single band at approximately 150 kDa and 200 kDa, respectively. Two bands were stained in the shark, sea bass, and trout. In the shark and sea bass these bands were in the molecular weight range of mammalian NF 150K and NF 200K. In the trout the upper band was approximately 150 kDa and the lower band 130 kDa. Our findings suggest an early origin of NF 150K and NF 200K in vertebrate phylogeny as well as considerable divergence in several species.

The distribution of somatostatin in the rat spinal cord was studied immunohistochemically with particular reference to the localization in the caudal centers that innervate the pelvic organs. For detailed studies of the laminar distribution of somatostatin the combination of immunohistochemistry and acetylcholinesterase enzyme histochemistry was employed. Deafferentation experiments were carried out to shed light on the origin of the somatostatin-containing axons. These experiments showed that the bulk of the spinal somatostatin has a spinal origin. The structures showing somatostatin immunoreactivity formed a distinct and detailed pattern. The marginal layer and particularly the substantia gelatinosa contained a dense immunoreactivity in terminallike structures. Such structures were also found in the reticular nucleus, along the medial border of the dorsal horn, and in the nucleus of the dorsolateral funiculus. In all of these regions somatostatin-positive cell bodies were also observed. In the intermediate gray matter stained terminals were present around the central canal in a varying number. The most prominent stainability was found in the lumbosacral transition zone. Many terminals were also observed in the sacral parasympathetic intermediolateral nucleus. In contrast, very few appeared in the sympathetic nuclei. Immunoreactive somata were present in the surroundings of the central canal at all levels. Moreover, positive neurons were found in the intermediolateral nucleus of the sacral cord. By combined retrograde tracing and immunohistochemistry the existence of somatostatin-containing parasympathetic visceromotoneurons was ascertained. Corresponding to this, somatostatin-positive terminals were seen in the major pelvic ganglion. The ventral horn generally contained few terminals, and the density was particularly low in the motoneuron neuropil. However, a dense somatostatin network was found in the sixth lumbar segment in relation to the neurons in Onuf's nucleus X complex, the nucleus that innervates the small pelvic muscles including the striated sphincters. It is concluded that somatostatin, besides being involved in the processing of sensory input, serves an important motor task, that of taking part in the complex control of the pelvic organs and their associated striated muscles.

With the indirect immunofluorescence technique of Coons, the overall distribution of the substance P (SP)-positive neuron system in the frog brain and spinal cord was explored. SP-positive cells were observed in more than ten areas, such as olfactory bulb, amygdaloid complex, septal area, bed nucleus of hippocampal commissure, hypothalamic periventricular zone, dorsal and ventral thalamus, infundibulum, torus semicircularis, optic tectum, the area dorsal to the interpeduncular nucleus, central gray matter of the mesorhombencephalon, and raphe region, etc. A dense network of SP-positive fibers was also widely distributed in the frog brain and spinal cord. SP-positive fibers were roughly divided into two types. One consisted of very fine SP-positive fibers and gave the region a diffuse appearance. The area medial to n. Bellonci, interpeduncular nucleus, n. isthmi, and optic tectum contained this type of SP-positive fibers. The other one consisted of clearly distinguishable varicose fibers. A number of SP-positive fibers located in the amygdaloid complex, striatal complex, hypothalamus, central gray matter of the mesorhombencephalon, trigeminal spinal nucleus, and posterior horn of the spinal cord belonged to this category. The functional role of the SP-positive neuron system in the central nervous system is also briefly discussed.

Immunocytochemical procedures have been used to examine the distributions of substance P (SP)-positive fibres within the intermediate zone of the thoracolumbar spinal cords of rabbits, cats, and monkeys. In all three species SP fibres were concentrated in areas known to contain sympathetic preganglionic neurones. These included the intermediolateral nucleus and the funiculus just lateral to it, the medial gray matter in the area of the nucleus intercalatus, and the paracentral region. The density of the SP innervation varied in a characteristic way both between these subpopulations of sympathetic neurones and in its overall input to different segmental levels. Generally the greatest accumulations of SP fibres were found in the T3-T5 and L2-L4 regions and these were concentrated in the intermediolateral nucleus (ILN). The highest densities of SP fibres in the lateral funiculus were in the upper thoracic and upper lumbar segments whereas SP fibres forming transverse bands, possibly in association with neurones in the nucleus intercalatus, were most prominent in T5-T8. Substance P fibres adjacent to the midline were more or less equally dense throughout the segments examined. Substance P-positive cell bodies situated immediately lateral to the central canal were present at a density of 200-300 per segment throughout the cat thoracolumbar cord. These neurones may be the cells of origin of at least some of the SP fibres in the intermediate zone. The close association of sympathetic preganglionic neurones with SP fibres, many of which are thought to be derived from cells in the medulla, suggests a role for SP-containing fibres in the modulation of sympathetic activity. The variation in input to different segments and classes of sympathetic neurones further suggests a specificity which may be related to the different functions of the neurones innervated.

The present studies in the rat employ spinal transections and hemisections, dorsal and/or ventral rhizotomies to determine whether enkephalin fibers in spinal sympathetic and parasympathetic nuclei are of supraspinal, intraspinal, or peripheral origin. Our results suggest enkephalin fibers in thoracolumbar sympathetic nuclei are of both supraspinal and intraspinal origin, whereas the enkephalin innervation of the sacral parasympathetic nucleus is primarily intraspinal in origin. Furthermore, the majority of descending enkephalin systems selectivity project to the intermediolateralis, pars principalis and pars funicularis, and the dorsal commissural sympathetic nuclei, whereas intraspinal enkephalin pathways appear to exist primarily in the intercalatus spinalis and intercalatus spinalis, pars paraependymalis sympathetic nuclei. These new observations suggest that specific patterns exist for supraspinal and intraspinal enkephalin pathways.

An analysis of vasoactive intestinal polypeptide immunoreactivity (VIP-IR) and substance P-IR in the cat spinal cord has revealed marked differences in the distribution of the two peptides. While substance P-IR was located at all levels of the cord, VIP-IR was most prominent in the sacral segments in Lissauer's tract and lamina I on the lateral edge of the dorsal horn. VIP-IR was also present in the sacral cord in (1) laminae V, VII, and X, (2) a thin band on the medial side of the dorsal horn, (3) the dorsal commissure, (4) the lateral band of the sacral parasympathetic nucleus, and (5) in a few animals in Onuf's nucleus. In other segments of the spinal cord VIP-IR was much less prominent but was present in Lissauer's tract and laminae I, II, and X. Substance P-IR was more uniformly distributed at all segmental levels in laminae I-III, V, VII, and X and in the dorsal commissure. In ventrolateral lamina I of the sacral spinal cord both VIP-IR and substance P-IR exhibited a distinctive periodic pattern in the rostrocaudal axis. The peptides were associated with bundles of dorsoventrally oriented axons and varicosities spaced at approximately 210-micron intervals center to center along the length of the spinal cord. The bundles in lamina I continued into lamina V where they further divided into smaller bundles that extended medially through laminae V and VII. The most prominent bundles of VIP axons passed ventrally from lateral laminae V and VII to enter lamina X and the ventral part of the dorsal gray commissure. On the other hand the majority of substance P axons in lamina V turned dorsally to join with axons on the medial side of the dorsal horn and to pass into the dorsal part of the dorsal gray commissure. Rostrocaudal VIP axons were present not only in Lissauer's tract but also in dorsolateral lamina I, in the lateral funiculus and in the ependymal cell layer of the central canal. Following unilateral transection of the sacral dorsal roots (2 weeks-22 months) the density of VIP axons and terminals was markedly reduced in ipsilateral Lissauer's tract and lateral laminae I and V; however, no change was detected in lamina X. Sacral deafferentation reduced substance P-IR in the dorsal gray commissure and in lateral laminae I and V.(ABSTRACT TRUNCATED AT 400 WORDS)

The distribution of neuropeptide Y [NPY]-immunoreactive material was examined in the spinal cord and dorsal root ganglia of rat, guinea-pig, cat, marmoset, and horse. Considerable concentrations of NPY and similar distribution patterns of immunoreactive nerve fibres were found in the spinal cord of all species investigated. The dorsal root ganglia of the cat and the horse contained numerous immunoreactive nerve fibres, but in these species, as in the other three studied [rat, guinea-pig, marmoset], no positively stained cell bodies were found. Neuropeptide Y-immunoreactive nerves were observed at all levels of the spinal cord, being most concentrated in the dorsal horn. In the rat, guinea-pig, and marmoset, there was a marked increase of NPY-immunoreactive fibres in the lumbosacral regions of the spinal cord, and this was reflected by a considerable increase of extractable NPY. Estimations of NPY-immunoreactive material in the various regions of the rat spinal cord were as follows: cervical, 13.8 +/- 1.0; thoracic, 21.1 +/- 2.5; lumbar, 16.3 +/- 2.9; sacral, 92.4 +/- 8.5 pmol/gm wet weight of tissue +/- SEM. In the ventral portion of the guinea-pig spinal cord they were as follows: cervical, 7.1 +/- 1.2; thoracic, 8.2 +/- 3.6; lumbar, 22.6 +/- 7.0; sacral, 36.7 +/- 9.5 pmol/gm wet weight of tissue +/- SEM. Analysis of spinal cord extracts by reverse phase high performance liquid chromatography [HPLC] demonstrated that NPY-immunoreactive material elutes in the position of pure NPY standard. No changes in the concentration and distribution of the NPY-like material in the rat spinal cord were observed following a variety of surgical and pharmacological manipulations, including cervical rhizotomy, sciatic nerve section and ligation, and local application of capsaicin [50 mM] to one sciatic nerve. It is therefore suggested that most of the NPY-immunoreactive material in the spinal cord is derived either from intrinsic nerve cell bodies or from supraspinal tracts.

The present paper is concerned with the arrangement of axons and synaptic terminals immunostained for calcitonin gene-related peptide (CGRP), a primary afferent marker, in the primate (Macaca fascicularis) dorsal horn. The CGRP axons and terminals are uniformly distributed in laminae I and II outer (o) but they are concentrated laterally and distributed intermittently in the reticulated region of lamina V. A prominent bundle of labeled axons is seen in the sacral cord dorsal to the central canal. Emphasis is given to the relation of CGRP-immunoreactive terminals to other terminals, both labeled and unlabeled, in laminae I and IIo. In this regard, adjacent CGRP-immunoreactive terminals are often united by puncta adhaerentia. Of particular interest is the observation that CGRP-immunoreactive terminals can be found presynaptic to other terminals which sometimes resemble central primary afferent endings. In addition CGRP-immunoreactive terminals end on other CGRP terminals. Both findings suggest that primary afferent terminals interact synaptically with other primary afferent terminals.