5-Hydroxytryptamine, substance P, and thyrotropin-releasing hormone in the adult cat spinal cord segment L7: immunohistochemical and chemical studies.

The terminal projections of the descending 5-hydroxytryptamine (5-HT) bulbospinal pathway and the coexistence among 5-HT-, substance P (SP)-, and thyrotropin-releasing hormone (TRH)-like immunoreactivities (LI) in fibers innervating the L7 segment in the cat spinal cord were studied quantitatively and semiquantitatively by use of the indirect double-staining immunofluorescence technique. The content of 5-HT, SP, and TRH in different parts of the spinal cord was determined by use of radioimmunoassay (RIA) (SP and TRH) and high-performance liquid chromatography with electrochemical detection (HPLC-ECD) (5-HT). For all three substances studied, immunoreactive (IR) axon terminals were found in all parts of the gray matter, but with clear regional variation in the density of innervation. Thus, all three substances showed a dense innervation in the motor nucleus, particularly in the ventral part of the nucleus, while the superficial dorsal horn was very densely innervated by SP-IR fibers (laminae I and II) and TRH-IR fibers (laminae II and III). In the motor nucleus, the studied substances coexisted to a very high degree, but some 5-HT-IR fibers (about 10%) lacked peptide-LI and some SP-IR fibers (about 10%) lacked 5-HT-LI while virtually all TRH-IR fibers also contained 5-HT-LI. In the superficial dorsal horn (laminae I-III), no coexistence was detected, while other parts of the gray matter displayed various degrees of coexistence in between those found in the motor nucleus and laminae I-III. The quantitative analysis of IR varicosities in the motor nucleus suggested that the unilateral L7 motor nucleus is innervated by about 55-110 x 10(6) 5-HT-IR nerve terminals, which may indicate as many as 4,000 boutons per descending 5-HT cell body in the brain stem only with this restricted projection. When combing these results with the biochemical data, it could be calculated that the concentration of 5-HT in IR varicosities is about 3-6 x 10(-3) M, while the corresponding figures for SP and TRH was 0.3-0.5 x 10(-3) M and 0.1-0.2 x 10(-3) M, respectively. In cats subjected to spinal cord transection at the lower thoracic level, all 5-HT-IR fibers in the L7 segment had disappeared 44 days after the lesion, indicating a strict suprasegmental origin of 5-HT-IR fibers in this segment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Origins of serotonergic projections to the lumbar spinal cord in the monkey using a combined retrograde transport and immunocytochemical technique

A newly developed technique employing the retrograde transport of horseradish peroxidase combined with immunocytochemistry is used to identify the cells of origin of descending spinal pathways and their putative neurotransmitters. With this technique the brainstem origins of descending serotonergic (5HT) pathways to the lumbar spinal cord have been determined in the monkey. Numerous 5HT stained neurons are found in the nucleus raphe obscurus and raphe magnus and in the adjacent reticular formation projecting to the lumbar spinal cord. The nucleus raphe pallidus contains relatively fewer descending 5HT neurons. In addition to the spinally projecting neurons containing 5HT, large multipolar shaped neurons within the raphe nuclei were found to project to the spinal cord, but these do not stain for 5HT immunoreactivity. These findings indicate that the raphe nuclear complex provides both serotonergic and non-serotonergic inputs to the spinal cord. The advantages and uses of the present double labeling method for localizing other neurotransmitter substances in identified neuronal pathways are discussed.     

Distribution of dopamine immunoreactivity in the rat,cat, and monkey spinal cord

In the present study, the distribution of dopamine (DA) was identified light microscopically in all segments of the rat, cat, and monkey spinal cord by using immunocytochemistry with antibodies directed against dopamine. Only fibers and (presumed) terminals were found to be immunoreactive for DA. Strongest DA labeling was present in the sympathetic intermediolateral cell column (IML). Strong DA labeling, consisting of many varicose fibers, was found in all laminae of the dorsal horn, including the central canal area (region X), but with the exception of the substantia gelatinosa, which was only sparsely labeled, especially in rat and monkey. In the motoneuronal cell groups DA labeling was also strong and showed a fine granular appearance. The sexually dimorphic cremaster nucleus and Onuf's nucleus (or its homologue) showed a much stronger labeling than the surrounding somatic motoneurons. In the parasympathetic area at sacral levels, labeling was moderate. The remaining areas, like the intermediate zone (laminae VI-VIII), were only sparsely innervated. The dorsal nucleus (column of Clarke) showed the fewest DA fibers, as did the central cervical nucleus, suggesting that cerebellar projecting cells were avoided by the DA projection. In all species, the descending fibers were located mostly in the dorsolateral funiculus, but laminae I and III also contained many rostrocaudally oriented fibers. It is concluded that DA is widely distributed within the spinal cord, with few differences between species, emphasizing that DA plays an important role as one of the monoamines that influences sensory input as well as autonomic and motor output at the spinal level. © 1996 Wiley-Liss, Inc.     

Thyrotropin-releasing hormone (TRH)-like immunoreactivity in the grey monkey (Macaca fascicularis) spinal cord and medulla oblongata with special emphasis on the bulbospinal tract.

The distribution of thyrotropin-releasing hormone (TRH)-like immunoreactivity (LI) has been studied in the grey monkey (Macaca fascicularis) spinal cord and medulla oblongata by the use of indirect immunofluorescence and the peroxidase-antiperoxidase (PAP) technique. Furthermore, double-labeling experiments were performed in order to study colocalization of 5-hydroxytryptamine (5-HT)- and substance P-LI. A dense innervation of TRH-immunoreactive (IR) varicose fibers was found in the ventral horn motor nuclei, in the region surrounding the central canal, in the intermediolateral cell column, and in the dorsal horn laminae II and III. In addition, cell bodies harboring TRH-LI were found in the dorsal horn laminae II-IV. In the ventral horn, many of the large cell bodies and their proximal dendrites were totally encapsulated by TRH-IR fibers. From double-labeled sections a high degree of coexistence could be established between TRH-/5-HT-LI, TRH-/substance P-LI, and 5-HT-/substance P-LI in fibers in the motor nuclei; as a consequence, a large proportion of these fibers should harbor TRH-/5-HT-/substance P-LI. A coexistence between TRH-/5-HT-LI could also be demonstrated in the intermediolateral cell column. However, no unequivocal coexistence could be found between TRH-/substance P-LI and 5-HT-/substance P-LI in this region. In the dorsal horn, no clear coexistence could be encountered for any of the above indicated combinations. Electron microscopic analysis of material from the lumbar lateral motor nucleus demonstrated TRH-IR terminals making synapses with large cell bodies and dendrites. In addition, contacts lacking synaptic specializations could also be verified. In the medulla oblongata, with the use of the PAP technique, a large number of cell bodies containing TRH-LI were encountered in the midline raphe nuclei and in nucleus reticularis lateralis. A similar distribution pattern could be found for 5-HT-LI, but no cell bodies containing substance P-LI could be seen in these regions. Chemical analysis of specimens from cervical, thoracic, and lumbar spinal cord revealed higher concentrations of TRH- and 5-HT-LI in the ventral quadrants, whereas substance P-LI dominated in the dorsal quadrants. Thus, the concentrations of TRH-, 5-HT-, and substance P-LI was in accordance with the observed regional variation in density of IR-fibers and varicosities found in the spinal cord. We have shown that TRH-LI has a distribution in the monkey spinal cord and medulla oblongata similar to that previously demonstrated in other species.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cellular localization and expression of the serotonin transporter in mouse brain

The high-affinity serotonin (5-HT) transporter (5-HTT) plays an important role in the removal of extracellular serotonin, thereby modulating and terminating the action of this neurotransmitter at various pre- and post-synaptic serotonergic receptors and heteroreceptors. In order to characterize the anatomical distribution of the 5-HTT in mouse brain, in situ hybridization histochemistry using ³⁵S-labeled riboprobes was performed. These results were compared with 5-HTT binding site distribution as evaluated by [¹²⁵I]RTI-55 autoradiography. High levels of 5-HTT mRNA were detected in all brain stem raphe nuclei, with variations in labeling among the various subnuclei. Those brain areas known to possess serotonergic cell bodies stained intensely for both 5-HTT mRNA and 5-HTT binding sites. In contrast to previous findings in rat brain, the highest densities of 5-HTT sites were found in areas outside the raphe complex, particularly in the substantia nigra, globus pallidus, and superior colliculi.     

Differential effects ofp-chlorophenylalanine on indoleamines in brainstem nuclei and spinal cord of rats. II. Identification of immunohistochemically stained structures using computer-assisted image enhancement techniques

Following intraperitoneal injection ofp-chlorophenylalanine (PCPA, 400 and 600 mg/kg) on 3 consecutive days, the brainstem and lumbar cord of rats were removed, frozen-sectioned and immunohistochemically stained (PAP method) for serotonin (5-HT). Using computer-assisted image analysis, the density of 5-HT staining in control, 400 and 600 mg/kg PCPA groups was determined. The mean number of pixels (representing 5-HT staining) was determined in 6 areas in the brainstem containing 5-HT cell bodies (nuclei raphe pallidus, raphe obscurus, rostral and caudal raphe magnus, raphe dorsalis and paragigantocellularis lateralis) and in the dorsal and ventral spinal cord. The resultssuggest a differential depletion of 5-HT within brainstem nuclei following PCPA treatment in that the most marked dose-related reductions were observed in nucleus raphe obscurus and caudal magnus. Furthermore, a computer program designed to isolate terminal structures in the spinal cord identified a differential depletion of 5-HT terminals in the dorsal horn versus the ventral horn. The present study describes 3 analytical approaches combining immunohistochemistry with the computer-assisted image analysis technique and allows comparison between groups of animals which received the same or different drug treatments.     

On the Distribution of GAP-43 and its Relation to Serotonin in Adult Monkey and Cat Spinal Cord and Lower Brainstem

By use of a monoclonal antibody, the distribution of growth-associated protein (GAP)-43-like immunoreactivity (LI) has been studied in the spinal cord of adult grey monkeys ( Macaca fascicularis ) and adult cats by use of immunofluorescence and peroxidase-antiperoxidase techniques. The brainstem was also studied with in situ hybridization histochemistry. In both monkeys and cats, a dense innervation of GAP-43-immunoreactive (IR) fibres was seen in close apposition to large cell bodies and their processes in the motor nucleus of the ventral horn. Double-labelling experiments revealed a high degree of coexistence between GAP-43- and 5-hydroxytryptamine (5-HT, serotonin)-LI in the monkey motor nucleus, while in the cat no such colocalization could be verified. At the electron microscopic level, GAP-43 labelling was seen as a coating of vesicles and axolemma inside the terminals. In both monkey and cat, cell bodies expressing mRNA encoding GAP-43 were demonstrated in the medullary midline raphe nuclei. A similar location was also encountered for mRNA for aromatic l -amino acid decarboxylase, an enzyme found in both catecholamine- and serotonin-containing neurons. The present results suggest that GAP-43 is present in the 5-HT bulbospinal pathway of the monkey. In the cat, GAP-43 mRNA-expressing cell bodies were demonstrated in areas where descending 5-HT neurons are located, but no convincing colocalization of 5-HT- and GAP-43-LI was found at spinal cord levels, despite the existence of extensive fibre networks containing either of the two compounds. Possible explanations for this species discrepancy are discussed. The function of GAP-43 in nerve terminals impinging on the motoneurons is unknown. However, it may play a role in transmitter release and/or plasticity, since such roles have been proposed for this protein in other systems.     

Substance P innervation of the rat and cat thalamus. II. Cells of origin in the spinal cord.

Evidence in the preceding paper suggests that fibers and terminals immunopositive for substance P (SP) in somatosensory thalamic nuclei are part of the spinothalamic tract (STT). In this paper, more direct evidence on this point is provided by immunocytochemistry for SP on the cervical spinal cord, alone or combined with the retrograde transport of colloidal gold-labeled wheat germ agglutinin conjugated to enzymatically inactive horseradish peroxidase (WGAapoHRP-Au). In cats and rats pretreated with colchicine and/or anterolateral chordotomy (to increase SP content in cell bodies), many small to large cell bodies are SP-immunopositive especially in laminae I and V, but also in more ventral laminae of the upper cervical cord. SP neurons are also present in the dorsolateral funiculus (in the lateral spinal nucleus, LSN, in rats) but not in the lateral cervical nucleus or in the internal basilar nucleus. In both species there is a considerable degree of overlap in the distribution of SP-positive neurons and that of STT neurons. SP immunocytochemistry in rats after WGAapoHRP-Au injection in the somatosensory thalamus reveals SP-positive STT neurons in LSN, in lamina I and in lamina V, and, to a lesser extent, in more ventral laminae. These results demonstrate that SP is a marker and/or neuromediator for some STT neurons. Together with the evidence discussed in the preceding paper, the results also suggest that SP-positive neurons may be involved in the transmission of nociceptive input.     

Nerve growth factor receptor-like immunoreactivity in nerve fibers in the spinal and medullary dorsal horn of the adult monkey and cat: correlation with calcitonin gene-related peptide-like immunoreactivity

The distribution of nerve growth factor receptor (NGFr)-like immunoreactivity and of calcitonin gene-related peptide (CGRP)-like immunoreactivity was studied in nerve fibers of the spinal and medullary dorsal horn of the monkey and the cat, using double-labelling immunohistochemistry with polyclonal CGRP antiserum and a monoclonal antibody against human NGFr. In both species there was an extensive distribution overlap between NGFr-LI and CGRP-LI. However, lack of NGFr-LI in many CGRP-immunoreactive axons suggests that not all CGRP-positive primary afferent fibers possess NGFr.     

Immunohistochemical detection of calcium/calmodulin-dependent protein kinase II in the spinal cord of the rat and monkey with special reference to the corticospinal tract

Calcium/ calmodulin-dependent protein kinase II is a prominent enzyme in the mammalian brain that phosphorylates a variety of substrate proteins. In the present study, monoclonal antibodies that specifically recognize either the α or the β isoforms of this enzyme were used to determine the distribution of these isoforms within the rat and monkey spinal cord. In the rat, the corticospinal tract consists of two components: the dorsal corticospinal tract, which occupies the ventralmost aspect of the dorsal funiculus; and the ventral corticospinal tract, which occupies an area adjacent to the ventral median fissure. Both dorsal and ventral corticospinal tract fibers were strongly immunopositive for the α-antibody. Unilateral ablation of the sensorimotor cortex of the rat eliminated the α-immunoreactive staining in the contralateral dorsal corticospinal tract. The neuropil in the superficial laminae of the dorsal horn (Rexed's laminae I and II) was densely stained with the α-antibody, whereas the neuropil in laminae IV-X was immunonegative. Dense α-immunopositive neurons were also distributed in the head of the dorsal horn (laminae I-IV). In contrast to the strong α-immunoreactivity seen in the dorsal corticospinal tract fibers, only very weak β-immunoreactivity was observed in this tract. Moderate β-immunoreactive products were distributed homogenously throughout the neuropil of the gray matter, although the neuropil of the superficial laminae of the dorsal horn (laminae I and II) was stained more strongly than the other regions of the gray matter (laminae III-X). Neuronal components in all laminae were immunopositive for the β-antibody. Thus, motoneurons in the ventral horn, which were immunonegative for the α-antibody, were immunopositive for the β-antibody. This selective distribution pattern of immunoreactivity of α- and β-antibodies in the rat was also present in the monkey spinal cord, although the α-immunopositive corticospinal tract fibers in the monkey descended in the lateral funiculus as the lateral corticospinal tract instead of passing through the dorsal funiculus, as is the case in the rat. The differential distribution of immunoreactivity in the spinal cord suggests that these two isoforms of calcium/ calmodulin-dependent protein kinase II may have different functional roles in the spinal cord. © Wiley-Liss, Inc.     

Microglial activation, emergence of ED1-expressing cells and clusterin upregulation in the aging rat CNS, with special reference to the spinal cord

With advancing age, the incidence of neuronal atrophy and dystrophy increases and, in parallel, behavioural sensorimotor impairment becomes overt. Activated microglia has been implicated in cytotoxic and inflammatory processes in neurodegenerative diseases as well as during aging. Here we have used immunohistochemistry and in situ hybridization to examine the expression of OX42, ED1, ED2, GFAP and clusterin in CNS of young adult and behaviourally tested aged rats (30-month-old), to study the occurrence of activated microglia/ED1 positive macrophages in senescence and to what extent this correlates with astrogliosis and signs of sensorimotor impairment among the individuals. The results show a massive region-specific increase in activated microglia and ED1 expressing cell profiles in aged rats. The infiltration was most prominent in the spinal cord dorsal columns, including their sensory relay nuclei, and the outer portions of the lateral and ventral columns. At such sites the occurrence of macrophages coincided with increased levels of GFAP and positive correlations were evident between the labeling for, on the one hand, OX42 and, on the other, GFAP and ED1. Also, the ventral and dorsal roots were heavily infiltrated by ED1 positive cells. The signs of gliosis were most pronounced among aged rats with advanced sensorimotor impairment. In contrast, the grey matter of aged rats showed very few activated microglia/ED1 labeled cells despite signs of focal astrogliosis. ED2 expression was confined to perivascular cells and leptominges with a similar labeling pattern in young and aged rats. In aged rats increased expression of clusterin was observed in GFAP-immunoreactive profiles of the white matter only. It is suggested that this increase may reflect a response to degenerative/inflammatory processes.     

The structure of the brainstem and cervical spinal cord in lungless salamanders (family plethodontidae) and its relation to feeding

We present an HRP study of the sensory tracts and motor nuclei associated with feeding (especially use of the tongue) in plethodontid salamanders (mainly Batrachoseps attenuatus, Bolitoglossa subpalmata, Desmognathus ochrophaeus, Eurycea bislineata, and Plethodon jordani). The nerves studied are VII (ramus hyomandibularis only), IX, X, XI, the first spinal nerve (hypoglossus), and the second spinal nerve. Two types of sensory projections are universally found in the brainstem: superficial somatosensory projections of VII, IX, and X, and deeper visceral sensory projections of IX and X to the fasciculus soltarius. The first spinal nerve and the spinal accessory nerve (XI) have no sensory projections, but the second spinal nerve has typical projections along the dorsal funiculus of the spinal cord. The motor nuclei of VII ramus hyomandibularis, IX, and X form a combined nucleus situated at the level of the IX/X root complex. The nucleus of the first spinal nerve is well separated from the combined nucleus and is situated rostral and caudal to the obex. The rostral part of the motor nucleus of the second spinal modestly overlaps that of the first. The motor nucleus of the spinal accessory nerve is more or less restricted to the region of the second spinal nerve. Its fibers leave the brain through the last root of the IX/X complex and the related ganglion. Bolitoglossine and nonbolitoglossine differ in the architecture of the spinal nuclei. Two distinct types of motor neurons occur in spinal nuclei of nonbolitoglossine species--some of those with tongue projection--but only one type is found among the tongue-projecting bolitoglossine group.     

Distribution of enkephalin-immunoreactive cell bodies in relation to serotonin-containing neurons in the raphe nuclei of the cat: Immunohistochemical evidence for the coexistence of enkephalins and serotonin in certain cells

In this study we have examined the distribution of enkephalin-like immunoreactive (ELI) cell bodies in the rat raphe nuclei pallidus (NRP), obscurus (NRO), magnus (NRM), pontis and dorsalis (NRD) after intratissular or intraventricular administration of colchicine. All the raphe nuclei examined were observed to contain ELI cell bodies along their whole caudorostral extent. By comparing consecutive sections treated separately with anti-5-HT and enkephalin-antiserum it was observed that certain 5-HT cells in each raphe nucleus contain ELI material. A quantitative estimation was attempted. In NRP and NRo approximately half of the total immunoreactive neuronal population appeared to be immunoreactive for both 5-HT and the enkephalins. In NRM the proportion would be one-third, whereas it seemed almost negligible in NRD. Among the 5-HT cells, approximately two-thirds might be ELI in NRP and NRO, and one-half in NRM.     

Parabrachial nucleus neurons projecting to the lower brain stem and the spinal cord. A study in the cat by the Fink-Heimer and the horseradish peroxidase methods

Direct projections from the parabrachial nucleus (PBN) to the lower brain stem and the spinal cord were examined in the cat by the Fink-Heimer and the horseradish peroxidase (HRP) methods. After placing lesions in the PBN, many fine degenerated fibers were seen contralaterally in the ventromedial portions of the caudal pontine reticular formation, and ipsilaterally in the lateral portions of the facial nucleus, the regions around the hypoglossal nucleus, and the regions around the ambiguus nucleus; some degenerated fibers were traced ipsilaterally down to the spinal cord. Subsequently, HRP injections were attempted into these regions where many fine degenerated fibers were observed. In cats injected with HRP into the lateral portions of the facial nucleus, the regions around the hypoglossal nucleus, the regions around the ambiguus nucleus, or the first cervical cord segment, many HRP-labeled neurons were seen in the ventral portions of the PBN. The mean of the average soma diameters of the PBN neurons labeled with HRP injected into the regions around the hypoglossal nucleus or the first cervical cord segment was significantly larger than that of the PBN neurons labeled with HRP injected into the lateral portions of the facial nucleus or the regions around the ambiguus nucleus.     

Inhibition of morphine tolerance and dependence by diazepam and its relation to μ-opioid receptors in the rat brain and spinal cord

We have recently observed that concomitant administration of diazepam to morphine pellet implanted rats results in the inhibition of the development of morphine tolerance and dependence. We have now analyzed μ-opioid receptors in rats treated with morphine and diazepam for 5 days by using -DAMGO for binding studies. Male Sprague–Dawley rats were made tolerant and dependent by subcutaneous (s.c.) implantation of six morphine pellets (two pellets on the first day, and four on the second day). Diazepam (0.25 mg/kg b.wt) was injected once daily intraperitoneally (i.p.) for 5 days. Control rats were implanted with placebo pellets and injected once daily with saline or diazepam (i.p.). Animals were administered s.c. naloxone (10 mg/kg) to induce naloxone-precipitated withdrawal syndrome on the final day of the experiment (day 5). There was an up-regulation of μ-receptor (B_max increased) in the spinal cord of morphine tolerant (+139%) and dependent (+155%) rats compared to saline treated animals. Diazepam treatment abolished the up-regulation of μ-receptors in spinal cord of morphine treated rats. In the cortex, B_max was not affected in morphine tolerant or dependent rats but it decreased by 38% in morphine tolerant and 65% in morphine dependent rats treated with diazepam. The K_d of μ-receptors increased in the cortex, striatum and hypothalamus of morphine dependent rats. Diazepam treatment decreased the K_d of μ-receptors in the cortex of morphine tolerant and hypothalamus of morphine-dependent rats. These results suggest that diazepam treatment antagonizes the up-regulation of CNS μ-receptors observed in morphine tolerant rats. In addition, morphine tolerance and dependence may be associated with conversion of μ-opioid receptors to μ*-constitutive opioid receptors that are less active, and this conversion is prevented in the brain of animals treated with diazepam.     

Projections from the brain stem reticular formation to laminae I and II of the spinal cord. Studies using light and electron microscopic techniques in the North American opposum

The horseradish peroxidase and autoradiographic methods show that laminae I and outer II are innervated by the nucleus reticularis gigantocellularis pars ventralis, and the nucleus reticularis pontis pars ventralis. Both areas contain neurons of the indolamine type and probably account for the indolamine-like varicosities which are present within laminae I and II. Degeneration material prepared for electron microscopy showed that axons from the above nuclei end on small dendritic shafts and spines as well as on vesicle-filled profiles. The terminals identified formed asymmetrical contacts and contained clear as well as dense-cored vesicles. No terminals were present within glomuruli. A projection to laminae I and outer II also arises within the dorsolateral pons and several lines of evidence suggest that it is catecholaminergic. The electron microscope revealed that axons from the dorsolateral pons are fairly numerous within laminae I and II, but that terminal contacts are relatively rare. Those present are asymmetrical and alternate with intermediate-sized dendrites. They contain clumps of clear, spherical vesicles as well as larger vesicles with a variety of dense cores.     

Distribution of 125I-galanin binding sites, immunoreactive galanin, and its coexistence with 5-hydroxytryptamine in the cat spinal cord: biochemical, histochemical, and experimental studies at the light and electron microscopic level.

The distribution of galanin-like immunoreactivity (GAL-LI) in the spinal cord of the cat was studied by use of indirect histochemistry and the peroxidase-antiperoxidase (PAP) technique. In the ventral horn GAL-immunoreactive (IR) axonal fibers and terminals were most frequent in the ventral part of the motor nucleus. The GAL-IR axons also contained 5-hydroxytryptamine (5-HT)-LI, and they disappeared after spinal cord transection. It was concluded that these GAL-IR fibers belong to the serotoninergic bublospinal pathway. In the medulla oblongata from normal cats, scattered GAL-IR cell bodies were encountered within the nucleus raphe obscurus and nucleus raphe pallidus. Electron microscopic observations revealed that the fine structure of the GAL-IR axonal boutons in the motor nucleus was similar to that of 5-HT-IR boutons with a varying number of immunoreactive large dense core vesicles. The postsynaptic element in all cases studied was a dendrite. A dense GAL-IR axonal plexus was found in the superficial laminae I-II of the dorsal horn. Coexistence was found between the GAL- and substance P-LI in fibers within the dorsal horn plexus. Spinal cord transection did not alter the pattern of GAL-LI in the dorsal horn, while the vast majority of GAL-IR axonal swellings disappeared following dorsal root sectioning. Electron microscopic observations in lamina II (substantia gelatinosa) revealed that the GAL-IR axonal terminals could be divided into two main groups. One with small to medium-sized axonal boutons formed synaptic contacts with both dendritic and axonal profiles. The other formed the central axon terminals of glomeruli, suggesting that GAL-LI may be present in C-type primary afferents. Numerous small GAL-IR cell bodies were encountered in laminae II and III. GAL-IR cell bodies were also observed in lamina X. The dorsal root ganglia contained a low but consistent number of small to medium-sized GAL-IR cell bodies, which all contained immunoreactive calcitonin gene-related peptide (CGRP). Following peripheral sciatic nerve transection, the number and the labeling intensity of GAL-IR cell bodies in the corresponding dorsal root ganglia showed a moderate increase. Radioimmunoassay revealed that the concentration of GAL-LI increased along the rostrocaudal axis of the normal spinal cord, and was about three times higher in the dorsal than in the ventral regions. The concentration in the dorsal root ganglia was intermediate to those seen in the corresponding dorsal and ventral cord regions.(ABSTRACT TRUNCATED AT 400 WORDS)