Immunohistochemical studies on the spinal dorsal horn of the turtle Chrysemys d'orbigny.

Immunohistochemical methods were used to characterize some of the systems of nerve fibers occurring in the spinal dorsal horns of the turtle Chrysemys d'orbigny. Substance P (SP), calcitonin gene-related peptide (CGRP) and leuenkephalin (Enk) immunoreactive fibers were found concentrated in the superficial horn region, termed here synaptic field Ia. From this zone the immunoreactive fibers project to deeper dorsal horn regions. Comparison with histological images obtained after HRP labeling of dorsal root axons indicates that SP-, CGRP- and Enk-immunoreactive fibers are small-diameter primary sensory fibers entering the cord via synaptic field Ia. It is posulated here that these three substances may coexist in the same fibers. Enk-positive fibers also occur randomly scattered in the lateral funiculi, showing a conspicuous increase in density at the perimedullary plexus level. Tyrosine hydroxylase (TH) immunoreactive fibers were found in the more compact dorsal horn neuropil (synaptic field II) and also forming bilateral conspicuous bundles in the lateral funiculi. TH-immunoreactive cell bodies were found in the epithelium lining the central canal. Taking into account data derived from Golgi impregnated material it is proposed that they represent epithelial cells undergoing neural differentiation.     

Neural organization of the lamina neuropil of the larva of the tiger beetle (Cicindela chinensis)

Six neural elements, viz., retinular axons, a giant monopolar axon, straight descending processes (type I), lamina monopolar axons (type II), processes containing clusters of dense-core vesicles (type III), and processes coursing in various directions with varicosities (type IV), have been identified at the ultrastructural level in the lamina neuropil of the larval tiger beetle Cicindela chinensis. Retinular axons make presynaptic contact with all other types of processes. Type I and II processes possess many pre-and postsynaptic loci. Type II processes presumably constitute retinotopic afferent pathways. It remains uncertain whether type I processes are lamina monopolar axons or long retinular axons extending to the medullar neuropil. Type III processes may be efferent neurons or branches of afferent neurons contributing to local circuits. A giant monopolar axon extends many branches throughout the lamina neuropil; these branches are postsynaptic to retinular axons, and may be nonretinotopic and afferent. Type IV processes course obliquely in the neuropil, being postsynaptic to retinular axons, and presynaptic to type I processes.     

Distribution of synapses on two types of ascending interneurons in the crayfish,Procambarus clarkii

Summary About 60 pairs of ascending interneurons are present in the terminal ganglion of the crayfish Procambarus clarkii (Girard). Some of these interneurons have been impaled intracellularly, characterized physiologically, and then labeled with horseradish peroxidase (HRP) to examine the distribution and ultrastructure of synapses. A close relationship between ultrastructure and physiological properties has been found between two types of interneurons, which either have a pre-motor effect upon motor neurons or have no such effect. In one interneuron with a pre-motor effect (6D2), input and output synapses are intermingled on thicker branches, whereas only input synapses are found on small diameter branches. Only input synapses have been observed on the branches in another interneuron with-out a pre-motor effect (6B1). No differences in branch morphology are found in these two interneurons. Interneuron 6D2 contains large numbers of small round agranular vesicles, but the same type of synaptic vesicles is rarely seen in interneuron 6B1, which has no output synapses. Our results indicate a good correlation between the synaptic distribution and pre-motor effects of interneurons in the terminal ganglion.Abbreviations A6, 7 Sixth and seventh abdominal segment of the terminal ganglion - AVC anterior ventral commissure - DC I dorsal commissure I - DIT dorsal intermediate tract - DMT dorsal medial tract - eLG extra lateral giant interneuron - LVT lateral ventral tract - LG lateral giant interneuron - LVT lateral ventral tract - MDT median dorsal tract - MG medial giant interneuron - MoG motor giant neuron - MVT median ventral tract - PVC posterior ventral commissure - R1s sensory fiber tract of nerve root 1 - R3m motor fiber tract of nerve root 3 - R4–7 nerve roots 4–7 - SC I,II sensory commissure I,II - VC I,III ventral commissure I, III - VIT ventral intermediate tract - VLT ventral lateral tract - VMT ventral medial tract     

Golgi study of the anterior dorsal ventricular ridge in a lizard. I. neuronal typology in the adult

Using Golgi techniques we have studied neuronal cell types in the anterior dorsal ventricular ridge (ADVR) of the adult lizard Gallotia galloti. Multipolar, bitufted, and juxtaependymal neuronal forms were found. The multipolar and bitufted neurons are present in both the periventricular and central ADVR zones. Multipolar neurons can be subdivided into multipolar neurons with polygonal somata and four to six main dendritic trunks and multipolar neurons with pyramidal somata and three or more dendritic trunks. The former are the cells most frequently impregnated in the ADVR. In the population of bitufted neurons, we distinguish subtypes I, II, and III according to the number of dendritic trunks that emerge from the somata. Juxtaependymal neurons are restricted to a cell-poor zone, adjacent to ependymal cells. Their dendrites either are orientated parallel to the ventricular surface or extend into the periventricular zone. The dendrites of ADVR neurons have pedunculated spines with knob-like tips. However, such spines do not appear on the somata or on the primary dendritic trunks. The number of spines is scarce or moderate. The periventricular neuronal clusters contain two to five cells. The morphology of these neurons is mainly multipolar, but we also found some bitufted neurons.     

Fos Protein Expression in Sacral Spinal Cord in Relation to Early Phase of Cauda Equina Syndrome in Dogs

1. The aim of the present study is to map the incipient phase of Fos expression in the sacral spinal cord neuronal pools of multiple cauda equina constrictions canine model.2. Fos-positive neurons were found bilaterally in the lateral portion of superficial dorsal horn layers (Laminae I–III) and along the lateral edge of the dorsal horn accompanied by the lateral collateral pathway, fibers of Lissauer's tract, terminating at the sacral parasympathetic nucleus. Similarly, high Fos expression was detected in the ventral portion of the dorsal sacral commissure and in the dorsomedial portion of the anterior horns at S₁–S₃ segment level. Finally, a clearly expressed Fos-positivity was disclosed bilaterally in the neuropil of the nucleus Y in the anterior horn.3. Data from the present study show that continuous stimulation of the central fibers of sacral dorsal root ganglia neurons, i.e., fibers of sacral primary afferents, unlike those using various stimulations of the peripheral fibres offers an unusual pattern of Fos-like immunoreactivity.     

Cauda equina syndrome and nitric oxide synthase immunoreactivity in the spinal cord of the dog

The development of the cauda equina syndrome in the dog and the involvement of spinal nitric oxide synthase immunoreactivity (NOS-IR) and catalytic nitric oxide synthase (cNOS) activity were studied in a pain model caused by multiple cauda equina constrictions. Increased NOS-IR was found two days post-constriction in neurons of the deep dorsal horn and in large, mostly bipolar neurons located in the internal basal nucleus of Cajal seen along the medial border of the dorsal horn. Concomitantly, NOS-IR was detected in small neurons close to the medioventral border of the ventral horn. High NOS-IR appeared in a dense sacral vascular body close to the Lissauer tract in S1-S3 segments. Somatic and fiber-like NOS-IR appeared at five days post-constriction in the Lissauer tract and in the lateral and medial collateral pathways arising from the Lissauer tract. Both pathways were accompanied by a dense punctate NOS immunopositive staining. Simultaneously, the internal basal nucleus of Cajal and neuropil of this nucleus exhibited high NOS-IR. A significant decrease in the number of small NOS immunoreactive somata was noted in laminae I-II of L6-S2 segments at five days post-constriction while, at the same time, the number of NOS immunoreactive neurons located in laminae VIII and IX was significantly increased. Moreover, high immunopositivity in the sacral vascular body persisted along with a highly expressed NOS-IR staining of vessels supplying the dorsal sacral gray commissure and dorsal horn in S1-S3 segments. cNOS activity, based on a radioassay of compartmentalized gray and white matter regions of lower lumbar segments and non-compartmentalized gray and white matter of S1-S3 segments, proved to be highly variable for both post-constriction periods.     

Patterning and organization of motor neuron dendrites in the Drosophila larva

Precise patterns of motor neuron connectivity depend on the proper establishment and positioning of the dendritic arbor. However, how different motor neurons orient their dendrites to selectively establish synaptic connectivity is not well understood. The Drosophila neuromuscular system provides a simple model to investigate the underlying organizational principles by which distinct subclasses of motor neurons orient their dendrites within the central neuropil. Here we used genetic mosaic techniques to characterize the diverse dendritic morphologies of individual motor neurons from five main nerve branches (ISN, ISNb, ISNd, SNa, and SNc) in the Drosophila larva. We found that motor neurons from different nerve branches project their dendrites to largely stereotyped mediolateral domains in the dorsal region of the neuropil providing full coverage of the receptive territory. Furthermore, dendrites from different motor neurons overlap extensively, regardless of subclass, suggesting that repulsive dendrite-dendrite interactions between motor neurons do not influence the mediolateral positioning of dendritic fields. The anatomical data in this study provide important information regarding how different subclasses of motor neurons organize their dendrites and establishes a foundation for the investigation of the mechanisms that control synaptic connectivity in the Drosophila motor circuit.     

Avesicular intercellular contacts in the neuropil of the ganglia of the subpharyngeal complex and in the nerves of the edible snail

Intercellular interconnections in neuropil of dorsal ganglia in the subpharyngeal complex and nerves have been studied in a mature snail (Helix pomatia), dimensions of the shell--3.0 X 3.5 sm, after double fixation (glutaraldehyde and osmium tetraoxide) and treatment with phosphoric tungsten acid. Zones of the neuropil in the left pleural and parietal ganglia have been investigated--where the nerves leave ganglia and in the area of giant cells; neuropil of the central part of both parietal ganglia; central part of the visceral ganglion and nerves--left palial, intestinal and left pleural commissure. The most spread forms of avesicular intercellular contacts in the neuropil are symmetrical. Besides the latter, processes of the neuropil form fissural and desmosomal junctions. Localization of the avesicular contacts supposes presence of dendro-dendritic junctions of desmosomal and fissural types and axo-axonal ones--of desmosomal type. Various types of the contacts differ from each other by their ultrastructural organization.     

Distribution of 5HT-immunoreactivity in the pedal ganglion of Mytilus galloprovincialis

Summary Serotonergic cell bodies and fibers were identified in the pedal ganglia of Mytilus gattoprovincialis with a serum raised against serotonin and the unlabelled peroxidase-antiperoxidase pre- and post-embedding methods. Examination of Vibratome and serial semithin sections showed that most reactive perikarya are located in the ganglionic cortex, being mainly concentrated at the medial aspect of the postero-dorsal portion of the ganglia. Immunoreactive fibers form a dense network in the neuropil, extend throughout the commissure and run parallel in the nerves and connective tracts. The morphology of serotonin-positive cells compared with that of Golgi-impregnated neurons allows the identification of a main population of unipolar, probably projecting neurons and of smaller multipolar cells likely representing local circuit elements. The ultrastructure of labelled neurons is comparable to that of serotonergic cells described in both vertebrate and invertebrate nervous systems.Supported by Ministero Pubblica Istruzione (40%)     

Intrinsic organization of the medial cerebral cortex of the lizard Lacerta pityusensis: A golgi study

The morphology of cells and the organization of axons were studied in Golgi-Colonnier and toluidine blue stained preparations from the medial cerebral cortex of the lizard Lacerta pityusensis. In the medial cortex, six strata were distinguished between the superficial glial membrane and the ependyma. Strata I and II formed the outer plexiform layer, stratum III formed the cellular layer, and strata IV go VI the inner plexiform layer. The outer plexiform layer contained smooth bipolar neurons; their dendrites were oriented anteroposteriorly and their axons were directed towards the posterior zone of the brain. Five neuronal types were observed in the cellular layer. The spinous pyramidal neurons had well-developed apical dendrites and poorly developed basal ones. Their axons entered the inner plexiform layer and gave off collaterals oriented anteroposteriorly. The small, sparsely spinous pyramidal neurons had poorly developed dendrites and their axons entered the inner plexiform layer. The spinous bitufted neurons had well-developed apical and basal dendritic tufts. Their axons gave off collaterals that reached the outer and inner plexiform layers of both the dorsomedial and dorsal cortices. The sparsely spinous horizontal neurons had dendrites restricted to the outer plexiform layer. Their axons entered the inner plexiform layer. The sparsely spinous, multipolar neurons had their soma close to stratum IV and their axons entered the outer plexiform layer. In stratum V of the inner plexiform layer were large, spiny polymorphic neurons; they had dendrites with long spines, and their axons reached the cellular layer. On the basis of these results, we have subdivided the medial cortex into two subregions: the superficial region, which contains the neurons of the cellular layer and their dendritic domains, and the deep region, strata V and VI, which contains the large, spiny polymorphic neurons. The neurons in the medial cortex of these lizards resembles those in the area dentata of mammals. On this basis, the superficial region may be compared to the dentate gyrus and the deep region to the hilar region of the hippocampus of mammals.     

Position and size of the axon hillock in various groups of neurons

The origin of the axon was studied in Golgi-Kopsch impregnated specimens prepared from the spinal cord and brain of adult rats. Five types of neurons were sampled: large ventral horn neurons, neurons in the intermediate zone and ventral horn of the spinal cord, antenna-type neurons in the spinal dorsal horn, neurons in the thalamus, and neurons in the hypothalamus. The axon originated from the perikaryon in 76% of the large ventral horn neurons and in 64% of the neurons in the thalamus. In contrast, the axon emerged from one of the dendrites in 75% of the neurons in the intermediate zone and the ventral horn of the spinal cord and in 68% of the neurons in the hypothalamus. In the case of the antenna-type neurons in the spinal dorsal horn, the axon often originated from one of the dendrites, but never from a dorsally oriented dendrite. The mean distance of the axon hillock of dendritic origin was the longest in the neurons in the intermediate zone and the ventral horn of the spinal cord. The size of the axon hillock was proportional to the size of the perikaryon. The impregnated portion of the axon was longest in the large ventral horn neurons.     

Distribution of dipeptidyl peptidase II (Dpp II) in rat spinal cord

The histochemical localization of dipeptidyl peptidase II (Dpp II; E.C. 3.4.14.2) activity was demonstrated at the light microscope level in the rat spinal cord. Prominent staining was observed in motoneurons of the ventral horn and in medium to large neurons in the deep laminae of the dorsal horn, the intermediate gray, and in lamina X surrounding the spinal canal. Within neurons, Dpp II was localized largely in cell perikarya and large primary dendrites with no staining observed in cell nuclei. Neurons in the superficial dorsal horn lack Dpp II enzyme activity. Nonneuronal elements which also stained prominently were pericytes associated with blood vessels and ependymal cells lining the lumen of the spinal canal. A few oligodendrocytes and astrocytes were also stained, but they represented a minor component of the total amount of Dpp II activity. Following ventral root injury, Dpp-II-containing motoneurons degenerate; some glial cells in the region of degenerating neurons become Dpp II positive. The localized distribution of Dpp II in spinal cord neurons suggests that this proteolytic enzyme may play a role in the metabolism of an unidentified neuropeptide.     

Ultrastructural localization of immunolabeled substance P and methionine-enkephalin-octapeptide in the surface layer of the dorsal horn of rat spinal cord

Summary A preembedding dual immunolabeling technique and electron microscopy were utilized to demonstrate the localization of immunoreactive substance P and methionine-enkephalin-octapeptide (Enk-8) in ultrathin sections of the surface layer (laminae I and II) of rat spinal dorsal horn. The immunoreaction of Enk-8 was visualized as goldtoned silver particles and that of substance P as diaminobenzidine reaction products. Axonal terminals with immunoreactive substance P, and also unlabeled axonal terminals, formed synaptic junctions with the perikarya and dendritic processes of Enk-8-containing neurons. Dendritic profiles immunolabeled for substance P were synaptically linked with unlabeled axons but not with Enk-8-positive ones. Furthermore, it was found that Enk-8 axons and substance P axons terminated synaptically in juxtaposition to one another on the same immunonegative dendrites. Among the Enk-8-containing neurons axonal profiles also appeared to be synaptically associated with immunoreactive Enk-8 dendritic processes.     

Ultrastructure of orexin-1 receptor immunoreactivities in the spinal cord dorsal horn

The ultrastructural properties of orexin 1-receptor-like immunoreactive (OX1R-LI) neurons in the dorsal horn of the rat spinal cord were examined using light and electron microscopy techniques. At the light microscopy level, the most heavily immunostained OX1R-LI neurons were found in the ventral horn of the spinal cord, while some immunostained profiles, including nerve fibers and small neurons, were also found in the dorsal horn. At the electron microscopy level, OX1R-LI perikarya were identified containing numerous dense-cored vesicles which were more heavily immunostained than any other organelles. Similar vesicles were also found within the axon terminals of the OX1R-LI neurons. The perikarya and dendrites of some of the OX1R-LI neurons could be seen receiving synapses from immunonegative axon terminals. These synapses were found mostly asymmetric in shape. Occasionally, some OX1R-LI axon terminals were found making synapses on dendrites that were OX1R-LI in some cases and immunonegative in others. The synapses made by OX1R-LI axon terminals were found both asymmetric and symmetric in appearance. The results provide solid morphological evidence that OX1R is transported in the dense-cored vesicles from the perikarya to axon terminals and that OX1R-LI neurons in the dorsal horn of the spinal cord have complex synaptic relationships both with other OX1R-LI neurons as well as other neuron types.     

Retinopetal neuronal system in the brain of an air-breathing teleost fish,Channa punctata

Summary Retinopetal neurons were visualised in the telencephalon and diencephalon of an air-breathing teleost fish, Channa punctata, following administration of cobaltous lysine to the optic nerve. The labelled perikarya (n=45–50) were always located on the side contralateral to the optic nerve that had received the neuronal tracer. The rostral-most back-filled cell bodies were located in the nucleus olfactoretinalis at the junction between the olfactory bulb and the telencephalon. In the area ventralis telencephali, two groups of telencephaloretinopetal neurons were identified near the ventral margin of the telencephalon. The rostral hypothalamus exhibited retrogradely labelled cells in three discrete areas of the lateral preoptic area, which was bordered medially by the nucleus praeopticus periventricularis and nucleus praeopticus, and laterally by the lateral forebrain bundle. In addition to a dorsal and a ventral group, a third population of neurons was located ventral to the lateral forebrain bundle adjacent to the optic tract. The dorsal group of neurons exhibited extensive collaterals; a few extended laterally towards the lateral forebrain bundle, whereas others ran into the dorsocentral area of the area dorsalis telencephali. A few processes extended via the anterior commissure into the telencephalon ipsilateral to the optic nerve that had been exposed to cobaltous lysine. However, the ventral cell group did not possess collaterals. In the diencephalon, retinopetal cells were visualised in the nucleus opticus dorsolateralis located in the pretectal area; these were the largest retinopetal perikarya of the brain. The caudal-most nucleus that possessed labelled somata was the retinothalamic nucleus; it contained the largest number of retinopetal cells. The limited number of widely distributed neurons in the forebrain, some with extensive collaterals, might participate in functional integration of different brain areas involved in feeding, which in this species is influenced largely by taste, not solely by vision.     

Anatomy and physiology of neurons composing the commissural ring nerve of the cricket, Acheta domesticus

The commissural ring nerve (RN) of the cricket Acheta domesticus links together the two cercal motor nerves of the terminal abdominal ganglion. It contains the axons of almost 100 neurons including two bilateral clusters of eight to 13 ventrolateral neurons and approximately 75 neurons with midline somata within the terminal abdominal ganglion. The ventrolateral neurons have an ipsilateral dendritic arborization within the dorsal neuropil of the ganglion and their axons use the RN as a commissure in order to enter the contralateral nerves of the tenth ganglionic neuromere. In contrast, most midline neurons have bifurcating axons projecting bilaterally into the neuropil of the ganglion as well as into the RN where they often branch extensively before entering the contralateral tenth nerves. Most RN neurons have small, non-spiking somata with spike initiation zones distant from the soma. Many midline neurons also produce double-peaked spikes in their somata, indicative of multiple spike initiation zones. Spontaneous neuronal activity recorded extracellularly from the RN reveals several units, some with variable firing patterns, but none responding to sensory stimuli. The RN is primarily composed of small (50 nm diameter) axon profiles with a few large (0.5-1 microm diameter) profiles. Occasionally, profiles of nerve terminals containing primarily small clear vesicles and a few large dense vesicles are observed. These vesicles can sometimes be clustered about an active zone. We conclude that the primary function of the RN is to serve as a peripheral nerve commissure and that its role as a neurohemal organ is negligible. J. Exp. Zool. 286:350-366, 2000.Copyright 2000 Wiley-Liss, Inc.     

Structure and development of the larval central complex in a holometabolous insect,the beetle Tenebrio molitor

Summary The neuroarchitecture of the central complex, a prominent neuropil in the midbrain of the holometabolan, Tenebrio molitor, is described throughout larval development. The analysis is based on classical silver impregnations and on fate-mapping of identified neurons using antisera against serotonin and FMRF-amide. In T. molitor, the central body is present in the first larval instar, and is formed by side branches of contralaterally projecting neurons. Glial cells surround eight neuropil compartments in the first larval instar. These subdivisions in the organization of the fan-shaped body are maintained throughout development. Intrinsic interneurons are found from the 5th larval instar onwards. In the last larval stage, the central complex consists of the fan-shaped body, the protocerebral bridge, and the anlage of the ellipsoid body. The cellular architecture of the fan-shaped body of the last larval instar resembles the basic structural characteristics of the adult. Serotonin-immunoreactive neurons and FMRF-amide immunoreactive neurons in the midbrain of the first larval instar show the basic structural features of the respective imaginal cells. The structural organizations of larval and adult midbrain are compared.Abbreviations a Anterior - AGT antenno-glomerular tract - aL -lobus - AL antennal lobe - AP anterior protocerebrum - bL -lobe - BSN bilateral symmetrical - FMRF amide-immunopositive neurons - CA calyx - CL1-CL4 serotonin-immunopositive neurons cluster 1–4 - d dorsal - DAB diaminobenzidine tetrahydrochloride - DC dorsal commissure - DCFB dorsal commissure of the fan-shaped body - DHT dorsal horizontal tract - DLTR dorsal lateral triangle - DMLP dorsal medial lateral protocerebrum - DN serotonin-immunopositive deuterocerebral neuron - EB ellipsoid body - en1, en2 extrinsic neurons connecting two FB-subcompartments - esn extrinsic subcompartmental neuron - l lateral - FB fan-shaped body - FN serotonin-immunopositive fan-shaped neuron - fs1, fs2 fanshaped neurons of type 1 and 2 - GC great commissure - HF horizontal fibres - in intrinsic neuron connecting two FB-subcompartments - isn intrinsic subcompartmental neuron - IT isthmus tract - LF large-field neurons - LFASC lateral fascicle - LMFASC lateral median fascicle - MB median bundles - MLP medial lateral protocerebrum - p posterior - P pedunculus - PB protocerebral bridge - pb-fb protocerebral bridge-fan-shaped body connection - PBS phosphate-buffered saline - PDC posterio-dorsal commissure - PTX phosphate-buffered saline containing Triton X-100 - SU suboesophageal ganglion - SVT small ventral triangles - TN 1,2 tritocerebral serotonin-immunoreactive neuron 1,2 - v ventral - VB ventral body - VBC ventral body commissure - VCBC ventral central body commissure - VCFB ventral commissure of the fan-shaped body     

Internuclear connections between the pretectum and the accessory optic system in Salamandra salamandra

Summary Application of horseradish peroxidase into the posterior thalamic and basal optic neuropils of Salamandra salamandra (L.) revealed strong reciprocal connections between the pretectum and the accessory optic system. Pretectal neurons located within the periventricular gray matter project to the basal optic neuropil distributing their terminals over the whole extent of this neuropil. A well developed nucleus of the basal optic neuropil, with its neurons within and medial to this neuropil, projects to the posterior thalamic neuropil. Its terminals appear to be located selectively within the core of the posterior thalamic neuropil which receives no ipsilateral retinal afferents.The pretectum and the accessory optic system are reciprocally connected to a ventral tegmental nucleus, which has not previously been described in urodeles. This nucleus is located immediately dorsal to the oculomotor and trochlear nuclei and extends from the oculomotor root to the middle of the trochlear nucleus.Dendrites of the nucleus of Darkschewitsch reach the posterior thalamic neuropil but mainly enter the rostral tegmental neuropil, while the dendrites of the nucleus of the medial longitudinal fasciculus ramify within the basal optic neuropil and the anterior tegmental neuropil with minor branches in the caudal posterior thalamic neuropil.     

Differential localization of neuronal nitric oxide synthase immunoreactivity and NADPH-diaphorase activity in the cat spinal cord

The distributions of neuronal nitric oxide synthase immunoreactivity (NOS-IR) and NADPH-diaphorase (NADPH-d) activity were compared in the cat spinal cord. NOS-IR in neurons around the central canal, in superficial laminae (I and II) of the dorsal horn, in the dorsal commissure, and in fibers in the superficial dorsal horn was observed at all levels of the spinal cord. In these regions, NOS-IR paralleled NADPH-d activity. The sympathetic autonomic nucleus in the rostral lumbar and thoracic segments exhibited prominent NOS-IR and NADPH-d activity, whereas the parasympathetic nucleus in the sacral segments did not exhibit NOS-IR or NADPH-d activity. Within the region of the sympathetic autonomic nucleus, fewer NOS-IR cells were identified compared with NADPH-d cells. The most prominent NADPH-d activity in the sacral segments occurred in fibers within and extending from Lissauer's tract in laminae I and V along the lateral edge of the dorsal horn to the region of the sacral parasympathetic nucleus. These afferent projections did not exhibit NOS-IR; however, NOS-IR and NADPH-d activity were demonstrated in dorsal root ganglion cells (L7-S2). The results of this study demonstrate that NADPH-d activity is not always a specific histochemical marker for NO-containing neural structures.