Development of Calbindin-D28k Immunoreactive Neurons in the Embryonic Chick Lumbosacral Spinal Cord

The development of immunoreactivity for the calcium-binding protein calbindin-D28k (CaB) was investigated in the embryonic and hatched chick lumbosacral spinal cord. CaB-immunoreactive neurons were revealed in the dorsal and ventral horns as well as in the intermediate grey matter from early stages of neuronal development. CaB immunoreactivity was first detected in large neurons in the presumptive dorsal horn at embyronic day 5, while small neurons in the lateral dorsal horn were the last to appear, at embryonic day 10. We have identified and traced the morphological maturation of six CaB-immunoreactive cell groups, three in the dorsal horn and three in the ventral horn. In the dorsal horn these groups were (1) large neurons in the lateral dorsal horn (laminae I and IV), (2) small neurons in the lateral dorsal horn (lamina II), and (3) small neurons in the medial dorsal horn (lamina III). All three groups were present throughout the entire length of the lumbosacral spinal cord and showed persistent CaB immunoreactivity. In the ventral horn, CaB-immunoreactive neurons were classified into the following three categories: (1) Neurons dorsal to the lateral motor column (lamina VII). These neurons were present exclusively in the upper lumbosacral segments (LS1 – 3), and they showed steady CaB immunoreactivity during their maturation. (2) Neurons at the dorsomedial aspect of the lateral motor column (at the border of laminae VII and IX). This population of neurons was characteristic of the lower segments of the lumbosacral cord (LS5 – 7) and presented transient CaB expression. (3) Neurons within the lateral motor column (lamina IX). These neurons were dispersed throughout the length of the lumbosacral spinal cord. They were three to four times more numerous in the upper than in the lower lumbosacral segments, and their numbers declined throughout LS1 – 7 as the animal matured. The characteristic features of the development of neurons immunoreactive for CaB are discussed and correlated with previous neuroanatomical and physiological studies concerning sensory and motor functions of the developing chick spinal cord.     

Transient expression of GABA immunoreactivity in the developing rat spinal cord.

The development of GABAergic neurons in the spinal cord of the rat has been investigated by immunocytochemical staining of frozen sections with anti-gamma-aminobutyric acid (GABA) antiserum. In the cervical cord, GABA-immunoreactive fibers first appeared at embryonic day (E) 13 in the presumptive white matter within the ventral commissure, ventral funiculus, and dorsal root entrance zone, and in the ventral roots. There were no GABA-immunoreactive cell bodies detected at this age. By E14, motoneurons, the earliest generated spinal cells, were the first cell population to become GABA-immunoreactive at the cell body level. Thereafter, GABA-immunoreactive neurons increased progressively in number and extended from ventral to dorsal regions. GABA-immunoreactive relay neurons within lamina I of the dorsal horn were initially detected at E17. Interneurons in the substantia gelatinosa, the latest generated cells in the spinal cord, were also the last to express the GABA immunoreactivity at E18. Immunoreactive neurons peaked in intensity and extent at E18 and 19. GABA immunoreactivity was only detectable in neurons within the intermediate and marginal zones 1-3 days after they withdrew from the cell cycle. This contrasts to glutamate decarboxylase immunoreactivity, which is detected in precursor cells in the ventricular zone prior to, or during, withdrawal from the cell cycle. Toward the end of gestation, GABA immunoreactivity declined in intensity and extent. This regression began in the ventral horn of the cervical region and ended in the dorsal horn of the lumbosacral region. During the first week after birth, immunoreactivity in motoneurons and in many other neurons within the ventral horn, intermediate gray, and deeper layers of the dorsal horn disappeared, and only in those neurons predominantly within the superficial layers of the dorsal horn did it persist into adulthood. Thus, the expression and regression of GABA immunoreactivity in the spinal cord followed ventral-to-dorsal, rostral-to-caudal, and medial-to-lateral gradients. These observations indicate that the majority of embryonic spinal neurons pass through a stage of transient expression of GABA immunoreactivity. The functional significance of this transient expression is unknown, but it coincides with the period of intense neurite growth of motoneurons, sensory neurons, and interneurons, and of neuromuscular junction formation, suggesting that the transient presence of GABA may play an important role in the differentiation of sensorimotor neuronal circuits.     

Three types of GABA-immunoreactive cells in the lamprey spinal cord

Polyclonal antisera raised against conjugated GABA were used to study the distribution of GABAergic neurons in the spinal cords of lampreys (Lampetra fluviatilis and Ichtyomyzon unicuspis) using immunofluorescence and peroxidase-antiperoxidase techniques. Three morphologically distinct types of GABA-immunoreactive (GABA-ir) cell bodies were observed, multipolar neurons in the lateral grey cell column, apparently bipolar cells in the ventral aspect of the dorsal horn, and small liquor-contacting cells surrounding the central canal. A high density of immunoreactive fibers of spinal origin were present in the lateral and ventral funiculi, whereas the dorsal column had a relatively low density. Dense GABA-ir plexuses were situated in the lateral spinal margin, and in the dorsal part of the dorsal horn. A chronic lesion of the rostral spinal cord did not result in any observable loss of GABA-ir fibers below or above the lesion, suggesting that the 3 types of segmental GABA-ir neurons are the main sources of the GABAergic innervation of the lamprey spinal cord.     

Ontogenic changes of the GABAergic system in the embryonic mouse spinal cord

Numerous studies have demonstrated an excitatory action of GABA early in development, which is likely to play a neurotrophic role. In order to better understand the role of GABA in the mouse spinal cord, we followed the evolution of GABAergic neurons over the course of development. We investigated, in the present study, the ontogeny of GABA immunoreactive (GABA-ir) cell bodies and fibers in the embryonic mouse spinal cord at brachial and lumbar levels. GABA-ir somata were first detected at embryonic day 11.5 (E11.5) exclusively at brachial level in the marginal zone. By E13.5, the number of GABAergic neurons sharply increased throughout the extent of the ventral horn both at brachial and lumbar level. Stained perikarya first appeared in the future dorsal horn at E15.5 and progressively invaded this area while they decreased in number in the presumed ventral gray matter. At E12.5, E13.5 and E15.5, we checked the possibility that ventral GABA-ir cells could belong to the motoneuronal population. Using a GABA/Islet-1/2 double labeling, we did not detect any double-stained neurons indicating that spinal motoneurons do not synthesize GABA during the course of development. GABA-ir fibers also appeared at the E11.5 stage in the presumptive lateral white matter at brachial level. At E12.5 and E13.5, GABA-ir fibers progressively invaded the ventral marginal zone and by E15.5 reached the dorsal marginal zone. At E17.5 and postnatal day 0 (P0), the number of GABA-ir fibers declined in the white matter. Finally, by P0, GABA immunoreactivity that delineated somata was mainly restricted to the dorsal gray matter and declined in intensity and extent. The ventral gray matter exhibited very few GABA-ir cell bodies at this neonatal stage of development. The significance of the migration of somatic GABA immunoreactivity from ventral to the dorsal gray matter is discussed.     

Contribution of brainstem GABAergic circuitry to descending antinociceptive controls: I. GABA-immunoreactive projection neurons in the periaqueductal gray and nucleus raphe magnus

The fact that GABA receptor agonists and antagonists influence nociceptive thresholds when microinjected into the rostroventral medulla or in the spinal cord may reflect the involvement of GABAergic neuronal elements in endogenous antinociceptive pathways. In the present study we used immunocytochemistry and retrograde tract tracing to investigate the contribution of GABAergic projection neurons to the antinociceptive network linking the midbrain periaqueductal gray matter (PAG), the nucleus raphe magnus (NRM), and the spinal cord dorsal horn. The tracer, WGAapoHRP-Au was injected into either the NRM or the spinal cord and the distribution of labeled neurons in sections of the PAG and medulla, respectively, was studied. The same sections were immunostained to demonstrate GABA-immunoreactive neurons. Although GABA-immunoreactive neurons were abundant in the PAG, only 1.5% were retrogradely labeled from the NRM. Similarly, very few GABA-immunoreactive neurons within the cytoarchitectural boundaries of the NRM were retrogradely labeled from the spinal cord. A much higher proportion of GABA-immunoreactive neurons in the region lateral to the NRM, however, were retrogradely labeled from the spinal cord. Eighteen percent of GABA-immunoreactive neurons were retrogradely labeled in the nucleus reticularis paragigantocellularis; conversely, 15% of the retrogradely labeled neurons in this region were GABA-immunoreactive. These results indicate that GABAergic projections constitute a very minor component of the PAG-NRM-spinal cord pathway; however, there is a significant contribution of GABAergic neurons to the spinal projections that originate lateral to the NRM. The majority of GABAergic neurons in the PAG and NRM are presumed to be inhibitory interneurons that directly or indirectly regulate activity in efferent pathways from these regions.     

Coexistence of NADPH diaphorase with GABA,glycine, and acetylcholine in rat spinal cord

The enzyme NADPH diaphorase is present in many spinal neurons, and is thought to correspond to nitric oxide synthase. In order to determine which types of neuron in the spinal cord contain this enzyme, we have carried out a combined enzyme histochemical and immunocytochemical study with antibodies to GABA, glycine, and choline acetyltransferase. Two hundred and twenty-four NADPH diaphorase-positive neurons in midlumbar spinal cord from four rats were tested for GABA- and glycine-like immunoreactivity. The majority of these neurons (207/224) were GABA-immunoreactive and 139 were also glycine-immunoreactive. NADPH diaphorase-positive neurons in laminae I and II generally showed both types of immunoreactivity, while those in deeper laminae of the dorsal horn and around the central canal either showed both types or else were only GABA-immunoreactive. Since GABA and acetylcholine are thought to coexist in spinal neurons, NADPH diaphorase staining was combined with immunostaining for choline acetyltransferase. Immunoreactive neurons in laminae III and IV were all NADPH diaphorase-positive, while only some of those around the central canal and in the deeper laminae of the dorsal horn were positive. Choline acetyltransferase-immunoreactive neurons in the intermediolateral cell column (presumed sympathetic preganglionic neurons) were often NADPH diaphorase-positive, whereas those in the ventral horn (presumed motorneurons) were not. NADPH diaphorase-positive cells in the intermediolateral cell column were not immunoreactive with GABA or glycine antibodies. These results suggest that NADPH diaphorase is largely restricted to GABAergic neurons in the lumbar spinal cord, and that it is mainly present in those neurons in which GABA coexists with glycine or acetylcholine. Since nitric oxide has been implicated in pain processing and hyperalgesia, while GABA, glycine, and acetylcholine are thought to be involved in analgesia and prevention of hyperalgesia, it is likely that nitric oxide synthase-containing GABAergic neurons in dorsal horn have dual actions in transmission of nociceptive information. © 1993 Wiley-Liss, Inc.     

Expression of the glycinergic system during the course of embryonic development in the mouse spinal cord and its co-localization with GABA immunoreactivity

To understand better the role of glycine and gamma-aminobutyric acid (GABA) in the mouse spinal cord during development, we previously described the ontogeny of GABA. Now, we present the ontogeny of glycine-immunoreactive (Gly-ir) somata and fibers, at brachial and lumbar levels, from embryonic day 11.5 (E11.5) to postnatal day 0 (P0). Spinal Gly-ir somata appeared at E12.5 in the ventral horn, with a higher density at the brachial level. They were intermingled with numerous Gly-ir fibers reaching the border of the marginal zone. By E13.5, at the brachial level, the number of Gly-ir perikarya sharply increased throughout the whole ventral horn, whereas the density of fibers declined in the marginal zone. In the dorsal horn, the first Gly-ir somata were then detected. From E13.5 to E16.5, at the brachial level, the density of Gly-ir cells remained stable in the ventral horn, and after E16.5 it decreased to reach a plateau. In the dorsal horn, the density of Gly-ir cells increased, and after E16.5 it remained stable. At the lumbar level, maximum expression was reached at E16.5 in both the ventral and dorsal horn. Finally, the co-localization of glycine and GABA was analyzed, in the ventral motor area, at E13.5, E15.5, and E17.5. The results showed that, regardless of developmental stage studied, one-third of the stained somata co-expressed GABA and glycine. Our data show that the glycinergic system matures 1 day later than the GABAergic system and follows a parallel spatiotemporal evolution, leading to a larger population of glycine cells in the ventral horn.     

Propriospinal neurons in the C1-C2 spinal segments project to the L5-S1 segments of the rat spinal cord

Physiological studies indicate that neurons in the upper cervical spinal cord have descending projections to the lumbosacral spinal cord and mediate inhibition of dorsal horn neurons activated from afferent input. In the present study, retrograde tracing techniques were used to examine the distribution of propriospinal neurons in C1-C2 spinal segments that project to lumbosacral spinal segments. Fluorogold or horseradish peroxidase were injected unilaterally or bilaterally into the L5-S1 spinal segments. After 2–4 days, rats were perfused with fixative and C1-C2 spinal segments were processed for retrograde labeling. Numerous neurons were found in the C1-C2 segments. In unilaterally and bilaterally injected rats, retrogradely labeled neurons were located on both the ipsilateral and contralateral sides. Retrogradely labeled neurons were located in the following locations: lateral cervical and spinal nuclei, nucleus proprius, ventral horn and the central gray region (area X). These studies demonstrate a descending projection from C1-C2 segments to the lower lumbar and sacral spinal cord. We hypothesize that many of these C1-C2 propriospinal neurons are important in modulating responses of spinal neurons at lower segmental levels to various peripheral stimuli.     

Immunocytochemical approach of GABAergic innervation of the mouse spinal cord using antibodies to GABA

The distribution of gamma-amino-butyric acid containing neurons in the Mouse spinal cord has been studied at both the light and electron microscope levels using antibodies against GABA and revelation by the Fab-peroxidase technique. At the light microscope level immunoreactive profiles of perikarya and neuronal processes were particularly abundant in the superficial laminae (I-IV) of the dorsal horn. Scattered soma profiles were found in the other layers and more particularly in the lamina X where Liquor contacting immuno-reactive neurons could be detected. GABAergic cell bodies were very sparse in the ventral horn. Electron microscopic observations confirmed the light microscope results: terminals constituted synaptic symmetrical contacts that provide a morphological basis for inhibition in the dorsal horn and for post-synaptic inhibition of motoneurons in the ventral horn.     

Somatostatin immunoreactive structures in the developing rat spinal cord

Somatostatin immunoreactive (SOM-I) perikarya were first observed in the ventral horn at E12, in the presumptive intermediate gray area at E14, and in the alar plate of the rostral spinal cord at E14. In general, after their initial appearance, their density increased and then decreased during development. A moderate density of SOM-I varicosities became obvious in the superficial laminae of the E20 dorsal horn. By E12 a few SOM-I perikarya, interpreted to be dorsal root ganglia, were observed lateral to the spinal cord, and by E13, SOM immunoreactivity was visualized within the central and peripheral processes of dorsal root ganglion axons. In the marginal zone, SOM-I fibers were first demonstrable in the ventral funiculus at E14, and in the lateral funiculus at E15. After their initial appearance, their density increased and then decreased with age, with the exception of the dorsal part of the lateral funiculus where it increased at the early stages of development to an apparently stable level. The early detection of SOM immunoreactivity in specific spinal regions corresponds well with the birth dates of cells in those regions. This indicates that the SOM-I cells are capable of synthesizing the substance at least as early as they have entered their final cell division.     

Distribution of cholinergic neurons in the chick spinal cord during embryonic development. Comparison of ChAT immunocytochemistry with AChE histochemistry.

The location of cholinergic neurons was studied during the development of the chick embryo spinal cord. A comparison between choline acetyltransferase (ChAT) immunocytochemistry and acetylcholinesterase (AChE) histochemistry was performed. ChAT-positive neurons could be detected only from embryonic day 9 (E9) onwards by the FITC technique and from E12 onwards by the PAP technique. These neurons were located mainly in the medial and lateral motor columns in the ventral horn of the gray matter and some of them were observed in the intermediate region of the spinal cord. AChE-containing cell bodies were much more numerous than the ChAT immunoreactive ones and were distributed in the ventral horn of the gray matter, the intermediate gray region and mostly off the apical part of the dorsal horn. ChAT should provide a reliable and specific marker for cholinergic neurons.     

Axosomatic synapses in the spinal cord of the shark

Previous published reports indicated that the shark central nervous system is atypical of other vertebrates with regard to its lack of internuncial neurons and the rare occurrence of axosomatic synapses. This study was undertaken to examine the general organization of the shark spinal cord and to examine the ventral horn with respect to the synaptic arrangement on motor horn neurons. Spinal cord segments from five juvenile Atlantic nurse sharks (Ginglymostoma cirratum) were removed at the pectoral fin level after mixed aldehyde perfusion and were subsequently processed for light and electron microscopy. Light microscopy of the spinal cord in transverse section revealed an oval cord with relatively discrete dorsal and ventral gray horns connected only by finger-like projections of gray matter. The absence of a definitive intermediate gray zone was the most distinctive structural peculiarity observed. The ventral motor neurons exhibited characteristic neuronal cyto-architecture, and electron microscopy revealed abundant synapses on both ventral horn cell perikarya and primary dendrites. These observations indicate that the shark spinal cord is prototypic of “higher” vertebrate species.     

GABA-immunoreactive neurons in the nematode Ascaris

gamma-Aminobutyric acid (GABA) immunoreactive neurons in the cephalic, somatic, and caudal regions of the Ascaris nervous system were visualized with serial section and whole-mount GABA immunocytochemistry. In the ventral and dorsal nerve cords, GABA-like immunoreactivity (GLIR) is localized to the neurites and cell bodies of identified inhibitory motor neurons and to two fibers, one in each cord, that arise from neurons in the nerve ring. GLIR is absent from identified excitatory motor neurons and from ventral cord interneurons. In neurons containing GLIR, immunoreactivity was present throughout the cell, which argues against an exclusive localization of GABA at conventional synapses. In whole mounts, ten GABA-immunoreactive neurons were present in the cephalic region. These include four nerve ring-associated cells (the RME-like cells), two bilaterally symmetrical pairs of lateral ganglia neurons (the amphid-GABA and deirid-GABA cells) and one bilaterally symmetrical pair of ventral ganglion cells (the VG-GABA cells). In sections, the RME-like cells and the VG-GABA cells were consistently stained through the cephalic region. However, anti-GABA staining of the lateral ganglia cells in sections was light, thus suggesting that they contain less GLIR than the other more intensely stained GABA-immunoreactive neurons. In the caudal region, a single GABA-immunoreactive neuron was present in the dorsal rectal ganglion. Our data suggest that these ten cephalic neurons, and a single dorsal rectal ganglion neuron, use GABA as a neurotransmitter.     

Ontogeny of gamma-aminobutyric acid-immunoreactive neurons in the rhombencephalon and spinal cord of the sea lamprey

The development of neurons expressing gamma-aminobutyric acid (GABA) in the rhombencephalon and spinal cord of the sea lamprey (Petromyzon marinus) was studied for the first time with an anti-GABA antibody. The earliest GABA-immunoreactive (GABAir) neurons appear in late embryos in the basal plate of the isthmus, caudal rhombencephalon, and rostral spinal cord. In prolarvae, the GABAir neurons of the rhombencephalon appear to be distributed in spatially restricted cellular domains that, at the end of the prolarval period, form four longitudinal GABAir bands (alar dorsal, alar ventral, dorsal basal, and ventral basal). In the spinal cord, we observed only three GABAir longitudinal bands (dorsal, intermediate, and ventral). The larval pattern of GABAir neuronal populations was established by the 30-mm stage, and the same populations were observed in premetamorphic and adult lampreys. The ontogeny of GABAergic populations in the lamprey rhombencephalon and spinal cord is, in general, similar to that previously described in mouse and Xenopus.     

Topography of choline acetyltransferase Immunoreactive Neurons and fibers in the rat spinal cord

The topography of choline acetyltransferase immunoreactivity was studied in the rat spinal cord with a monoclonal antibody. Cholinergic fibers were most prominent in lamina III of the dorsal horn and originated from cholinergic neurons within the spinal cord. Lamina X, which was rich in cholinergic neurons and fibers, provided cholinergic interconnections between the dorsal, intermediate and ventral gray. Within the ventral gray, choline acetyltransferase immunoreactive boutons were found on motor neurons. This study suggests that the cholinergic innervation of the spinal cord arises from neurons intrinsic to the spinal cord. The cholinergic neurons within the spinal cord may provide several, overlapping levels of regulation of spinal cord neurons.     

Substance P receptors in the human spinal cord: decrease in amyotrophic lateral sclerosis

The distribution of substance P receptors was examined by autoradiography at all levels of the human postmortem spinal cord using the ligand [125I]Bolton-Hunter substance P. Adjacent sections were used to localize substance P-like immunoreactivity by a radioimmunohistochemical technique. In the control spinal cord substance P-like immunoreactivity was found to be highly concentrated in the superficial layers of the dorsal horn, intermediolateral cell columns and lamina X, while lower levels of immunoreactivity were observed in other areas of the grey matter of the spinal cord. In contrast, high densities of substance P binding sites were localized not only to the substantia gelatinosa of the dorsal horn but also to other regions of the grey matter of the spinal cord, particularly in the area of the preganglionic sympathetic neurons in the intermediolateral cell column and in the region of the somatic motor neurons of the ventral horn. In 5 cases of amyotrophic lateral sclerosis we found a marked reduction of substance P binding, especially in the ventral horn associated with the loss of motor neurons. These results suggest a postsynaptic localization of substance P receptors to the motor neurons of the ventral horn in the human spinal cord and a role for substance P in the function of motor neurons.     

Propriospinal projections to the ventral horn of the rostral and caudal hindlimb enlargement in turtles

In limbed vertebrates, the capacity to generate rhythmic motor patterns for locomotion and scratching is distributed over spinal cord segments of the limb enlargement (e.g., lumbosacral segments), but within this region, rostral segments are more rhythmogenic than caudal segments. The underlying reasons for this rostrocaudal asymmetry are not clear. One possibility is that rostral and caudal segments receive distinct sets of propriospinal projections. To test this hypothesis, I injected horseradish peroxidase (HRP) into the ventral horn unilaterally in a rostral or caudal segment of the turtle hindlimb enlargement. I quantitatively assessed the distributions of retrogradely labeled neurons in six hindlimb enlargement and pre-enlargement segments. The cross-sectional distribution did not depend on which segment was injected. Ipsilateral labeling occurred predominantly in the deep dorsal horn, the lateral part of the intermediate zone, and the dorsal two-thirds of the ventral horn, while contralateral labeling occurred mainly in the medial part of the ventral horn and the lateral part of the intermediate zone. This cross-sectional distribution is similar to what has been seen in mammals. The rostrocaudal distribution of labeled cells, however, depended on which segment was injected. Rostral injections gave rise to rostrally skewed distributions, dominated by descending propriospinal neurons. Caudal injections gave rise to caudally skewed distributions, dominated by ascending propriospinal neurons. Thus, rostral segments of the hindlimb enlargement received more propriospinal inputs from immediately rostral than immediately caudal segments, while the reverse was true for inputs to caudal segments. This anatomical asymmetry may contribute to known functional asymmetries within the enlargement.     

Spinal projections of the A5, A6 (locus coeruleus), and A7 noradrenergic cell groups in rats

The pontine noradrenergic cell groups, A5, A6 (locus coeruleus), and A7, provide the only noradrenergic innervation of the spinal cord, but the individual contribution of each of these populations to the regional innervation of the spinal cord remains controversial. We used an adeno-associated viral (AAV) vector encoding green fluorescent protein under an artificial dopamine beta-hydroxylase (PRSx8) promoter to trace the spinal projections from the A5, A6, and A7 groups. Projections from all three groups travel through the spinal cord in both the lateral and ventral funiculi and in the dorsal surface of the dorsal horn, but A6 axons take predominantly the dorsal and ventral routes, whereas A5 axons take mainly a lateral and A7 axons a ventral route. The A6 group provides the densest innervation at all levels, and includes all parts of the spinal gray matter, but it is particularly dense in the dorsal horn. The A7 group provides the next most dense innervation, again including all parts of the spinal cord, but is it denser in the ventral horn. The A5 group supplies only sparse innervation to the dorsal and ventral horns and to the cervical and lumbosacral levels, but provides the densest innervation to the thoracic intermediolateral cell column, and in particular to the sympathetic preganglionic neurons. Thus, the pontine noradrenergic cell groups project in a roughly topographic and complementary fashion onto the spinal cord. The pattern of spinal projections observed suggests that the locus coeruleus might have the greatest effect on somatosensory transmission, the A7 group on motor function, and the A5 group on sympathetic function.     

The regional distribution of nitric oxide synthase activity in the spinal cord of the dog

The aim of this study was to examine the distribution of calcium-dependent nitric oxide synthase activity (cNOS) in the white and gray matter in cervical, thoracic, lumbar and sacral segments of the spinal cord and cauda equina of the dog. The enzyme's activity, measured by the conversion of [3H]arginine to [3H]citrulline revealed considerable region-dependent differences along the rostrocaudal axis of the spinal cord in general and in cervical (C1, C2, C4, C6 and C8) and lumbar (L1-L3, L4-L7) segments in particular. In the non-compartmentalized spinal cord, the cNOS activity was lowest in the thoracic and highest in the sacral segments. No significant differences were noted in the gray matter regions (dorsal horn, intermediate zone and ventral horn) and the white matter columns (dorsal, lateral and ventral) in the upper cervical segments (C1-C4), except for a significant increase in the ventral horn of C4 segment. In C6 segment, the enzyme's activity displayed significant differences in the intermediate zone, ventral and lateral columns. Surprisingly, extremely high cNOS activity was noted in the dorsal horn and dorsal column of the lowest cervical segment. Comparing the enzyme's activity in upper and lower lumbar segments of the spinal cord, cNOS activity prevailed in L4-L7 segments in the dorsal horn and in all the above mentioned white matter columns.