大鼠脊髓背角浅层神经元中神经肽Y Y1受体与生长抑素共存

用间接免疫荧光双标记方法,研究大鼠脊髓Ⅱ层神经元中神经肽Ｙ Ｙ１受体与生长抑素共存关系。结果显示在Ⅱ层深部许多神经元含Ｙ１受体－免疫反应性物质,只观察到少量的生长抑素免疫反应阳性神经元胞体,其中个别Ｙ１受体阳性神经元也含生长抑素免疫反应性物质;在局部使用秋水仙素后,大多数Ｙ１受体免疫反应阳性神经元含有生长抑素免疫反应性物质。推论脊髓背角Ⅱ层神经元中的Ｙ１受体可能与生长抑素在脊髓水平的功能有关。     

Cell type-specific postsynaptic effects of neuropeptide Y in substantia gelatinosa neurons of the rat spinal cord

Cellular mechanisms of antinociceptive action of neuropeptide Y were investigated in substantia gelatinosa (SG) neurons in rat spinal cord slices. Somatic and synaptic effects of NPY were compared in two subpopulations of cells with different firing patterns, tonic (TFNs), and delayed firing (DFNs) neurons. For the study, TFNs were selected on morphological basis: they had appearance of central and radial but not islet cells, and are likely excitatory interneurons in dorsal horn networks. In their turn, DFNs were classified as radial and vertical cells. 0.3 μM NPY via Y1 receptors activated hyperpolarizing postsynaptic current of GIRK type in majority of TFNs (～77%) but not DFNs (～8%). Miniature synaptic currents in all neurons were seen as a mixture of excitatory (mEPSCs) and inhibitory (mIPSCs), the frequency of the former being ～5 times greater. The mEPSCs were mediated by glutamate receptors of AMPA subtype, while the dominant part of mIPSCs--by glycine receptors. In all cell types, NPY moderately depressed the frequency of both mEPSCs and mIPSCs; the effects occurred via Y2 and Y1 receptors, respectively. The data suggest that behavioral NPY-evoked antinociception is achieved via postsynaptic hyperpolarization of majority of TFNs (assumingly, excitatory interneurons) via Y1 receptors and depression of the mEPSCs via Y2 receptors.     

Neuropeptide Y: regional distribution chromatographic characterization and immunohistochemical demonstration in post-mortem human brain

Using a neuropeptide Y (NPY)-directed radioimmunoassay the post-mortem stability of NPY was assessed in both rat and human brain samples. The regional distribution of NPY-like immunoreactivity in human brain was determined. The NPY-like immunoreactivity in human brain separated on Sephadex G-50 columns and ¹⁸C reverse phase at the position expected for NPY. Immunohistochemical staining using the NPY-directed antiserum revealed a characteristic population of cortical and striatal neurons containing NPY-like immunoreactivity.     

A human spinal cord cell promotes motoneuron survival and maturation in vitro

Primary cultures of motoneurons represent a good experimental model for studying mechanisms underlying certain spinal cord pathologies, such as amyotrophic lateral sclerosis and spinal bulbar muscular atrophy (Kennedy's disease). However, a major problem with such culture systems is the relatively short cell survival times, which limits the extent of motoneuronal maturation. In spite of supplementing culture media with various growth factors, it remains difficult to maintain motoneurons viable longer than 10 days in vitro. This study employs a new approach, in which rat motoneurons are plated on a layer of cultured cells derived from newborn human spinal cord. For all culture periods, more motoneurons remain viable in such cocultures compared with control monocultures. Moreover, although no motoneurons survive in control cultures after 22 days, viable motoneurons were observed in cocultures even after 7 weeks. Although no significant difference in neurite length was observed between 8-day mono- and cocultures, after 22 and 50 days in coculture motoneurons had a very mature morphology. They extended extremely robust, very long neurites, which formed impressive branched networks. Data obtained using a system in which the spinal cord cultures were separated from motoneurons by a porous polycarbonate filter suggest that soluble factors released from the supporting cells are in part responsible for the beneficial effects on motoneurons. Several approaches, including immunocytochemistry, immunoblotting, and electron microscopy, indicated that these supporting cells, capable of extending motoneuron survival and enhancing neurite growth, had an undifferentiated or poorly differentiated, possibly mesenchymal phenotype.     

Characterization and localization of immunoreactive dynorphin, alpha-neo-endorphin, Met-enkephalin and substance P in human spinal cord

By use of specific antisera, the distributions of immunoreactive dynorphin (ir-DYN), alpha-neo-endorphin (ir-alpha-NEO), Met-enkephalin (ir-MET) and substance P (ir-SP) were evaluated in discrete regions of human spinal cord and spinal ganglia. The relative concentrations of immunoreactive peptides in particular regions were as follows: sacral greater than lumbar greater than cervical greater than thoracic. Concentrations of ir-DYN, ir-alpha-NEO and ir-SP were 2-10-fold, but of ir-MET 1-2-fold, higher in the dorsal as compared to the ventral parts of cervical, lumbar and sacral cord. The concentrations of all peptides (when examined in discrete areas of thoracic cord) were found to be highest in the substantia gelatinosa. All peptides were present in the gray matter but only ir-MET was found in white matter. Gel-permeation chromatography of dorsal sacral spinal cord extracts revealed two major ir-DYN peaks. The smaller molecular weight peak, eluted at the position of synthetic dynorphin1-17. ir-alpha-NEO and ir-SP comigrated exactly with their respective synthetic marker peptides. Substantial amounts of ir-SP and also, as confirmed by high pressure liquid chromatography, ir-MET, were found in the dorsal and ventral roots and spinal ganglia, and very low concentrations of ir-DYN or ir-alpha-NEO were also detected in these tissue. These results suggest that dynorphin and alpha-neo-endorphin, in addition to enkephalins, may be involved in transmission of somatosensory information in the human spinal cord.     

Neuropeptide Y activates urocortin 1 neurons in the nonpreganglionic Edinger-Westphal nucleus

Central regulatory pathways promoting stress adaptation utilize various neurotransmitters/neuropeptides, such as urocortin 1 (Ucn1) and neuropeptide Y (NPY). Ucn1 is abundantly expressed in the nonpreganglionic Edinger-Westphal nucleus (npEW), where it is codistributed with NPY-immunoreactive (ir) terminals. A special role for both neuropeptides has been postulated in stress adaptation. Using double-labeling immunohistochemistry, we observed close appositions between NPY-ir terminals and neurons immunoreactive for Ucn1 in the rat, as well as in the human npEW. Therefore, we hypothesized that NPY might control the activity of Ucn1-positive neurons in the npEW. To test this hypothesis, NPY was injected into the lateral cerebral ventricle of rats, resulting in a strong activation of npEW Ucn1 neurons as revealed by Fos immunohistochemistry. Ucn1 mRNA was also upregulated in the npEW 2 hours after the injection of NPY. In a search for the type of NPY receptor that mediates this NPY-induced recruitment of npEW-Ucn1 cells, we found that the great majority of Ucn1 cells exhibited NPY Y5 receptor immunoreactivity, and only a few of the Ucn1 cells coexpressed the Y1 receptor. We concluded that NPY, via NPY Y5 and to a lesser extent via the Y1 receptors, exerts a stimulatory action on Ucn1 cells in the npEW. Further studies are currently in progress to elucidate the significance of this NPY-Ucn1 interaction in the npEW.     

Characterization and localization of immunoreactive dynorphin, α-neo-endorphin, met-enkephalin and substance P in human spinal cord

By use of specific antisera, the distributions of immunoreactive dynorphin (ir-DYN), α-neo-endorphin (ir-α-NEO), Met-enkephalin (ir-MET) and substance P (ir-SP) were evaluated in discrete regions of human spinal cord and spinal ganglia. The relative concentrations of immunoreactive peptides in particular regions were as follows: sacral > lumbar > cervical > thoracic. Concentrations of ir-DYN, ir-α-NEO and ir-SP were 2–10-fold, but of ir-MET 1–2-fold, higher in the dorsal as compared to the ventral parts of cervical, lumbar and sacral cord.The concentrations of all peptides (when examined in discrete areas of thoracic cord) were found to be highest in the substantia gelatinosa. All peptides were present in the gray matter but only ir-MET was found in white matter.Gel-permeation chromatography of dorsal sacral spinal cord extracts revealed two major ir-DYN peaks. The smaller molecular weight peak, eluted at the position of synthetic dynorphin_1–17. ir-α-NEO and ir-SP comigrated exactly with their respective synthetic marker peptides. Substantial amounts of ir-SP and also, as confirmed by high pressure liquid chromatography, ir-MET, were found in the dorsal and ventral roots and spinal ganglia, and very low concentrations of ir-DYN or ir-α-NEO were also detected in these tissue.These results suggest that dynorphin and α-neo-endorphin, in addition to enkephalins, may be involved in transmission of somatosensory information in the human spinal cord.     

Distribution and development of proenkephalin-like immunoreactivity in the Lumbar spinal cord of the chicken

Met5-enkephalin- (Met-ENK), Leu5-enkephalin-, Met5-enkephalin-Arg6-Phe7-, metorphamide- and BAM 22P-like peptides could be detected in the lumbar spinal cord of the chicken by immunocytochemistry and/or high performance liquid chromatography. However, a peptide identical to Met5-enkephalin-Arg6-Gly7-Leu8 could not be detected in the lumbar spinal cord of the chicken using an antiserum that was capable of detecting the octapeptide in mammalian tissues. Nerve fiber- and terminal-like processes containing proenkephalin-derived peptides were concentrated in the superficial laminae of the dorsal horn and along the midline rostral to the central canal. A lesser concentration of processes containing proenkephalin-derived peptides occurred in the medial and lateral motor columns of the ventral horn. The level of total radioimmunoassayable Met-ENK in the lumbar spinal cord of the chicken embryo increased more than 1000-fold between day 4.5 and day 18. A schedule of curare administration that had previously been shown to prevent naturally occurring somatic motoneuron death in the chicken lumbar spinal cord resulted in a two-fold increase in total radioimmunoassayable Met-ENK in the lumbar spinal cord.     

用免疫细胞化学方法观察胚胎脊髓组织移植后的细胞构筑特征

本文应用生长抑素、5-羟色胺、P物质,胶质酸性蛋白的抗体,通过免疫细胞化学反应,对胚胎脊髓组织移植后的细胞构筑特征进行了观察。结果发现,移植后2—6个月的胚胎脊髓组织内分布有与上述抗体相应的免疫阳性神经元及神经纤维。在移植组织与宿主脊髓融合差的情况下,移植界面可见胶质酸性蛋白免疫反应明显增强。研究表明,免疫细胞化学方法可反映移植组织的生长分化程度,并可显示移植部位的细胞构筑特征。     

The release of immunoreactive neuropeptide Y in the spinal cord of the anaesthetized rat and cat

The release of immunoreactive (ir-) neuropeptide Y (NYP) was studied in the anaesthetized rat and cat by means of microprobes bearing immobilized antibodies to the C terminus of NPY. An extensive basal release of ir-NYP was detected throughout the dorsal and upper ventral horn of the rat. This spontaneous release was not significantly altered by sectioning the spinal cord at the thoraco-lumbar junction nor by electrical stimulation of peripheral nerves. Since NPY is virtually absent in primary afferents it is probable that spontaneous release within the spinal cord comes from active NPY-containing intrinsic spinal neurones. In the spinal cat spontaneous release of ir-NPY was detected in the mid-dorsal horn and this was unaltered by peripheral noxious thermal or noxious mechanical stimuli. As in the rat, release from intrinsic spinal neurones is most probable. The extensive spontaneous release of ir-NPY in both species suggests a widespread role in spinal cord function.     

Characterization of forms of immunoreactive somatostatin in sensory neuron and normal and deafferented spinal cord

In order to determine the contribution made by primary sensory afferents and supraspinal projections to the immunoreactive somatostatin (IRS) content of the spinal cord, measurements were made of the concentration of IRS in the dorsal and ventral halves of the cord in cats subjected to unilateral lumbosacral dorsal rhizotomy (L1-S3) alone or combined with spinal cord transection. The molecular forms of IRS (characterized by gel chromatography) in L7 lumbar spinal cord, L6-S1 dorsal roots, ventral roots and dorsal root ganglia, and sciatic nerve were also determined. S14 was the predominant form in all tissues examined, but two additional molecular forms corresponding to S28 and S11.5 kdalton were present in dorsal root ganglia and spinal cord; S28 but not S11.5 kdalton was detected in both dorsal roots and sciatic nerves. These results indicate that S14 and S28 and S28 are transported along the central and peripheral processes of dorsal root ganglia, but that spinal cord S11.5 kdalton originates in the central nervous system. IRS in the dorsal horn was reduced by ca. 40% following dorsal root section. Neither disruption of descending pathways by spinal transection nor surgical isolation of the lumbar segements lowered cord somatostatin content below that produced by dorsal root section, indicating that most of the somatostatin within the cord arises from the dorsal root and from neurons in local spinal segments. Although the total content of IRS in the dorsal horn was reduced by ca. 40% following dorsal rhizotomy, the pattern of molecular forms was not changed accordingly. Since S14 and S28 but not S11.5 kdalton are transported via the dorsal root, the dorsal root section would be predicted to produce a relatively greater decrease in S14 and S28 than in S11.5 kdalton. Therefore, failure to find a selective loss of S14 and S28 suggests that dorsal rhizotomy affects dorsal horn IRS content not only by removing afferent input but possibly also by modifyinh the processing of IRS by the remaining somatostatinergic neurons.     

Immunohistochemical demonstration of some putative neurotransmitters in the lamprey spinal cord and spinal ganglia: 5-hydroxytryptamine-, tachykinin-, and neuropeptide-Y-immunoreactive neurons and fibers

The distribution of some putative neurotransmitters was investigated in the spinal cord and spinal ganglia of the lamprey, a primitive vertebrate, by using immunohistochemical methods. In the spinal cord a midline row of 5-hydroxytryptamine (5-HT)-immunoreactive neurons was present immediately ventral to the central canal over the entire length of the spinal cord. The ventral processes of these neurons formed a dense ventromedial plexus of varicosities. In the dorsal, lateral, and ventral spinal axon columns, several longitudinal 5-HT fibers were present. After chronic spinal transections the distribution of 5-HT fibers was unchanged; it is therefore concluded that there was no substantial descending 5-HT contribution and that the spinal 5-HT neurons supplied the regional 5-HT innervation. The spinal 5-HT cells sent fibers into the dorsal and ventral roots; 5-HT cell bodies and fibers were also present in the spinal dorsal root ganglia, in their dorsal, ventral, and lateral nerve branches, and in the dorsal and ventral branches of the ventral roots. Neurons and fibers containing peptides of the tachykinin (TK) family (to which, amongst others, substance P belongs) were found in the spinal cord. TK neurons in the spinal cord supplied the local TK innervation, as well as TK fibers in the dorsal and ventral roots. Fibers have been found containing either TK, or 5-HT, or both compounds. Neurons containing neuropeptide-Y (NPY)-immunoreactive material were present in a medial column just dorsal to the central canal. The NPY neurons have longitudinal, mainly descending, fibers that provide the local NPY innervation of the lamprey spinal cord. The present results provide evidence for local spinal systems containing 5-HT, TK, 5-HT and TK, or NPY, but in contrast to mammals, these compounds do not seem to arise from supraspinal neurons.     

Distribution and chromatographic characterization of neuromedin B-like immunoreactivity in the human spinal cord

The quantitative regional distribution of neuromedin B-like immunoreactivity in normal potmertem human spinal cord was studied by a specific radioimmunoassay. Beuromedin B-like immunoreactivity was found in highest concentration in the dorsal part of the sacral cord. Chromatographic analyses by gel permeation and reverse-phase high-pressue liquid chromatography revealed two major peaks of neuromedin B-like immunoreactivity and the prevalent molecular form approx. 90% of the total immunoreactivity, was chromatographically identical to synthetic porcine neuromedin B.     

Differential microglial regulation in the human spinal cord under normal and pathological conditions

As the primary intrinsic immune effector cells of the central nervous system, microglia are involved in virtually all pathological processes of the brain and spinal cord including inflammatory, neurodegenerative, traumatic, neoplastic and vascular diseases. Despite this important role, there is a lack of data concerning microglial distribution and protein expression in the human spinal cord. In this study, we immunohistochemically investigated 10 normal human spinal cords to establish reference data and compared these results with 15 pathological human spinal cords deriving from distinct pathologies. Each spinal cord was evaluated at eight different levels for three white and two grey matter areas for both constitutive (MHC-II, CD68, IL-16, AIF-1, LCA, CD4) and reactive (MRP-8, MRP-14) microglial antigens. Whereas previous studies revealed significant regional differences in microglial distribution and protein expression in human brain, normal spinal cord displayed a uniform expression pattern, reaching levels of up to 17% MHC-II positive cells of the total cell population. This datum formed the basis for the further evaluation of microglia expression levels in pathological spinal cords, where levels of up to 45% positive cells were observed. Our results represent important reference values for future neuropathological diagnostic and therapeutical approaches in spinal cord pathologies.     

神经肽Y受体在人垂体腺瘤中的基因表达

目的研究NPY受体在人垂体腺瘤中表达及其规律。方法收集2004年1月~2005年8月期间我院神经外科手术切除垂体腺瘤获取标本57例。通过巢式聚会酶链反应(N-PCR)测定NPY的Y1R和Y2R受体。结果不同类型垂体腺瘤中均有NPY的Y1R、Y2R mRNA表达。Y1R基因表达的差异无统计学意义(F=1.97,P=0.098);Y2R表达的差异有显著统计学意义(F=2.703,P=0.03)。NPY与Y2R呈正相关(r=0.414,P=0.003),但与Y1R无相关性(r=-0.123,P=0.405),且Y1R与Y2R之间也无相关性(r=0.158,P=0.284)。PRL腺瘤中Y2R的表达明显低于GH腺瘤和促性腺瘤。GH腺瘤中NPY与Y2R表达呈正相关(r=0.558,P=0.025)。结论垂体腺瘤中存在NPY和Y1R及Y2R表达;Y2R表达的差异有显著统计学意义。Y2R在GH腺瘤和促性腺细胞腺瘤中的表达水平明显高于PRL腺瘤,而Y1R的表达无显著性差异。NPY受体的表达差异可能与不同类型垂体腺瘤的发生、发展及其内分泌行为有关。     

Distribution of enkephalin in human fetus and infant spinal cord: An immunofluorescence study

The distribution of enkephalin-like immunoreactivity in the human fetus and infant spinal cord have been studied by indirect immunofluorescence. Enkephalin-like immunoreactive fibers were detectable in the lateral funiculus of fetal spinal cord as early as 10 weeks. At the other fetal ages examined, ranging from 12 to 28 weeks, and in infant, enkephalinlike immunoreactivity was found widely distributed throughout the whole spinal cord. In fetus spinal cord several enkephalin-like immunoreactive cells were sometimes seen scattered in the intermediate gray region. Most of the labeling was, however, represented by thin, varicose, immunofluorescent fibers mainly localized in the intermediate gray regions, in the ventral horn and in the superficial dorsal horn layers where they progressively increased in number. Further, the white matter exhibited enkephalin-like immunoreactive fibers particularly in the lateral funiculus where a dense punctiform immunofluorescence could be seen. On the whole, similar patterns were also visible in infant spinal cord. Thus, the superficial layers of the dorsal horn and the intermediolateral and reticular nuclei areas displayed dense plexuses of immunoreactive fibers. In contrast, the white matter showed only little labeling. In addition, no immunoreactivity was found in fetus and infant dorsal root ganglia. Our results emphasize the wide distribution of the enkephalin-like immunoreactivity in the fetus as in the infant spinal cord and further suggest its first appearance early in fetal life, possibly at the embryonic stage.     

Concurrent isolation and characterization of oligodendrocytes, microglia and astrocytes from adult human spinal cord

A cellular preparation of highly enriched oligodendrocytes was obtained from adult human spinal cord by Percoll gradient centrifugation followed by either differential adhesion or fluorescence-activated cell sorting after immunostaining with an antibody against galactocerebroside (Ol). The adherent and O1-negative cell fractions were 96% microglia. The non-adherent and O1-positive fractions were 96% positive for the oligodendrocyte markers O4 and O1, 0–2% positive for glial fibrillary acidic protein, and were devoid of neuronal or microglial markers. If the oligodendrocyte fraction was co-cultured with purified dissociated rat dorsal root ganglion neurons, the oligodendrocytes adhered to the axons and their numbers increased over a 4 week period. However, myelin sheaths were not produced around axons in these cultures. In contrast, if the oligodendrocyte cell fraction was grown alone in culture for 3 weeks, the number of oligodendrocytes decreased and a layer of astrocytes developed underneath the oligodendrocytes. The oligodendrocytes could be eliminated from these cultures by subsequent passaging, thus producing cultures of pure astrocytes. The astrocytes accumulated both K⁺ and glutamate with kinetic properties similar to those reported for rodent astrocytes. We suggest that these astrocytes arose in part from an O4/O1-positive precursor which did not initially express glial fibrillary acidic protein. These results define a relatively simple method by which highly enriched populations of oligodendrocytes, astrocytes and microglia can be obtained from adult human spinal cord.     

The microvasculature of the spinal cord in the human adult

The microvasculature of the human spinal cord have been studied with scanning electron microscopy (SEM) on the corrosion casts of blood vessels, and light microscopy on the transparent after intra-arterial injection of Indian ink and vascular staining specimens. On the surface of the cord, the density of the pial venous plexus was higher than the pial arterial plexus. The anterior spinal artery and vein gave off central arteries and veins, which ran posteriorly within the anterior median fissure. The number of the central arteries and veins per centimeter in length is highest in the lumbosacral cord, followed by the cervical and then the thoracic cord. In this study, the central arteries and veins were classified into types; type 1 dominated the arterial system and type 2 the venous system. The central arteries and veins sent long branches both rostrally and caudally along the axis of spinal cord. The order of their length was as follows: thoracic, cervical, lumbar and sacral cord. These blood vessels overlapped each other. The peripheral arteries terminated in the periphery of the gray matter while the peripheral veins originated within the different depth of the gray matter. The posterior spinal artery supplied the posterior horn and the substantia gelatinosa. The posteromedial veins arose in the deep posterior horns and posterior funiculus, and were occasionally connected with the anterior spinal vein within the cord. Overlapped zone which was supplied by both the central and peripheral vascular system was seen within the cord, and its extent was variable at different levels. The overlapped zone of the venous supply was broader than the arterial one. The peripheral veins appeared to drain blood not only from the white matter but also from the gray matter. The distribution of the capillary networks in the gray matter was slightly denser and showed more horizontal arrangement in the lumbosacral cord, and more sparse and longitudinal in the cervicothoracic cord. The distribution of the capillary networks in the white matter was parallel to nerve fibers which run longitudinal in the whole cord. Based on the result of present study, it was considered that the circulatory disturbances are prone to occur in the thoracic cord, and the circulatory block in the arterial system of the cord tends to cause a lesion in the gray matter, whereas the circulatory disturbances in the venous system tends to cause a lesion in the white matter.     

Co‐localized neuropeptide Y and GABA have complementary presynaptic effects on sensory synaptic transmission

We have examined the morphological relationship of neuropeptide Y (NPY) and GABAergic neurons in the lamprey spinal cord, and the physiological effects of NPY and GABA_B receptor agonists on afferent synaptic transmission. NPY-containing fibres and cell bodies were identified in the dorsal root entry zone. NPY immunoreactive (–ir) fibres made close appositions with primary afferent axons. Co-localization of NPY and GABA-ir was found in the dorsal horn and dorsal column. Fifty-two per cent of NPY-ir profiles showed immunoreactivity to GABA at the ultrastructural level. Electron microscopic analysis showed that NPY-immunoreactivity was present throughout the axoplasm, including over dense core vesicles, whereas GABA-immunoreactivity was mainly found over small synaptic vesicles. Synthetic lamprey NPY, and the related peptide, peptide YY, reduced the amplitude of monosynaptic afferent EPSPs in spinobulbar neurons. NPY had no significant effect on the postsynaptic input resistance or membrane potential, the electrical component of the synaptic potential, or the response to glutamate, but it could reduce the duration of presynaptic action potentials, suggesting that it was acting presynaptically. NPY also reduced the excitability of the spinobulbar neurons, suggesting at least one postsynaptic effect. Because NPY and GABA colocalize, we compared the effects of NPY and the GABA_B agonist baclofen. Both presynaptically reduced EPSP amplitudes, baclofen having a larger effect and a faster onset and recovery than NPY. The GABA_B antagonist phaclofen reduced the effect of baclofen, but not that of NPY. We conclude that NPY and GABA are colocalized in terminals in the dorsal spinal cord of the lamprey, and that they have complementary actions in modulating sensory inputs.     

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.