Distribution of NADPH-d and nNOS-IR in the thoracolumbar and sacrococcygeal spinal cord of the guinea pig

The distribution of NADPH-d staining and neuronal nitric oxide synthase (nNOS)-immunoreactivity in the spinal cord of the guinea pig was studied to evaluate the potential role of nitric oxide in lumbosacral afferent and spinal autonomic pathways and to compare the distribution of these two markers to that observed in other species. NADPH-d staining and nNOS-immunoreactivity were present in neurons and fibers in the superficial dorsal horn, dorsal commissure and in neurons around the central canal in all levels of the spinal cord examined. Sympathetic preganglionic neurons in the thoracic and rostral lumbar segments identified by choline acetyl transferase (ChAT) immunoreactivity exhibited prominent NADPH-d staining and nNOS-immunoreactivity; whereas the ChAT-immunoreactive parasympathetic preganglionic neurons in the sacral segments were not stained. The most prominent NADPH-d staining in the sacral segments occurred in fibers extending from Lissauer's tract through laminae I along the lateral edge of the dorsal horn to the region of the sacral parasympathetic nucleus (lateral collateral pathway of Lissauer). These fibers were prominent in the S1-S3 segments but not in adjacent (L5-L7 and Cx1) or thoracolumbar segments. These NADPH-d fibers were, for the most part, not nNOS-immunoreactive, but did overlap with a prominent fiber bundle containing vasoactive intestinal polypeptide immunoreactivity in the sacral spinal cord. These results indicate that nitric oxide may function as a transmitter in thoracolumbar sympathetic preganglionic neurons, but not in sacral parasympathetic preganglionic neurons. Although the functional significance of the NADPH-d positive, nNOS-negative fiber bundle on the lateral edge of the sacral dorsal horn remains to be determined, this fiber tract may represent, in part, visceral afferent projections to the sacral parasympathetic nucleus.     

Preferential immunohistochemical localization of vasoactive intestinal polypeptide (VIP) in the sacral spinal cord of the cat: light and electron microscopic observations

In the present study we have employed immunoperoxidase techniques to investigate the distribution of vasoactive intestinal polypeptide (VIP)-like immunoreactivity in the spinal cord and sensory ganglia of the cat. The spinal distribution of VIP-containing neuronal processes was also compared with that of substance P (SP), somatostatin (SOM), and cholecystokinin-8 (CCK) at lumbar, sacral, and coccygeal levels. At sacral levels, VIP was found to be contained in small and medium-sized primary sensory neurons and in dorsal rootlets. Deafferentation, by either ganglionectomy or dorsal rhizotomy, resulted in a nearly complete loss of VIP immunoreactivity in the spinal cord. The spinal distribution of VIP fibers and terminals was most dense and extensive in sacral segments. Forming a thin shell around the dorsal horn, collaterals, apparently originating from Lissauer's tract, projected either medially or laterally through lamina I. Laterally, many VIP axons terminated in lateral laminae V to VII. Others projected further through the neck of the dorsal horn to medial lamina V and the gray matter near the central canal. Medially, VIP axons descended through lamina I to expand into terminal fields in the posterior commissure and medial lamina V. At the ultrastructural level, VIP-like immunoreactivity was found in dense core vesicles within axonal enlargements containing both large dense core and smaller clear round vesicles. Synaptic connections were infrequently observed but, when encountered, were of the simple axodendritic type. The spinal distribution of VIP-containing fibers was remarkably similar to that reported for pelvic nerve visceral afferents, both in termination patterns within the spinal gray matter and in localization to the sacral cord. The density of SP-, SOM-, and CCK-containing fibers and terminals was constant at all levels examined (L4 to Co4). In marked contrast, the distribution of VIP fibers, much like that of pelvic nerve afferents, was mostly confined to sacral segments. Thus, although SP, SOM, and CCK may be contained within a population of sacral visceral afferents, they must be common to afferent systems in other segments as well. VIP, however, appears to be preferentially contained within pelvic visceral afferent fibers confined mostly to sacral segments.     

Localization of NADPH diaphorase in the lumbosacral spinal cord and dorsal root ganglia of the cat

The distribution of NADPH-d activity in the spinal cord and dorsal root ganglia of the cat was studied to evaluate the role of nitric oxide in lumbosacral afferent and spinal autonomic pathways. At all levels of the spinal cord NADPH-d staining was present in neurons and fibers in the superficial dorsal horn and in neurons around the central canal and in the dorsal commissure. In addition, the sympathetic autonomic nucleus in the rostral lumbar segments exhibited prominent NADPH-d cellular staining whereas the parasympathetic nucleus in the sacral segments was not well stained. The most prominent NADPH-d activity in the sacral segments occurred in fibers extending from Lissauer's tract through laminae I along the lateral edge of the dorsal horn to lamina V and the region of the sacral parasympathetic nucleus. These fibers were very similar to VIP-containing and pelvic nerve afferent projections in the same region. They were prominent in the S₁–S₃ segments but not in adjacent segments (L₆–L₇ and Cx₁) or in thoracolumbar and cervical segments. NADPH-d activity and VIP immunoreactivity in Lissauer's tract and the lateral dorsal horn were eliminated or greatly reduced after dorsal-ventral rhizotomy (S₁–S₃), indicating the fibers represent primary afferent projections. A population of small diameter afferent neurons in the L₇–S₂ dorsal root ganglia were intensely stained for NADPH-d. The functional significance of the NADPH-d histochemical stain remains to be determined; however, if NADPH-d is nitric oxide synthase then this would suggest that nitric oxide may function as a transmitter in thoracolumbar sympathetic preganglionic efferent pathways and in sacral parasympathetic afferent pathways in the cat. © 1994 Wiley-Liss, Inc.     

Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species

Calcitonin gene-related peptide (CGRP) immunoreactivity was found throughout the entire spinal cord of man, marmoset, horse, pig, cat, guinea pig, mouse, rat, and frog. CGRP-immunoreactive fibers were most concentrated in the dorsal horn. In the ventral horn of some species large immunoreactive cells, tentatively characterized as motoneurons, were present. Pretreatment of rats with colchicine enhanced staining of these large cells but did not reveal CGRP-immunoreactive cell bodies in the dorsal horn. In the dorsal root ganglia, CGRP immunoreactivity was observed in most of the small and some of the intermediate sized cells. Substance P immunoreactivity, where present, was co-localized with CGRP to a proportion of the small cells. In the cat the ratio of substance P-immunoreactive to CGRP-immunoreactive ganglion cells was 1:2.7 (p less than 0.001). The concentration of CGRP-immunoreactive material in tissue extracts was determined by radioimmunoassay. In the dorsal horn of the rat spinal cord the levels of peptide were found to range from 225.7 +/- 30.0 pmol/gm of wet weight in the cervical region to 340.6 +/- 74.6 pmol/gm in the sacral spinal cord. In the rat ventral spinal cord, levels of 15.7 +/- 2.7 to 35.1 +/- 10.6 pmol/gm were found. The concentration in dorsal root ganglia of the lumbar region was 225.4 +/- 46.9 pmol/gm. Gel permeation chromatography of this extractable CGRP-like immunoreactivity revealed three distinct immunoreactive peaks, one eluting at the position of synthetic CGRP and the others, of smaller size, eluting later. In cats and rats, rhizotomy induced a marked loss of CGRP-immunoreactive fibers from the dorsal horn of the spinal cord. In the cat, unilateral lumbosacral dorsal rhizotomy resulted in a significant (p less than 0.05) reduction of extractable CGRP from the ipsilateral lumbar dorsal horn (5.6 +/- 1.2 pmol/gm of wet weight) compared to the contralateral side (105.0 +/- 36.0 pmol/gm of wet weight). We conclude that the major origin of CGRP in the dorsal spinal cord is extrinsic, from afferent fibers which are probably derived from cells in the dorsal root ganglia. The selective distribution of CGRP throughout sensory, motor, and autonomic areas of the spinal cord suggests many putative roles for this novel peptide.     

Analysis of calcitonin gene-related peptide-like immunoreactivity in the cat dorsal spinal cord and dorsal root ganglia provide evidence for a multisegmental projection of nociceptive C-fiber primary afferents.

Recent studies have suggested that calcitonin gene-related peptide (CGRP) can be used as a marker for a subpopulation of nociceptive primary afferents. Consequently, CGRP-immunoreactive (CGRP-IR) primary afferents have been reported to project many segments rostral to their segment of entry and to send collaterals into the superficial and deep laminae of the dorsal horn. This study reports that some CGRP-IR primary afferents of sacral origin project rostral through the ipsilateral lumbar enlargement in the cat. The ultrastructure of these multisegmentally projecting primary afferent axons and terminals identified in a partially denervated cat was examined and compared to the ultrastructure of CGRP-IR afferents from an intact cat. Retrograde transport of wheatgerm agglutinin-colloidal gold injected into the cat L4 spinal cord resulted in labeling of primary afferent cell bodies in the ipsilateral L4-S1 dorsal root ganglia (DRG). Analysis of every fourth section through the ipsilateral S1 DRG revealed as many as 1,000 retrogradely labeled neuronal cell bodies. One third of these cell bodies were double labeled for CGRP-like immunoreactivity. The number of single- and double-labeled cells increased in ganglia closer to the injection site (L4-L7). At the ultrastructural level, in the lumbosacral dorsal spinal cord of a normal cat, most CGRP-IR axons were unmyelinated, while the rest were small myelinated axons. In both the superficial dorsal horn and lamina V, CGRP-IR varicosities were dome shaped, scallop shaped, or elongated. The CGRP-IR varicosities contained small agranular vesicles and frequently a few dense core vesicles. These labeled varicosities formed asymmetric synapses on unlabeled dendritic spines, shafts, or neuronal somata. One cat received multiple unilateral dorsal rhizotomies (S1-L4) and an ipsilateral hemisection (mid L4). CGRP-IR axons and terminals were found within each of the rhizotomized segments, although their density was greatly reduced compared to that in the intact animals. In Lissauer's tract the majority (greater than 90%) of CGRP-IR fibers were unmyelinated. In laminae I and V, the remaining CGRP-IR varicosities were mainly the dome-shaped varicosities morphologically similar to those observed in the normal spinal cords. They contained small agranular vesicles and a few dense core vesicles and formed asymmetric synapses on unlabeled dendritic shafts and spines. These data demonstrate that unmyelinated, presumably C-fiber nociceptive primary afferents and some small myelinated A-delta nociceptive primary afferents of sacral origin project rostral through the cat lumbar enlargement and make synaptic connections in both the superficial and deep laminae of the cat dorsal spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunolocalization and quantitation of a novel nerve terminal protein in spinal cord development

In the adult spinal cord, the neuron-specific protein NT75 is located in nerve terminals synapsing in the superficial laminae of the dorsal horn. The present study examines the occurrence of NT75 in the developing rat spinal cord. NT75 immunoreactivity is detectable in primary afferent axons at the dorsal root entry zone on embryonic day 15. Subsequently, staining of presumptive nerve terminals appears in the deeper laminae of the dorsal horn, expanding into the superficial laminae during the first postnatal week. NT75 staining also appears in developing corticospinal tract axons in the brainstem at birth, and at lumbosacral levels by postnatal day 5. As NT75-positive nerve terminals approach the adult distribution, staining of primary afferent and corticospinal axons decreases, becoming undetectable by postnatal day 30. Dense transient staining of presumed nerve terminals in the ventral horn is also apparent during early postnatal development. Quantitative analysis of developing spinal cord shows a low level of NT75 immunoreactivity at birth. NT75 activity then increases substantially, reaching values by the third and fourth postnatal weeks up to 2.5 times that seen in adults. The occurrence of NT75 immunoreactivity correlates with the reported time course of synaptic development in the spinal cord. In addition, the results suggest that NT75 immunoreactivity is maintained at high levels in the nerve terminals of certain neural pathways into adulthood, whereas in other systems NT75 immunoreactivity may be detectable only during development.     

The role of neuropeptides in the sacral autonomic reflex pathways of the cat

Immunohistochemical and pharmacological studies were conducted to examine the origin and function of peptidergic nerves in the sacral autonomic system of the cat. Leucine-enkephalin (L-Enk) immunoreactivity was identified in nerve terminals in peripheral ganglia on the surface of the urinary bladder and in the parasympathetic nucleus in the sacral spinal cord. In colchicine-treated animals L-Enk was also detected in sacral preganglionic neurons (sPGN) identified by retrograde transport of a fluorescent dye. L-Enk terminals in bladder ganglia are believed to arise from sPGN since the terminals were eliminated by transection of the sacral ventral roots. Pharmacological studies indicated that exogenous as well as endogenously released enkephalins have an inhibitory action at both ganglionic and spinal sites in the sacral outflow to the urinary bladder. Peptides were also associated with afferents nerves in the sacral autonomic system. The distribution of substance P, VIP and cholecystokinin in the sacral dorsal horn paralleled the distribution of visceral afferent projections as demonstrated with HRP techniques. Dye labeling combined with immunohistochemistry revealed that some dorsal root ganglion cells projecting to the pelvic viscera contain substance P or VIP.     

Central distribution of afferent pathways from the uterus of the cat.

Afferent pathways from the uterus of the cat were labeled by injections of horseradish peroxidase (HRP), wheat germ agglutinin-HRP, or fluorescent dyes into the uterine cervix and uterine horns. Afferent input to the uterus arises from small to medium size neurons (average size 31 x 28 microns) in dorsal root ganglia at many levels of the spinal cord (T12-S3). The segmental origin correlates with the location of the afferent terminal field in the uterus. Eighty-seven percent of the dorsal root ganglion cells (average, 822 on one side) innervating the cervix are located in sacral ganglia, whereas 97% of the cells innervating the uterine horn (average 479 on one side) are located in lumbar ganglia. Double dye labeling experiments indicate that a small percentage (average 15%) of lumbar neurons innervating the uterine cervix also innervate the uterine horn. The majority (70-80%) of afferent input to the uterine cervix passes through the pelvic nerve and the remainder through the pudendal nerve, whereas afferent input to the uterine horn must travel in sympathetic nerves. Ovariectomy (10-14 days) did not change significantly the number, sizes, or segmental distribution of uterine afferent neurons. In some cats (25%) injections of WGA-HRP into the uterine cervix labeled neurons (90-125 per animal) in lamina VII in the S2 spinal segment in the region of the sacral parasympathetic nucleus. Central projections of uterine horn afferent neurons were not labeled; however, afferent projections from the cervix were detected in the sacral spinal cord. The most prominent labeling was present in Lissauer's tract and in lamina I and outer lamina II on the lateral edge of the dorsal horn. From this region some labeled axons extended through lamina V into the dorsal gray commissure. Very few afferents were labeled on the medial side of the dorsal horn. These results are discussed in regard to the physiological function of uterine afferents and the possible transmitter role of vasoactive intestinal polypeptide, which is present in a large percentage (70%) of cervical afferent neurons.     

Primary afferent and propriospinal fibers in the rat dorsal and dorsolateral funiculi

The purpose of this study is to determine the numbers of primary afferent and propriospinal fibers in the dorsal and dorsolateral funiculi of the rat. The reason for concentrating on these areas is that they contain large numbers of unmyelinated axons. Our data are axonal numbers from the S2 segment of spinal cord in animals that had unilateral dorsal rhizotomies or spinal cord isolations. The major conclusions are 1) that 23% of the primary afferent fibers in the dorsal funiculus are unmyelinated; 2) that there are approximately 12,500 unmyelinated primary afferent fibers in the dorsolateral funiculus, which is more than the number of primary afferent fibers in the dorsal funiculus and tract of Lissauer combined, and 3) that approximately 25% of the axons in the dorsal funiculus and 44% of the axons in the dorsolateral funiculus are propriospinal. These data modify and extend previous ideas of the organization of spinal white matter.     

Distribution and ultrastructure of tachykinin-like immunoreactivity in the frog (Rana esculenta) spinal cord, notably, the dorsal horn

Tachykinins are involved in pain transmission at the spinal level. In frog, at least four tachykinins [TK] have been isolated from the brain, but their organization in the dorsal horn of the spinal cord is still poorly known. We have reexamined TK distribution by immunocytochemistry using an antibody recognizing the sequence common to all tachykinins in the spinal cord and dorsal root ganglia of the green frog Rana esculenta. A dense tachykinin-like immunoreactivity (TK-LI) was observed in the dorsolateral fasciculus or Lissauer's tract running ventromedial to the entry of the dorsal root and in numerous small and medium-sized dorsal root ganglion cells showing a primary afferent origin for part of TK-LI of the dorsal horn. The observation of numerous cell bodies in the dorsal horn, in addition, suggested a local or propriospinal origin. One group of cells was localized at the entrance of the Lissauer's tract TK-LI fibers into the dorsal horn, and another group was localized in the upper dorsal horn, a region with a low density of TK-LI fibers. It was suggested that the latter group may correspond to neurokinin B. Electron microscopic examination of the Lissauer's tract showed numerous immunoreactive axons, some located at the center of glomerular-like arrangements, suggesting that the information brought by these fibers may be transmitted and most probably modulated before their entry in the dorsal horn. In conclusion, the functional organization of tachykinins in the frog spinal cord seems to be similar to that of mammals, albeit with a different morphological organization.     

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.     

Quantitation of propriospinal fibers in the tract of lissauer of the rat

The present study uses a spinal cord isolation procedure to remove extrinsic axons but leave intrinsic axons intact. The isolation is done by sectioning the spinal cord in two places and then cutting all dorsal roots between the two sections. The axons that survive the isolation procedure are thought to be propriospinal axons. Following isolation, approximately one-third of the axons in sacral tracts of Lissauer in the rat survive. Thus approximately one-third of the axons in sacral tracts of Lissauer in the rat are propriospinal. Proportionately more myelinated than unmyelinated axons are lost. There are approximately equal numbers of surviving axons in the medial as opposed to the lateral part of the tract. This implies that there is, as yet, no morphological basis for dividing the tract into medial and lateral halves. The fact that the number of propriospinal axons in the tract of Lissauer has been quantitated offers more precision in our thinking about the organization of the dorsal horn.     

Expression of receptors for glial cell line-derived neurotrophic factor family ligands in sacral spinal cord reveals separate targets of pelvic afferent fibers

Nerve growth factor has been proposed to mediate many structural and chemical changes in bladder sensory neurons after injury or inflammation. We have examined the expression of receptors for the glial cell line-derived neurotrophic factor (GDNF) family within sensory terminals located in the sacral spinal cord and in bladder-projecting sacral dorsal root ganglion neurons of adult female Sprague-Dawley rats. Nerve fibers immunolabelled for GFRalpha1 (GDNF receptor), GFRalpha2 (neurturin receptor), or GFRalpha3 (artemin receptor) showed distinct distribution patterns in the spinal cord, suggesting separate populations of sensory fibers with different functions: GFRalpha1-labeled fibers were in outer lamina II and the lateral-collateral pathway and associated with autonomic interneurons and preganglionic neurons; GFRalpha2-labeled fibers were only in inner lamina II; GFRalpha3-labeled fibers were in lamina I, the lateral-collateral pathway, and areas surrounding dorsal groups of preganglionic neurons and associated interneurons. Immunofluorescence studies of retrogradely labelled bladder-projecting neurons in sacral dorsal root ganglia showed that approximately 25% expressed GFRalpha1 or GFRalpha3 immunoreactivity, the preferred receptors for GDNF and artemin, respectively. After cyclophosphamide-induced bladder inflammation, fluorescence intensity of GFRalpha1-positive fibers increased within the dorsal horn, but there was no change in the GFRalpha2- or GFRalpha3-positive fibers. These studies have shown that GDNF and artemin may target bladder sensory neurons and potentially mediate plasticity of sacral visceral afferent neurons following inflammation. Our results have also revealed three distinct subpopulations of sensory fibers within the sacral spinal cord, which have not been identified previously using other markers.     

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)     

Co-localization of endomorphin-2 and substance P in primary afferent nociceptors and effects of injury: a light and electron microscopic study in the rat

Endomorphin-2 (EM2) is a tetrapeptide with remarkable affinity and selectivity for the mu-opioid receptor. In the present study, we used double-fluorescence and electron microscopic immunocytochemistry to identify subsets of EM2-expressing neurons in dorsal root ganglia and spinal cord dorsal horn of adult rats. Within the lumbar dorsal root ganglia, we found EM2 immunoreactivity mainly in small-to-medium size neurons, most of which co-expressed the neuropeptide substance P (SP). In adult rat L4 dorsal root ganglia, 23.9% of neuronal profiles contained EM2 immunoreactivity and ranged in size from 15 to 36 microM in diameter (mean 24.3 +/- 4.3 microM). Double-labelling experiments with cytochemical markers of dorsal root ganglia neurons showed that approximately 95% of EM2-immunoreactive cell bodies also label with SP antisera, 83% co-express vanilloid receptor subtype 1/capsaicin receptor, and 17% label with isolectin B4, a marker of non-peptide nociceptors. Importantly, EM2 immunostaining persisted in mice with a deletion of the preprotachykinin-A gene that encodes SP. In the lumbar spinal cord dorsal horn, EM2 expression was concentrated in presumptive primary afferent terminals in laminae I and outer II. At the ultrastructural level, electron microscopic double-labelling showed co-localization of EM2 and SP in dense core vesicles of lumbar superficial dorsal horn synaptic terminals. Finally, 2 weeks after sciatic nerve axotomy we observed a greater than 50% reduction in EM2 immunoreactivity in the superficial dorsal horn. We suggest that the very strong anatomical relationship between primary afferent nociceptors that express SP and EM2 underlies an EM2 regulation of SP release via mu-opioid autoreceptors.     

Swelling of frog dorsal root ganglion and spinal cord produced by afferent volley of impulses

Electrical stimulation of the frog sciatic nerve was found to produce rapid, transient swelling of the 8th and 9th dorsal root ganglia followed by prolonged swelling of the spinal cord. Swelling of the ganglion is analogous to the rapid mechanical change observed in invertebrate axons during excitation. The mechanical change observed in the spinal cord is probably related to prolonged depolarization of the primary afferent fibers near their terminals.     

Vasoactive intestinal polypeptide in sacral primary sensory pathways in the cat.

Unmyelinated sensory axons in the sacral spinal cord may play a role in bladder reflexes under certain pathological conditions. Previous data suggested vasoactive intestinal polypeptide (VIP) might be contained exclusively in sensory C-fibers, some of which innervate the bladder. This study was undertaken to describe the morphology of these VIP fibers in the sacral cord of the cat. VIP immunoreactivity was confined to unmyelinated axons observed at several levels of the sensory pathway including the dorsal root ganglia, dorsal roots, Lissauer's tract, and the lateral collateral pathway. A combination of light and electron microscopic observations showed VIP-immunoreactive fibers with labeled varicosities and synaptic terminals in laminae I, IIo, V, VII, and X. VIP-immunolabeled varicosities had a mean diameter of 1.6 microm (range = 0.11-7.4 microm, S.D. = 1.01, n = 311) with a small percentage (8%) being relatively large (3-7.4 microm). VIP varicosities contained a mixture of small clear vesicles (CLV) and large dense core vesicles (LDV). Although most varicosities contained a moderate number of LDVs (14.86 LDVs/microm2), some varicosities contained a large number of LDVs, whereas others contained very few. Varicosities that possessed synaptic specializations were classed as terminals and were divided into three morphological classes. Two of these resembled Gray's Type I terminal, whereas a third was similar to the Gray's Type II terminal. There was no consistent relationship between vesicle content of the terminal and the type of synaptic contact it possessed. This study shows that in the sacral spinal cord of the cat, VIP terminals originate only from C-fibers, terminate primarily in laminae I and V, and exhibit a variety of morphologies consistent with heterogeneous origins and functions of the lower urinary tract.     

Peripheral nerve autografts to the rat spinal cord: Studies with axonal tracing methods

In young adult female rats, autologous sciatic nerve segments were transplanted to the thoracic region of the spinal cord. The grafts became well innervated but led to no obvious functional improvement. The origin and termination of axons in the grafts was studied by retrograde neuronal labeling with horseradish peroxidase (HRP) and radioautographic axonal tracing. Studies with HRP indicated that some axons in the grafts originated from intrinsic CNS neurons with their cell bodies in nearby segments of the spinal cord and that others arose from dorsal root ganglia at the level of the grafts and at least 7 segments distal to them. After tritiated amino acids were injected into lumbar dorsal root ganglia, labeled axons could be followed into the grafts but not into the rostral spinal cord stumps. Together with other experimental observations, these results demonstrate a correlation between success or failure of elongation of dorsal root fibers and peripheral or central ensheathment at the axonal tip. The corticospinal tract was studied both with radioautography and retrograde axonal transport of HRP but no extension of its axons into peripheral nerve grafts was detected under these experimental conditions. The findings implicate both neuroglial and axonal factors in the feeble regenerative response usually seen after injury to the spinal cord.     

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.