Demonstration of sensory-motor innervation of postmetamorphic forelimb regenerates of Triturus alpestris (Urodela) on cryotome serial sections using horseradish peroxidase

A method has been developed to obtain horseradish peroxidase-treated serial sections containing spinal cord as well as bilateral ventral and dorsal roots, dorsal root ganglia and spinal nerves. Young postmetamorphic newts (Triturus alpestris) served as experimental animals. After cryotome cross sectioning the forelimb region of the trunk, slices 80 microns in thickness were mounted serially with up to 15 sections per slide. This facilitated subsequent staining manipulations and made partial loss of sections less likely.     

Innervation of the dermatomes in the neck of the mouse

The dorsal rami of the cervical and thoracic spinal nerves were investigated using both the in situ cholinesterase staining technique and cholinesterase staining on serial sections of plastic-embedded embryos. In most cases only the dorsal rami of the 2nd to 5th cervical spinal nerve possess cutaneous branches. The area innervated by the cutaneous branch of the dorsal ramus of the 5th spinal nerve borders on an area innervated by the cutaneous branch of the dorsal ramus of the 1st thoracic spinal nerve. The dorsal rami of the cervical spinal nerves 6-8 show no cutaneous branches. Therefore the gap in the series of the dorsal cutaneous branches is due only to the middle part of the nerves of the brachial plexus, which range from the 5th cervical nerve to the 1st thoracic nerve.     

Immunocytochemical localization of neuromedin K (neurokinin B) in rat spinal ganglia and cord.

Specific antisera directed against substance P and neuromedin K (neurokinin B) have been used in double-label immunofluorescence studies to unambiguously localize these two neuropeptides of the tachykinin family in single tissue sections of rat spinal cord and dorsal root ganglia. Substance P-like immunoreactivity (SPLI) is present but neuromedin K-like immunoreactivity (NMKLI) is undetectable in dorsal root ganglia. Both peptides are present in the spinal cord, but NMKLI is largely restricted to the dorsal gray while SPLI shows a broader distribution. In the spinal gray, NMKLI coexists with SPLI in some, but not all, fibers. While substance P in the dorsal spinal cord is largely of primary afferent origin, neuromedin K appears to originate largely from intrinsic spinal neurons.     

Human dorsal root ganglion neurons from embryonic donors extend axons into the host rat spinal cord along laminin-rich peripheral surroundings of the dorsal root transitional zone

Following dorsal root crush, the lesioned axons regenerate in the peripheral compartment of the dorsal root, but stop at the boundary between the peripheral and the central nervous system, the dorsal root transitional zone. We have previously shown that fibres from human fetal dorsal root ganglia grafted to adult rat hosts are able to grow into the spinal cord, but were not able to specify the route taken by the ingrowing fibres. In this study we have challenged the dorsal root transitional zone astrocyte boundary with human dorsal root ganglion transplants from 5–8-week-old embryos. By tracing immunolabelled human fibres in serial sections, we found that fibres consistently grow around the dorsal root transitional zone astrocytes in laminin-rich peripheral surroundings, and extend into the host rat spinal cord along blood vessels, either into deep or superficial laminae of the dorsal horn, or into the dorsal funiculus. Human fibres that did not have access to blood vessels grew on the spinal cord surface. These findings indicate, that in spite of a substantial growth capacity by axons from human embryonic dorsal root ganglion cells as well as their tolerance to non-permissive factors in the mature mammalian CNS, these axons are still sensitive to the repellent effects of astrocytes of the mature dorsal root transitional zone. Furthermore, this axonal ingrowth is consistently associated with laminin-expressing structures until the axons reach the host spinal cord.     

Autoradiographic distribution of I-galanin binding sites in the rat central nervous system

Galanin (GAL) binding sites in coronal sections of the rat brain were demonstrated using autoradiographic methods. Scatchard analysis of ¹²⁵I-GAL binding to slide-mounted tissue sections revealed saturable binding to a single class of receptors with a Kd of approximately 0.2 nM. ¹²⁵I-GAL binding sites were demonstrated throughout the rat central nervous system. Dense binding was observed in the following areas: prefrontal cortex, the anterior nuclei of the olfactory bulb, several nuclei of the amygdaloid complex, the dorsal septal area, dorsal bed nucleus of the stria terminalis, the ventral pallidum, the internal medullary laminae of the thalamus, medial pretectal nucleus, nucleus of the medial optic tract, borderline area of the caudal spinal trigeminal nucleus adjacent to the spinal trigeminal tract, the substantia gelatinosa and the superficial layers of the dorsal spinal cord. Moderate binding was observed in the piriform, periamygdaloid, entorhinal, insular cortex and the subiculum, the nucleus accumbens, medial forebrain bundle, anterior hypothalamic, ventromedial, dorsal premamillary, lateral and periventricular thalamic nuclei, the subzona incerta, Forel's field H₁ and H₂, periventricular gray matter, medial and superficial gray strata of the superior colliculus, dorsal parts of the central gray, peripeduncular area, the interpeduncular nucleus, substantia nigra zona compacta, ventral tegmental area, the dorsal and ventral parabrachial and parvocellular reticular nuclei. The preponderance of GAL-binding in somatosensory as well as in limbic areas suggests a possible involvement of GAL in a variety of brain functions.     

Neurokinin A-like immunoreactivity in rat primary sensory neurons; coexistence with substance P

Rat spinal cord, dorsal root ganglia and skin were investigated employing immunohistochemical technique with specific antisera to neurokinin A and substance P. Neurokinin A-like immunoreactivity was detected in the spinal dorsal horn and skin with a similar distribution pattern as that of substance P-like immunoreactivity. After dorsal root transection a parallel decrease of neurokinin A and substance P-like immunoreactivity was observed in the dorsal horn. Using colchicine pretreatment a population of neurokinin A positive cell bodies was seen in the dorsal root ganglia, and by comparison of consecutive sections of the same cells stained for substance P it was revealed that these neurons also display substance P-like immunoreactivity. However, substance P-, but not neurokinin A-, immunoreactive cells were also observed. It is concluded that neurokinin A- and substance P-like immunoreactivity coexist in a population of rat primary sensory neurons.     

Neurokinin A-like immunoreactivity in rat primary sensory neurons; coexistence with substance P

Summary Rat spinal cord, dorsal root ganglia and skin were investigated employing immunohistochemical technique with specific antisera to neurokinin A and substance P. Neurokinin A-like immunoreactivity was detected in the spinal dorsal horn and skin with a similar distribution pattern as that of substance P-like immunoreactivity. After dorsal root transection a parallell decrease of neurokinin A and substance P-like immunoreactivity was observed in the dorsal horn. Using colchicine pretreatment a population of neurokinin A positive cell bodies was seen in the dorsal root ganglia, and by comparison of consecutive sections of the same cells stained for substance P it was revealed that these neurons also display substance P-like immunoreactivity. However, substance P-, but not neurokinin A-, immunoreactive cells were also observed. It is concluded that neurokinin A- and substance P-like immunoreactivity coexist in a population of rat primary sensory neurons.     

Procion yellow staining of motoneurones in the frog.

Intracellular recording and subsequent staining of spinal motoneurones in the frog was made by procion-filled micropipettes. Spike discharges in response to dorsal root (DR) and ventral root (VR) volleys as well as to direct current injections were studied. Reconstruction of the dendritic tree of the cell stained after recording was made from photomicrographs taken from frozen serial sections of the spinal cord. Migration of the dye into a neighbouring unimpaled cell was observed. The advantages of the procion injection technique in studying the frog's spinal cord physiology are discussed.     

Short-term changes of NADPH diaphorase-exhibiting neuronal pools in the spinal cord of rabbit after repeated sublethal ischemia

The aim of this study was the histochemical characterization of NADPH diaphorase-positive neuronal pools in the rabbit lumbosacral segments using a model of single, repeated and multiple sublethal spinal cord ischemia. Following a single 8-min sublethal spinal cord ischemia and 1-hour reperfusion, the staining of NADPH diaphorase-exhibiting neurons in the dorsal horn, pericentral region, dorsal gray commissure and sacral parasympathetic nucleus was comparable with the control sections. In contrast to the foregoing sublethal ischemia, a regionally different somatic NADPH diaphorase (NADPHd) staining was found after multiple sublethal spinal cord ischemia. Whereas an almost complete loss of the staining of large NADPHd-exhibiting somata in the pericentral region was detected, the staining of the NADPHd-exhibiting neuronal pools in the deep dorsal horn and sacral parasympathetic nucleus was fully preserved. Concomitantly, a prominent reduction of small NADPH diaphorase-positive neurons was noted in the superficial dorsal horn layers of lower lumbar and sacral segments.     

Differential Changes in Neuronal Excitability in the Spinal Dorsal Horn After Spinal Nerve Ligation in Rats

Previous studies demonstrated that peripheral nerve injury induced excessive neuronal response and glial activation in the spinal cord dorsal horn, and such change has been proposed to reflect the development and maintenance of neuropathic pain states. The aim of this study was to examine neuronal excitability and glial activation in the spinal dorsal horn after peripheral nerve injury. We examined noxious heat stimulation-induced c-Fos protein-like immunoreactivity (Fos-LI) neuron profiles in fourth-to-sixth lumbar (L4–L6) level spinal dorsal horn neurons after fifth lumbar spinal nerve ligation (L5 SNL). Immunofluorescence labeling of OX-42 and GFAP was also performed in histological sections of the spinal cord. A significant increase in the number of Fos-LI neuron profiles in the spinal dorsal horn at the L4 level was found at 3 days after SNL, but returned to a level similar to that in sham-operated controls by 14 days after injury. As expected, a decrease in the number of Fos-LI neuron profiles in the spinal dorsal horn at the L5 level was found at 3 days after SNL. However, these profiles had reappeared in large numbers by 14 and 21 days after injury. Immunofluorescence labeling of OX-42 and GFAP indicated sequential activation of microglia and astrocytes in the spinal dorsal horn. We conclude that nerve injury causes differential changes in neuronal excitability in the spinal dorsal horn, which may coincide with glial activation. These changes may play a substantial role in the pathogenesis of neuropathic pain after peripheral nerve injury.     

Biochemical and autoradiographic studies of TRH receptors in sections of rabbit spinal cord

Receptors for thyrotropin-releasing hormone (pGlu-His-Pro-NH2, TRH) on thaw-mounted sections of rabbit spinal cord have been identified biochemically and visualized by light microscopic autoradiography. Binding of [3H] [3-Me-His2]TRH to 20 microns sections exhibited high apparent affinity and a pharmacological specificity almost identical to that previously demonstrated for spinal TRH receptors in membranes. In autoradiograms, the highest density of TRH receptors appeared in the substantia gelatinosa of the dorsal gray and around the central canal, with intermediate levels in the ventral gray.     

Binding Patterns of Lectins with GalNAc specificity in the mouse dorsal root ganglia and spinal cord

To localize membrane glycoconjugates in neurons of the mouse spinal cord and dorsal root ganglia (DRG), cryostat sections of newborn (P0), 7 day-old (P7), P14, P21 and P31 animals were stained with ten FITC-conjugated plant lectins, the majority of them recognizing N-acetyl-D-galactosamine (GalNAc) terminal sugar residues. In the dorsal root ganglia of P0 animals, the different lectins showed distinct patterns of labeling in either cells of the nervous system, including neurons, or other structures such as nerves or blood vessels. Moreover, some of these lectins showed important changes in their pattern of labeling during postnatal development. This was especially relevant for lectins that label a subpopulation of small-sized cells that have been previously identified as the nociceptive cells of the DRG. Enzymatic digestion of sections with neuraminidase removes sialic acid from the carbohydrate chains of glycoconjugates thus exposing novel sugar residues. When this treatment was applied to DRG sections from postnatal animals the pattern of lectin staining was either changed or eliminated and heterogeneous subsets of glycoconjugates normally masked by this sugar were exposed. In the spinal cord of PO animals, none of the lectins labeled cells in the central gray matter. However, after the enzymatic digestion of sections with neuraminidase, spinal cord motoneurons and some other cells were labeled by two of the lectins suggesting that GalNAc residues present in these cells are normally masked by terminal sialic acid. Altogether, these results show important changes in the temporal and spatial expression of glycoconjugates that may be relevant for the postnatal development of the CNS and PNS of mice.     

Electrophysiological analysis of the organization of somatosensory thalamic inputs in the frog

Potentials produced in the frog thalamus by electrical stimulation of the peripheral nerves were investigated by sink and current source-density analysis. Sinks, which are viewed as potential generation sites, were located in three regions: the cell-free zone of the ventral thalamus adjoining the ventrolateral nucleus, the ventromedial and ventrolateral nuclei, and the caudal section of the dorsal thalamus. Evoked activity was recorded in individual neurons in the area of the second and third of these sinks. The first sink failed to form after section of the dorsal tracks of the spinal cord, while the remaining two only appeared after a considerably extended latency. It is suggested that nuclei of the ventral and caudal sections of the dorsal thalamus receive somatic impulses through the systems connected with the dorsal as well as the ventrolateral columns of the spinal cord. The direct projections of the primordial nuclei of dorsal columns may be involved in afferentation the ventral thalamus.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 687–695, September–October, 1986.     

Nerve growth factor in glia and inflammatory cells of the injured rat spinal cord

Nerve growth factor (NGF) is crucial for the development of sympathetic and small-diameter sensory neurons and for maintenance of their mature phenotype. Its role in generating neuronal pathophysiology is less well understood. After spinal cord injury, central processes of primary afferent fibers sprout into the dorsal horn, contributing to the development of autonomic dysfunctions and pain. NGF may promote these states as it stimulates sprouting of small-diameter afferent fibers and its concentration in the spinal cord increases after cord injury. The cells responsible for this increase must be identified to develop a strategy to prevent the afferent sprouting. Using immunocytochemistry, we identified cells containing NGF in spinal cord sections from intact rats and from rats 1 and 2 weeks after high thoracic cord transection. In intact rats, this neurotrophin was present in a few ramified microglia and in putative Schwann cells in the dorsal root. Within and close to the lesion of cord-injured rats, NGF was in many activated, ramified microglia, in a subset of astrocytes, and in small, round cells that were neither glia nor macrophages. NGF-immunoreactive putative Schwann cells were prevalent throughout the thoracolumbar cord in the dorsal roots and the dorsal root entry zones. Oligodendrocytes were never immunoreactive for this protein. Therapeutic strategies targeting spinal cord cells that produce NGF may prevent primary afferent sprouting and resulting clinical disorders after cord injury.     

Central core in the spinal grey matter.

Golgi--Kopsch impregnated spinal cord sections of adult cats revealed a regular orientation of the dendrites in the intermediate region forming a circular or elliptical grey matter. The orientation of the dendrites is strictly transverse in the central core. A new interpretation of the spinal grey matter is proposed by viewing the dorsal, lateral and ventral horns (or columns) as crest-like appendages attached to the rod-like central core. A structural and possibly functional similarity between the central core and the brain stem reticular formation is emphasized.     

Synaptic organization of substance P, glutamate and GABA-immunoreactive boutons on functionally identified neurons in cat spinal deeper dorsal horn

In order to determine how nociceptive input conveyed by the C-fibers terminating in superficial lam-inae of the spinal cord reaches the wide dynamic range (WDR) cells in deeper dorsal horn, which functions as ascend-ing projection pathway, the morphological features of some WDR cells in the deeper dorsal horn of the cat lumbar spinal cord were studied by intracellular injection of horseradish peroxidase and physiological characterization. One of the fully stained neurons with somata in lamina V and dendrites that entered lamina Ⅱ were examined by electron mi-croscopy. Immunogold staining of ultrathin sections through the labeled proximal dendrites in lamina Ⅱ revealed that these dendrites received numerous synapses from substance P and glutamate immunoreactive (IR) axons, which were considered originating from C-fibers. In addition, many GABA-IR terminals were found presynaptic to the labeled dendrites. The results, therefore, suggest that the information carried by primary afferent can be sent from t     

Changes of parvalbumin expression in the spinal cord after peripheral inflammation

Parvalbumin (PV) is a calcium-binding protein that is expressed by numerous neuronal subpopulations in the central nervous system. Staining for PV was often used in neuroanatomical studies in the past. Recently, several studies have suggested that PV acts in neurons as a mobile endogenous calcium buffer that affects temporo-spatial characteristics of calcium transients and is involved in modulation of synaptic transmission. In our experiments, expression of PV in the lumbar dorsal horn spinal cord was evaluated using densitometric analysis of immunohistological sections and Western-blot techniques in control and arthritic rats. There was a significant reduction of PV immunoreactivity in the superficial dorsal horn region ipsilateral to the arthritis after induction of the peripheral inflammation. The ipsilateral area and intensity of PV staining in this area were reduced to 38 % and 37 %, respectively, out of the total PV staining on both sides. It is suggested that this reduction may reflect decreased expression of PV in GABAergic inhibitory neurons. Reduction of PV concentration in the presynaptic GABAergic terminals could lead to potentiation of inhibitory transmission in the spinal cord. Our results suggest that changes in expression of calcium-binding proteins in spinal cord dorsal horn neurons may modulate nociceptive transmission.     

Decreased Uptake and Release of D-Aspartate in the Guinea Pig Spinal Cord After Dorsal Root Section

This study attempts to determine if L-glutamate and/or L-aspartate may be transmitters of dorsal sensory neurons. The uptake and the electrically evoked release of D-[3H]aspartate, a putative marker for L-glutamate and L-aspartate, were measured in the cervical enlargement (segments C4-T1) of the guinea pig spinal cord before and after cutting dorsal roots C5-T1 on the right side. The uptake and the release of gamma-aminobutyric acid (GABA) also were measured as indices of the integrity of GABAergic neurons in the spinal cord. The cervical enlargement was excised and divided into left and right halves, then into dorsal and ventral quadrants. Quadrants from unlesioned animals took up D-aspartate and GABA, achieving concentrations in the tissues which were 14-25 times that in the medium. Subsequently, electrical stimulation evoked a Ca2+-dependent release of D-aspartate and of GABA. The uptake and release of D-aspartate and GABA were similar in tissues taken from intact and sham-operated animals. However, dorsal rhizotomy, without damage to dorsal radicular or spinal blood vessels, depressed the uptake (by 22-29%) and the release (by 50%) of D-aspartate only in quadrants ipsilateral to the lesion. The uptake and the release of GABA were unchanged. In transverse sections of the cervical enlargement, stained to reveal degenerating fibers, by far the heaviest loss of axons occurred in the cuneate fasciculus and in the gray matter ipsilateral to the cut dorsal roots. These findings suggest that the synaptic endings of dorsal sensory neurons probably mediate the uptake and the release of D-aspartate and, therefore, may use L-glutamate or L-aspartate as a transmitter. When spinal blood vessels were damaged during dorsal rhizotomy, the deficits in D-aspartate uptake and release were larger than those in the absence of vascular damage and were accompanied by deficits in GABA uptake and release. These findings imply that vascular damage results in the loss of intraspinal neurons, some of which probably mediate the uptake and release of D-aspartate and, therefore, may use L-glutamate and/or L-aspartate as a transmitter.     

Finite Element Study of the Mechanical Response in Spinal Cord during the Thoracolumbar Burst Fracture

Background

The mechanical response of the spinal cord during burst fracture was seldom quantitatively addressed and only few studies look into the internal strain of the white and grey matters within the spinal cord during thoracolumbar burst fracture (TLBF). The aim of the study is to investigate the mechanical response of the spinal cord during TLBF and correlate the percent canal compromise (PCC) with the strain in the spinal cord.

Methodology/Principal Findings

A three-dimensional (3D) finite element (FE) model of human T12-L1 spinal cord with visco-elastic property was generated based on the transverse sections images of spinal cord, and the model was validated against published literatures under static uniaxial tension and compression. With the validated model, a TLBF simulation was performed to compute the mechanical strain in the spinal cord with the PCC. Linear regressions between PCC and strain in the spinal cord show that at the initial stage, with the PCC at 20%, and 45%, the corresponding mechanical strains in ventral grey, dorsal grey, ventral white, dorsal white matters were 0.06, 0.04, 0.12, 0.06, and increased to 0.14, 0.12, 0.23, and 0.13, respectively. At the recoiled stage, when the PCC was decreased from 45% to 20%, the corresponding strains were reduced to 0.03, 0.02, 0.04 and 0.03. The strain was correlated well with PCC.

Conclusions/Significance

The simulation shows that the strain in the spinal cord correlated well with the PCC, and the mechanical strains in the ventral regions are higher than those in the dorsal regions of spinal cord tissue during burst fracture, suggesting that the ventral regions of the spinal cord may susceptible to injury than the dorsal regions.     

Changes in NADPH-diaphorase activity in the rat dorsal horn following an acute experimental myositis

Previous neurophysiological experiments have shown that in rats with an acute myositis of the gastrocnemius-soleus muscle, dorsal horn neurones exhibit an increase in responsiveness to peripheral stimulation and in background activity. The present study investigated the possible correlation between changes in NADPH-diaphorase activity and neurophysiological alterations. In the animals used for the electrophysiological experiments the diaphorase activity in sections of the lumbar spinal cord was determined with the NADPH-nitroblue tetrazolium reaction. The main finding was a massive reduction in the number of diaphorase-positive cells in the superficial dorsal horn in animals with a myositis. The staining intensity in the remaining neurones was unchanged. The results are interpreted as indicating that the myositis in addition to the surgical operations represents a supramaximal input to the dorsal horn causing neurotoxic effects in diaphorase- positive neurones.