Localization of sulfated glucuronyl glycolipids in human dorsal root and sympathetic ganglia.

Sulfated glucuronyl glycolipids (SGGLs) in human dorsal root ganglion (DRG) and sympathetic ganglion (SG) were analyzed biochemically and immunohistochemically. SGGLs were enriched in human DRG (1.02 +/- 0.23 micrograms/mg protein), whereas much lower concentrations of these glycolipids (0.043 +/- 0.23 micrograms/mg protein) were detected in SG. Myelin within DRG and SG was immunostained by anti-SGGL antiserum, although only a few myelinated fibers were seen in SG. Nerve cell bodies or unmyelinated fibers were not immunostained. Subcellular fractionation study of human DRG demonstrated that these glycolipids were not only enriched in myelin but also in the axolemma-enriched fraction. These data are consistent with the view that SGGLs may be expressed on myelinated fibers in myelin and axolemma, suggesting that these compounds may play an important role in regulating myelinogenesis.

Neurochemical and immunocytochemical studies on the distribution of N-acetyl-aspartylglutamate and N-acetyl-aspartate in rat spinal cord and some peripheral nervous tissues.

HPLC analysis of rat spinal cord revealed a uniform distribution of N-acetyl-aspartate (NAA) across both longitudinal and dorsoventral axes. In contrast, ventral cord N-acetyl-aspartylglutamate (NAAG) levels were significantly higher than those measured in dorsal halves of cervical, thoracic, and lumbar segments. Immunocytochemical studies using an affinity-purified antiserum raised against NAAG-bovine serum albumin revealed an intense staining of motoneurons within rat spinal cord. Along with the considerable NAAG content in ventral roots, these results suggest that NAAG may be concentrated in motoneurons and play a role in motor pathways. NAAG was also present in other peripheral neural tissues, including dorsal roots, dorsal root ganglia, superior cervical ganglia, and sciatic nerve. It is interesting that NAA levels in peripheral nervous tissues were lower than those in CNS structures and that NAA levels in ventral roots and sciatic nerve were lower than NAAG levels. These findings further document a lack of correlation between NAAG and NAA levels in both central and peripheral nervous tissues. Taken together, these data demonstrate the presence of NAAG in nonglutamatergic neuronal systems and suggest a more complex role of NAAG in neuronal physiology than previously postulated.

Axonal GABA receptors are selectively present on normal and regenerated sensory fibers in rat peripheral nerve.

A sucrose gap chamber was used to study the effect of gamma-aminobutyric acid (GABA) on normal and regenerating rat peripheral nerve fibers. Sciatic nerves and dorsal roots were depolarized by GABA and the GABAA receptor subtype agonist muscimol, but not by the GABAB receptor subtype agonist baclofen. Ventral root fibers were not affected by these agents, suggesting a selective presence of axonal GABA receptors on sensory fibers of mammalian peripheral nerves. Regenerating dorsal and ventral root fibers were studied 13 to 20 days following nerve crush. The regenerated dorsal root fibers were depolarized by GABA or muscimol, but the regenerated ventral root fibers were not. These results indicate that GABA receptors are selectively present on normal mammalian sensory axons, and are reestablished on regenerated sensory axons.

Regeneration of primary afferent neurons containing substance P-like immunoreactivity.

We compared changes in levels of substance P-like immunoreactivity (SPLI) in L4-6 dorsal root ganglia (DRG), L4-6 dorsal roots, sciatic nerve, tibial nerve and hind foot skin in rats following resection or crush injury of the sciatic nerve. The initial depletion of SPLI, which occurred in all areas sampled, was similar after either type of lesion. In DRG and dorsal roots, recovery to control values occurred in SPLI levels 35-45 days after sciatic crush, but not after resection. In sciatic nerve proximal to the injury, a partial recovery in SPLI content to about 60% of control occurred following crush injury, but not following resection. Distal to the injury, tibial nerve levels recovered rapidly following crush injury, consistent with the previously observed rapid regeneration of SPLI-containing axons. After resection, no recovery was observed until after 35 days, when it appeared that some axons succeeded in crossing the resection zone and regaining the distal nerve stump. Delayed and poor recovery of SPLI levels was observed in foot skin, even after crush injury. This correlated with the poor recovery of the plasma extravasation reaction, a functional index of SP-innervation of skin. In contrast, reinnervation by high-threshold mechanoreceptors was more rapid and complete, in agreement with a previous study. We conclude that although SPLI-containing axons regenerate rapidly, they appear to reinnervate skin less successfully than other afferents. Axon regeneration is associated with a recovery of SPLI levels which fell after axotomy: no recovery occurs if regeneration is prevented. Recovery was almost complete in DRG and roots, but incomplete in sciatic nerve. This peptide transmitter in afferent neurons thus behaves in a similar fashion to previously studied low-molecular weight transmitters and related materials in efferent neurons. Since recovery of SPLI levels begins before there is evidence for target reinnervation, it seems that axon regeneration is a sufficient condition for reversal of some axotomy-induced changes in these neurons. Further studies on substance P synthesis and on the response of individual DRG neurons to axotomy and regeneration will be required to explain fully the discrepancy between partial recovery of SPLI levels in sciatic nerve and full recovery in DRG and dorsal roots.

Structural properties of spinal nerve roots: protein composition.

The protein compositions of dorsal and ventral spinal nerve roots were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein readily soluble in sodium dodecyl sulfate (SDS) sample buffer made up 8.1% of the wet weight of dorsal and ventral roots. Spinal root protein samples consisted predominantly (60%) of myelin-associated proteins. Other major proteins including those tentatively identified as tubulin, actin, nuclear histones, and others accounted for the remainder of recovered protein. The protein constituents of nerve roots were similar to those of peripheral nerve but differed from those of spinal cord. Nerve roots and peripheral nerve were characterized by fewer major protein bands but greater concentrations of myelin proteins. Collagen which did not readily solubilize in SDS sample buffer was estimated by assaying for hydroxyproline. Nerve roots consisted of approximately 0.4% collagen by weight which was only one-fifth the amount estimated for nerve but six times more than spinal cord. It was apparent that the biomechanical frailty of roots compared with peripheral nerve might be explained by differences in the relative collagen contents of these tissues. The protein constituents of nerve roots after unilateral nerve crush were relatively stable compared with the profound changes seen in ipsilateral nerve and modest changes seen in contralateral uninjured nerve.

Regional distribution of substance P, neurokinin alpha and neurokinin beta in rat spinal cord, nerve roots and dorsal root ganglia, and the effects of dorsal root section or spinal transection.

The regional distribution of 3 mammalian tachykinins (substance P, neurokinin alpha and neurokinin beta) in the rat spinal cord and related structures was investigated using a method of radioimmunoassay combined with high performance liquid chromatography. Substance P and neurokinin alpha were found to be distributed in a very similar manner with fairly constant molar ratios i.e. ratios of substance P to neurokinin alpha were 3.69 in the dorsal root ganglia, 3.49 in the dorsal root and 3.09 in the dorsal horn of the cervical spinal cord. On the other hand, the distribution of neurokinin beta was different from other tachykinins; although concentrated in the dorsal horn, neurokinin beta in the dorsal root ganglia or in the dorsal roots was negligibly small in amount. When the cervical dorsal roots were sectioned unilaterally, substance P and neurokinin alpha were decreased in a parallel fashion in the dorsal horn, whereas neurokinin beta was not. In addition neurokinin alpha was selectively and significantly decreased in the dorsal horn of the intact side when compared to that in the unoperated control rat. Since the magnitude of a decrease of neurokinin alpha in molar basis was approximately the same as a decrease of substance P, these findings suggest that the neurokinin alpha and substance P-containing primary afferent fibres could project partly to the contralateral dorsal horn as well. When the thoracic spinal cord was transected, substance P (and neurokinin alpha) was decreased in the ventral part of the lumbar spinal cord, suggesting the presence of tachykinin(s)-containing descending fibres.(ABSTRACT TRUNCATED AT 250 WORDS)

Nerve growth factor supply for sensory neurons: site of origin and competition with the sympathetic nervous system.

Nerve growth factor (NGF) levels in the sensory nervous system were measured by a highly sensitive two-site enzyme immunoassay for NGF. Dorsal root ganglia and the adjacent spinal nerves contained 2.8 +/- 0.3 and 1.7 +/- 0.4 ng NGF/g wet wt., respectively, whereas no NGF was detectable in dorsal roots and spinal cord (less than 0.05 ng NGF/g wet wt.). It is concluded that sensory neurons are supplied with NGF exclusively from their peripheral and not from their central field of projection. Two days after treatment with 6-hydroxydopamine, which destroys sympathetic nerve terminals and thereby prevents the removal of NGF by sympathetic neurons, the NGF content of dorsal root ganglia and trigeminal ganglia increased to 285% and 161% of control, respectively. This indicates that in peripheral target tissues sensory and sympathetic neurons compete for NGF.

Astrocytic proteins in the dorsal and ventral roots in amyotrophic lateral sclerosis and Werdnig-Hoffmann disease.

Abnormalities were detected by two-dimensional gel electrophoresis in the protein composition of both the dorsal and ventral roots of three of six patients who succumbed to amyotrophic lateral sclerosis (ALS). The abnormalities consisted of a cascade of acidic protein spots on silver-stained gels which were shown by immunoblotting to react with an antiserum to human glial fibrillary acidic protein (GFAP). They were found distal to the normal central nervous system/peripheral nervous system (CNS/PNS) transition zone and were undetected in cervical and lumbar root segments taken at the same distances from the spinal cord of eight control patients. Similar changes were observed in the dorsal and ventral roots of one patient with Werdnig-Hoffmann disease (WHD), while a second patient with WHD had the changes in only the ventral roots. The abnormalities probably reflect the presence of radicular glial bundles, which are pathological extensions of glial cells into the spinal roots, indicating that subclinical changes occurred in the sensory nerves of the affected ALS and WHD patients. While no other qualitative abnormalities were noted on gels of ALS and WHD spinal roots, some quantitative changes may be present.

Spinal cord acetylcholine content: the consequence of transection or treatment with the neurotoxin 6-aminonicotinamide.

The distribution of choline (Ch) and acetylcholine (ACh) within the rat spinal cord was found to be rather uniform with values for Ch of about 280 pmol/mg protein and for ACh of about 250 pmol/mg protein. Cord transection at the T10-11 level did not reduce ACh below the lesion but there was about a 30% decrease of ACh in the thoracic region suggesting the presence of cholinergic fibers that ascend and terminate within the cord. Treatment of rats with the neurotoxin 6-aminonicotinamide resulted in spastic hindlimb paralysis and a loss of ACh in the lumbar region of the cord.

Projections of hindlimb dorsal roots to lumbosacral spinal cord of cat.

Single dorsal roots of spinal nerves that contribute to the cat lumbosacral plexus (L3-S2) were cut to evoke degeneration of centrally projecting axons. Serial sections throughout lumbosacral cord levels were impregnated by the Fink-Heimer method (20) to permit charting of the distribution patterns of segmental dorsal root afferent fibers. Afferent fibers that enter a single dorsal root have an extensive distribution to multiple cord segments; their longitudinal extent varies with entry level and with laminar targets. Afferent projections to the ventral horn reach motor nuclei only in their entry segment and the adjacent segments just above and below their entry. Those afferent fibers projecting to intermediate gray (laminae VI and VII) have the most extensive spinal distribution of any types; they may, from a single dorsal root, reach as many as 13 or 14 cord segments. Dorsal horn projections of single roots are also longitudinally expansive. Small-diameter afferent fibers course rostrally and caudally in Lissauer's tract (LT) for up to 9-10 segments. They appear to terminate in at least laminae I and II in and near their entry segment; their endings are difficult to demonstrate at greater distances where they are probably less dense. Larger caliber axons entering the dorsal horn generate a somatotopically organized projection, especially to laminae III and IV. Collaterals of these fibers appear to course longitudinally within the gray matter and they distribute to as many as six to seven segments.

Receptors for thyrotropin-releasing hormone (TRH) in rabbit spinal cord.

Specific binding of 3H-labeled [3-Me-His2]TRH ([3H]MeTRH) to membranes of rabbit spinal cord (thoracic) involved a homogeneous population of high-affinity sites (Kd = 2.7 +/- 0.17 (n = 5) nM, Bmax = 204 +/- 12(5) fmol/mg protein). TRH analogs competed for the binding in the following rank order of potency: MeTRH greater than TRH greater than TRH-Gly-NH2 greater than Ser-His-Pro-NH2 greater than Thr-His-Pro-NH2 greater than pGlu-His-Pro-NH-C2H5 greater than TRH-free acid. Competition experiments with rat amygdala, run in parallel with rabbit spinal cord, revealed a closely similar pattern of activity. These properties help identify binding sites in the rabbit spinal cord as physiological receptors for TRH. The binding sites resemble receptors previously demonstrated in pituitary and CNS tissues of other species. The densities of [3H]MeTRH binding sites in different segments were generally similar, although density in the thoracic segment appeared to be somewhat higher. In all segments, binding seemed to be enriched in the dorsal gray matter. Dorsal roots and their associated ganglia, however, displayed little or no binding.

[Dyssymmetry of the external structure of the human spinal cord: radicular bases and segments (a morphometric study)]. / Dissimmetriia vneshnego stroeniia spinnogo mozga cheloveka: koreshkovye osnovaniia i segmenty (morfometricheskoe issledovanie).

In 101 preparations of spinal cords obtained from persons of various age, dyssymmetry in level position and extent of the radicular bases, interradicular spaces on the right and left halves of the spinal cord segments has been studied macro- and microscopically. Quantitative treatment of the results obtained has demonstrated that certain elements of asymmetry in structure of the human spinal cord are revealed already in fetuses; with age, the dyssymmetry in the right and left halves of the segment increases. Indices of dyssymmetry are especially large at the level of the thoracic segments and on the posterior surface of the spinal cord.

Distribution of soluble proteins within spinal motoneurons: a quantitative two-dimensional electrophoretic analysis.

Soluble protein fractions obtained from bovine lumbar spinal motoneuron cell bodies, ventral gray matter, and ventral and dorsal roots were analyzed by two-dimensional gel electrophoresis. Each extract was separated into Coomassie blue-stained patterns of up to 350 polypeptides ranging in isoelectric point from pH 4 to 8 and in molecular weight from 10,000 to 200,000. Visual inspection of the protein pattern of the isolated cell bodies showed it to be substantially different from those of ventral gray matter and the spinal roots, while the patterns obtained from ventral and dorsal roots were indistinguishable. Computer-assisted densitometry of the major soluble proteins from spinal roots showed no quantitative difference between the predominant proteins in ventral and dorsal root extracts. Differences of 10-fold or more were common when the major proteins of the isolated perikarya were compared with those of the other fractions. Since most of the soluble proteins extracted from ventral and dorsal roots were probably derived from the axoplasm of motor and sensory nerves, respectively, these results are interpreted to mean that large differences exist in the distribution of individual soluble proteins between the cell body and axon of spinal motoneurons, while the major soluble proteins of spinal motor and sensory axons are highly similar.

Quantitative changes in myelin proteins in a peripheral neuropathy caused by Tullidora (Karwinskia humboldtiana).

Peripheral nerve demyelination was induced in cats by oral administration of ether extracts of Tullidora (Karwinskia humboldtiana). Proteins from several hindlimb nerves, spinal roots, and dorsal columns of the spinal cord were subjected to slab gel electrophoresis and quantified by densitometry. In Tullidora-treated cats with severe motor disturbances, specific myelin proteins were reduced by at least 50% in motor nerves and less than 25% in cutaneous axons. There was a greater decrease of these proteins in the distal than in the cephalad segments of the sciatic nerve; no changes were detected either in the spinal roots or in the white matter of the spinal cord. Electron microscopy revealed intense demyelination in the motor nerves only. Both the density of the 100 A-thick neurofilaments and the relative proportion of a polypeptide with a molecular weight of 68,000 were considerably increased in the affected nerves. It is tentatively concluded that the active principles of Tullidora may enter the axons through the motor nerve terminals. The distal segments of the motor nerves would then be preferentially affected and demyelination could result from axonal damage.

The neuritogenicity and encephalitogenicity of P2 in the rat, guinea-pig and rabbit.

In inbred Lewis rats, P2 basic protein from bovine peripheral nervous system (PNS) myelin produced experimental allergic neuritis (EAN) without involvement of the brain or spinal cord. In guinea-pigs, bovine P2 did not produce EAN but large doses produced mild experimental allergic encephalomyelitis (EAE). In rabbits, bovine P2 produced both mild EAE and EAN. Human P2 produced severe EAN in the Lewis rat, but only mild EAN with quite marked EAE in the guinea-pig. Material cross-reacting with bovine P on immunodiffusion was identified in the extracts from the nerves of all three species but only in the spinal cord of the guinea-pig and rabbit, not in the rat spinal cord. The species differences in response to immunisation with P2 cannot be simply explained by the presence or absence of P2 in their PNS or CNS, but may reflect differences in the immune response.