The effect of unilateral dorsal root ganglionectomies or ventral rhizotomies on alpha 2-adrenoceptor binding to, and the substance P, enkephalin, and neurotensin content of, the cat lumbar spinal cord.

The density of alpha 2-adrenoceptor binding sites and the content of substance P, enkephalins, and neurotensin were determined in quadrants of the lumbar spinal enlargement of control cats and of cats upon which either unilateral dorsal root ganglionectomies or unilateral ventral rhizotomies had been performed. The performance of unilateral dorsal root ganglionectomies resulted in a significant decrease (45-55%) of substance P content in the ipsilateral dorsal horn 7 and 21 days postoperatively. The concentration of alpha 2-adrenoceptor binding sites ([3H]rauwolscine Bmax) in the ipsilateral dorsal horn was consistently and significantly decreased at these same postganglionectomy times (20% reduced relative to the contralateral dorsal horn). Enkephalin content 7 and 21 days after ganglionectomies was not significantly different from control, whereas the neurotensin content of the ipsilateral dorsal horn was significantly increased in the 21-day survival cats. The performance of unilateral ventral rhizotomies did not produce any statistically significant changes in the density of alpha 2-adrenoceptor binding sites or in the substance P or enkephalin content of any spinal quadrant. The neurotensin content of both the ipsilateral dorsal and ipsilateral ventral quadrants of the ventral rhizotomized cats was significantly increased. The significant decrease of alpha 2-adrenoceptor binding site concentration in the ipsilateral dorsal horn after unilateral dorsal root ganglionectomies suggests that approximately 20% of the alpha 2-adrenoceptors present within the cat lumbar spinal dorsal gray are located on the axons or terminals of primary sensory afferents. Consistent with this interpretation of the ganglionectomy results, we found significant levels of saturable [3H]rauwolscine binding to homogenates of the cat L4-Sl spinal dorsal root ganglia. Because alpha 2-adrenoceptor binding sites in the ipsilateral ventral lumbar spinal gray were not significantly reduced after unilateral ventral rhizotomies, our results provide no evidence for the location of alpha 2-adrenoceptor on lumbar spinal motoneurons.

Changes in intracellular free Ca ion concentration evoked by electrical activity in cat spinal neurons in situ.

In cats under allobarbitone anaesthesia, Ca2+-sensitive microelectrodes were inserted into the lumbosacral spinal neurons to measure intracellular free Ca2+ concentration [Ca]i. In 72 resting motoneurons, the global mean [Ca]i was 7.9 microM (SD +/- 25.9). In the 36 "best" cells (with resting and action potentials better than 60 mV), mean [Ca]i was 1.6 microM (SD +/- 1.64). Activation of motoneurons by antidromic or direct stimulation evoked mean increases in [Ca]i of about 90 nM when stimulating for 30 s at 10 Hz, and 170 nM at 20 Hz. The mean time to half-recovery was 23 s (SD +/- 14.5). Orthodromic stimulation consistently produced smaller increases in [Ca]i. Measurements in motor axons showed a comparable resting level of [Ca]i, but only minimal changes during stimulation, even at 100 Hz. Sensory axons (also recorded within the spinal cord) similarly failed to show any increase in [Ca]i during high frequency stimulation. In some interneurons, however, particularly large and rapid increases in [Ca]i could be evoked by dorsal root stimulation at 1-5 Hz. Unresponsive cells (presumably neuroglia), with a typically high and stable resting potential, had a variable [Ca]i giving a mean of 32 microM (SD +/- 63.0). A tentative theoretical analysis of the magnitude and time course of delta [Ca]i evoked in motoneurons by tetanic stimulation is consistent with remarkably slow apparent diffusion of intracellular Ca2+ (1/250 of rate of diffusion in water), such as might be expected in the presence of very efficient mechanisms of Ca2+ sequestration.

Excitatory amino acids and intracellular pH in motoneurons of the isolated frog spinal cord.

Double-barrelled pH-sensitive micro-electrodes were used to measure changes of intracellular and extracellular pH in and around motoneurons of the isolated frog spinal cord during application of excitatory amino acids. It was found that N-methyl-D-aspartate, quisqualate and kainate produced a concentration-dependent intracellular acidification. Extracellularly, triphasic pH changes (acid-alkaline-acid going pH transients) were observed during the action of these amino acids. The possible significance of such pH changes for the physiological and pathophysiological effects of excitatory amino acids are discussed.

Oxygen tension in spinal cord gray matter during exposure to hyperbaric oxygen.

Adult female rats were exposed to 60 psig of 100% oxygen for 60 minutes. Oxygen tensions in the gray matter of the lumbosacral enlargement of the spinal cord, electroenecephalograms, electrocardiograms, and respirations were monitored before, during, and after the compression periods. Oxygen tensions were found to rise sharply to as high as 1050 mm Hg during compression and remained at significantly high levels throughout the entire hour of exposure. These data support the hypothesis that spinal cord lesions induced by exposure to hyperbaric oxygen are the result of excessive tissue oxygenation.

An o-dianisidine method of horseradish peroxidase neurohistochemistry.

A new method of horseradish peroxidase (HRP) neurohistochemistry using o-dianisidine (OD) as the chromogen was described. In labelling neurones of the hypoglossal nucleus in rats and cats, the new OD method was as sensitive as Mesulam's method using tetramethyl benzidine (TMB) as the chromogen.

Serotonin-containing nerve fibers in the rat spinal cord: electron microscopic immunohistochemistry.

The ultrastructure of the serotonin (5HT) system in the spinal cord of rats was studied by an immunohistochemical peroxidase-antiperoxidase (PAP) method. Under the light microscope, 5HT immunoreactive staining was observed as brown-colored dots in the anterior horn, lateral horn, posterior horn and pericentral canal region. These positively staining dots were probably indicative of 5HT immunoreactive varicosities and nerve terminals. At the ultrastructural level, 5HT immunoreactive nerve fibers appeared as darkly stained varicosities with PAP positive large electron dense vesicles (80-100 nm), as well as small clear vesicles (30-40 nm) finely coated with PAP immunoreactive products. In the anterior horn, some of the 5HT immunoreactive structures were clearly nerve terminals forming asymmetric synaptic contact with soma or dendrites of the anterior horn cells. In the lateral horn, posterior horn and pericentral canal region, however, only 5HT positive varicosities were detected.

Localization of nRNP antigen in mammalian cells stained with peroxidase-labelled IgG fraction of anti-nRNP antibody obtained from a patient with mixed connective tissue disease (MCTD).

The peroxidase-labelled IgG fraction of anti-nRNP antibody obtained from a MCTD patient stained the nuclei of almost any kind of cells of man and rat. In addition, the cytoplasma of anterior horn cells of the rat spinal cord was exclusively reacted with this antibody. Both cytoplasmic and nuclear staining of the anterior horn cells were reduced after treatment of tissue sections with RNase.

Cytoskeletal abnormalities in motor neuron disease. An immunocytochemical study.

Immunocytochemistry with antibodies against cytoskeletal proteins has been used to search for molecular differences in the spinal cord from patients with motor neuron disease (MND) of amyotrophic lateral sclerosis type and normal spinal cord. Monoclonal antibodies which recognize phosphorylated neurofilament epitopes diffusely labelled a proportion of normal and MND anterior horn cells, but did not permit differentiation between normal and MND tissue. However, in some MND and control anterior horn cells, dense 'floccular' accumulations were labelled by antibodies recognizing phosphorylated neurofilament epitopes. These accumulations of phosphorylated neurofilaments suggest abnormalities of cytoskeletal regulation, but were neither a common nor a specific feature of MND. Axonal spheroids, which were as common in normal as in MND tissue, were labelled by all antineurofilament antibodies. Normal-appearing axons, but not spheroids, in MND and control tissue were identified by an antiactin antibody, indicating that actin may be absent from the cytoplasmic domain which gives rise to spheroids. In summary, we have not found specific posttranslational changes of cytoskeletal proteins in MND and, in particular, phosphorylated neurofilament epitopes are common to both MND and control anterior horn cells.

On the probable absence of GABA receptors on the terminations of motor axon collaterals in the cat spinal cord.

When administered microelectrophoretically, GABA and the GABA-mimetic piperidine-4-sulphonic acid (P4S) appear to have no direct hyperpolarizing or depolarizing effect on the terminations of motor axon collaterals excited electrically in the ventral horn of the lumbar spinal cord of the cat. This lack of effect on axon terminals of motoneurones, which contrasts with the bicuculline-sensitive depolarization by P4S of the spinal terminals of primary afferent fibres, is consistent with previous reports of the probable absence of pharmacologically detectable GABA receptors on the spinal terminals of other central excitatory neurones, namely those of the red and lateral vestibular nuclei.

Immunocytochemical study of Onuf's nucleus in amyotrophic lateral sclerosis.

The sacral segments of the spinal cord in 15 cases of amyotrophic lateral sclerosis and 7 control cases were investigated using peptides immunocytochemistry. A rich supply of somatostatin-, enkephalin- and substance P-immunoreactive fibers and terminals were found in common with the intermediolateral cell column in Onuf's nucleus, but not with the motor neurons in ventral horn. Furthermore, there was no significant difference with peptides innervation between ALS and control cases. Onuf's nucleus, though located in the ventral horn of the upper sacral segment, has a rich supply of peptidergic innervation which suggests that this nucleus does not represent merely somatic motor neurons, but also represents autonomic neurons. This may be related to sparing of Onuf's nucleus along with the autonomic sacral intermediolateral nucleus in ALS.

Glutamate decarboxylase activities in single vertebrate neurons.

An enzymatic microassay method for glutamate decarboxylase (GAD) and gamma-aminobutyric acid (GABA) was improved to a degree yielding high sensitivity and low blank. Single cell bodies of anterior horn cells and dorsal root ganglion cells were dissected out from the freeze-dried sections of rabbit and chicken spinal cords and Purkinje cell bodies from those of rabbit cerebellum. A minute amount of GABA, present in single neurons or synthesized by GAD in single neurons, was enzymatically converted to NADPH. The NADPH was amplified 10,000-350,000-fold and measured, using an enzymatic amplification reaction (NADP cycling). GAD was contained in all Purkinje cell bodies and its average activity was four- to fivefold higher than those of the molecular and granular layers of rabbit cerebellum. The GABA concentration was threefold higher in Purkinje cell bodies than in these layers. GAD activity, at a level similar to that in the cerebellar layers, was found in almost all the cell bodies of anterior horn cells from rabbit and chicken. GABA was detected in 40% of rabbit neurons and not in chicken neurons. Dorsal root ganglion cells from both species contained no measurable GAD or GABA.