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Anatomical organization of motoneurons and interneurons in the mudpuppy (Necturus maculosus) brachial spinal cord: the neural substrate for central pattern generation.

The isolated brachial spinal cord of the mudpuppy is useful for studies of neural networks underlying forelimb locomotion, but information about its anatomy is scarce. We addressed this issue by combining retrograde labeling with fluorescent tracers and confocal microscopy. Remarkably, the central region of gray matter was aneural and contained only a tenuous meshwork of glial fibers and large extracellular spaces. Somata of motoneurons (MNs) and interneurons (INs), labeled retrogradely from ventral roots or axons in the ventro-lateral funiculus, respectively, were confined within a gray neuropil layer abutting the white matter borders, while their dendrites projected widely throughout the white matter. A considerable fraction of labeled INs was found contralaterally with axons crossing beneath a thick layer of ependyma surrounding the central canal. Dorsal roots (DRs) produced dense presynaptic arbors within a restricted dorsal region containing afferent terminations, within which dorsally directed MN and IN dendrites mingled with dense collections of synaptic boutons. Our data suggest that a major fraction of synaptic interactions takes place within the white matter. This study provides a detailed foundation for electrophysiological experiments aimed at elucidating the neural circuits involved in locomotor pattern generation.

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The organization of the motoneurons innervating the axial musculature of vertebrates. I. Goldfish (Carassius auratus) and mudpuppies (Necturus maculosus).

The motoneurons innervating different regions of the myomeres in goldfish and mudpuppies were examined by applying HRP to the musculature or to branches of spinal nerves. In goldfish, the populations of motoneurons innervating epaxial or hypaxial muscle occupied similar positions in the motor column and had similar size distributions. There was no relationship between the size or location of a motoneuron in the motor column and the dorsoventral location of the muscle it innervated in the myomeres. Instead, different populations of motoneurons innervated the functionally different red and white musculature. The red muscle was innervated only by small motoneurons that occupied the ventral portion of the motor column. Their small axons passed lateral to the Mauthner axon in the cord, and most of them traveled in a separate branch of each spinal nerve that ran in the horizontal septum to the red muscle. The white muscle was innervated by a population of motoneurons that did not innervate red. They were large and they occupied a characteristic position in the extreme dorsal part of the motor column. Their large axons traveled medial to the Mauthner axon in the cord and entered branches of spinal nerves running deep in the epaxial or hypaxial muscle. The white muscle was probably also innervated by some smaller motoneurons similar to those innervating red; however, these may have been motoneurons whose axons ran through white muscle to reach other muscle. The large motoneurons innervating only white muscle are similar to the primary motoneurons identified in developmental studies in teleosts (Myers: Soc. Neurosci. Abstr. 9:848, '83); the smaller ones, innervating both red and white, are like secondary motoneurons. Therefore, in goldfish, motoneurons having different morphology and developmental history also innervate different regions in the myomeres. The motor column in mudpuppies was, in general respects, similar to the column in goldfish. There were large primary motoneurons and small secondary ones. Though there were slight differences in the locations of motoneurons filled from nerves entering epaxial and hypaxial muscle, their distributions in the cord overlapped substantially. The motor columns in these two anamniotes differ substantially from the motor columns in those amniotes that have been studied. In amniotes, the motoneurons innervating epaxial and hypaxial muscles are spatially segregated in the cord (Smith and Hollyday: J. Comp. Neurol. 220:16-28, '83; Fetcho: J. Comp. Neurol. 249:551-563, '86).(ABSTRACT TRUNCATED AT 400 WORDS)

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[Effects of salicylate on the electrical properties of the proximal convoluted tubule of Necturus maculosus].

The effects of peritubular salicylate for chloride substitution were studied in the isolated perfused Necturus kidney. This substitution resulted in changes of cell membrane p.d., varying from tubule to tubule; the withdrawal of the test-anion invariably produced a steep and prolonged depolarization. Exposure of the tissue to salicylate brought about, in addition, electrical uncoupling of junctional membranes, which was not related to concomitant changes of membrane p.d.

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Morphological changes in tight junctions of Necturus maculosus proximal tubules undergoing saline diuresis.

Tight junctions between epithelial cells are believed to control the paracellular diffusion of substances across epithelia. Epithelia in which tight junctions are poorly developed display a higher paracellular electrical conductance, while those with extensive tight junctions show lower conductance values. We described here a particular epithelium, that of the proximal tubules of the Necturus kidney, in which the development of the tight junctions varies in parallel with a change of paracellular electrical conductance. In control conditions, tight junctions between epithelial cells of the proximal tubules are more developed than in tubules undergoing saline diuresis, a situation which increases the conductance across the paracellular shunt pathway.

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Distribution of monoamine oxidase activity in tissues of the urodeles Ambystoma tigrinum (tiger salamander) and Necturus maculosus (mudpuppy).

1. Monoamine oxidase (MAO) activity was determined fluorometrically in tissues of adult mudpuppies, and pre- (young) and post- (adult) metamorphic tiger salamanders. 2. From responses to specific inhibitors it was determined that 95% activity was MAO type A in all tissues. 3. In young salamanders MAO activity was greater in brain and intestine of males than of females, and was considerably higher in kidney of both sexes and in intestine of males compared to adults. 4. MAO activity was distributed differently in the mudpuppy compared to the salamander. Intestine and liver contained high activity and brain had relatively little MAO activity compared to salamander. 5. The apparent Michaelis constant of MAO activity in the different groups and tissues was generally similar, suggesting a similarity of the MAO molecule.

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Histamine and histamine receptors: behavioral thermoregulation in the salamander Necturus maculosus.

Low doses (0.01, 0.1 mg/kg, i.p.) of histamine (HA) caused selection of significantly lower temperatures, and higher doses (0.5, 1.0 mg/kg) increased temperatures by mudpuppies in linear thermal gradients. Injection of the HA precursor, L-histidine (500 mg/kg) produced an increase in the temperatures selected. Results from injections of HA H1-receptor agonist (2-pyridylethylamine) and antagonist (pyrilamine), and H2-receptor agonist (dimaprit) and antagonist (cimetidine) had significant effects on thermoregulation; H1-receptors may mediate behavioral hyperthermia and H2-receptors behavioral hypothermia. Responses to these histaminic compounds are significantly influenced by the time of day at which the responses are measured and by season and acclimation temperature. The equivalent behavioral responses in both endotherms and ectotherms to agents which produce physiological hyperthermia and hypothermia are probably behavioral hypothermia ("cold seeking") and behavioral hyperthermia ("heat seeking"), respectively.

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Electron microscopic immunocytochemistry of glutamate-containing nerve fibers in the taste bud of mudpuppy (Necturus maculosus).

The presence of glutamate immunoreactivity (glu-IR) in the nerve fibers of the mudpuppy taste bud was investigated by electron microscopy. Pre-embedding staining with avidin-biotin-peroxidase complex (ABC) and post-embedding staining with 5 nm colloid gold conjugates were used separately to identify immuno-stained structures. We have found the following: 1) the majority of the nerve fibers innervating the mudpuppy taste bud are unmyelinated; 2) about 85% of nerve fibers located at the base of the taste bud and about 60% of the nerve fibers located between the taste cells show glu-IR by pre-embedding staining; 3) there is a preferential staining of the glu-IR in the nerve fibers of the mudpuppy taste bud; and 4) the distribution of the colloidal gold particles in the nerve fibers is 1.5 to 2 times denser than that of the staining in the connective tissue background or cellular profiles of taste cells. From the distribution and pattern of the nerve fibers obtained in the thick and thin sections, we conclude that the mudpuppy taste bud is innervated by glutamate-containing unmyelinated nerve fibers.

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Dye-coupling in taste buds in the mudpuppy, Necturus maculosus.

Electrical coupling in taste buds and in non-taste lingual epithelium in the mudpuppy was examined by injecting cells with a fluorescent dye, Lucifer yellow. Lucifer yellow coupling has been shown to indicate the presence of electrical junctions between cells. Lucifer yellow-filled taste cells usually have an elongate shape. Cells were an average of 111 microns long and were 13 microns in diameter at the widest region (nucleus). In taste buds, from a sample of 105 impalements we detected Lucifer yellow coupling in 21 cases: dye-coupled pairs of cells were observed in 17 cases, and trios of cells in 4 cases. Larger subsets of coupled cells (greater than 3) were not observed. Dye-coupled cells were usually equally intensely stained. In non-taste epithelium, we examined dye-coupling in the superficial and basal layers. Extensive Lucifer yellow coupling was found in the basal layer (15/15 cases). The number of cells coupled to the dye-injected cell varied from 3 to 5. In the superficial epithelium, dye-coupling was rare (1/45 cases). No dye-coupling was observed between epithelial cells and taste cells at the taste pore region. We conclude that strong electrical coupling in groups of 2-3 cells occurs in the mudpuppy taste buds. Coupling may occur selectively between identical types of taste cells (dark, light, etc.), but this remains to be determined. Electrical coupling also exists among basal epithelial cells but not in the superficial epithelial layers.

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