Methodological optimization of applying neuroactive agents for the study of locomotor-like activity in the mudpuppies (Necturus maculatus).

We compared the effects of mode of delivery of neuroactive agents and the effects of dimethyl sulfoxide (DMSO), a vehicle for dissolving neuroactive agents, on locomotor-like activity in vitro. By superfusion, d-glutamate (0.3-0.9 mM) produced robust walking-like activity at superfusion rates 10-25 ml/min. In contrast, bolus application of the same or higher doses of glutamate (0.1-1.5 mM) failed to induce any rhythmic activity. Superfusion with AP-5, a NMDA receptor antagonist, produced dose-dependent inhibition of the ongoing walking-like activity induced by D-glutamate and completely blocked the activity at 20 microM. In contrast, bolus application of AP-5 did not block the walking-like activity at concentrations up to 120 microM. Similarly, superfusion of AP-5 inhibited the initiation of walking-like activity and completely blocked the initiation at 20 microM, while bolus application of AP-5 failed to do so at concentrations up to 120 microM. Superfusion of strychnine, a glycine receptor antagonist, blocked the walking-like activity at concentrations of 3-5 microM, while its bolus application altered NMDA-induced, but not glutamate-induced, walking-like activity to a synchronized pattern. DMSO significantly affected the walking-like activity in a dose-dependent manner at concentrations ranging 1-10% (v/v). These results demonstrate that the way by which the neuroactive agents are applied is a significant factor that determines the outcome of experiments on the neural control of locomotion. Also, the dose-dependent effects of DMSO on the activity of neural networks for locomotion should be taken into account in data interpretation.

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.

Morphology of brachial segments in mudpuppy (Necturus maculosus) spinal cord studied with confocal and electron microscopy.

The isolated brachial spinal cord of Necturus maculosus is useful for studies of neural networks underlying forelimb locomotion, but information about its cellular morphology is scarce. We addressed this issue by using confocal and electron microscopy. Remarkably, the central region of gray matter was aneural and consisted exclusively of a tenuous meshwork of glial fibers and large extracellular spaces. Somata of motoneurons (MNs) and interneurons (INs), labeled by retrograde transport of fluorescent tracers from ventral roots and axons in the ventrolateral funiculus, respectively, were confined within a gray neuropil layer abutting the white matter borders, whereas their dendrites projected widely throughout the white matter. About one-third of labeled INs were found contralaterally, with axons crossing ventral to a thick layer of ependyma surrounding the central canal. Lateral MN dendrites proliferated under the pial surface to form a dense, thin (1-2 microm) plexus immediately beneath a thin layer of glial fibrillary acidic protein-positive glia limitans. The latter contained arrays of unusual tubular structures (diameter 200-400 nm, length 3 microm) that resembled mitochondria but lacked double membranes or cristae. Dorsal roots (DRs) produced dense presynaptic arbors within a wedge-shaped afferent termination zone medial to the dorsal root entry, within which dendrites of MNs and INs 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 designing electrophysiological experiments to study the neural circuits involved in locomotor pattern generation.

Membrane properties of two types of basal cells in Necturus taste buds.

Necturus taste buds contain two types of basal cells: presumptive stem cells and Merkel-like basal cells. Both types of basal cells are small round cells located at the base of the taste bud, indistinguishable from each other with light microscopy. However, with electron microscopy, autoradiography, or immunocytochemistry, these two types of basal cells can be easily distinguished. We isolated basal cells from taste buds, characterized their voltage-dependent currents using gigaseal whole-cell recordings, and processed the cells for electron microscopy or immunocytochemistry. We were able to distinguish two cell types electrophysiologically and to correlate cell type with membrane properties. Isolated Merkel-like basal cells had several voltage-activated currents: transient, TTX-sensitive, inward Na+ current; sustained, saturating outward K+ current; and slowly inactivating inward Ca2+ current. These currents are similar to those observed in taste receptor cells. In contrast, presumptive stem cells from Necturus taste buds only had outward K+ currents.

Necturus gallbladder epithelial cell volume regulation and inhibitors of arachidonic acid metabolism.

Inhibition of the metabolism of arachidonic acid by the epoxygenase (cytochrome P-450) pathway with the inhibitor ketoconazole results in excessive cell swelling upon exposure to hyposmolality instead of the rapid and complete regulatory volume decrease (RVD) normally observed. NaCl entry from bathing solutions to cell interior was shown to cause this swelling, with Na influx occurring across the basolateral membrane and electrically silent Cl influx across the apical membrane. Ion substitution experiments show that the KCl efflux mediating RVD was unimpaired by ketoconazole, but was overwhelmed by the NaCl influx. Measurements of transepithelial fluid flux, Cl concentration, osmolality and pH showed that gallbladders treated with ketoconazole transiently secreted fluid rather than the normal absorption. We conclude that inhibition of arachidonic acid metabolism does not directly affect RVD by Necturus gallbladder, but that blockade of the epoxygenase pathway can have a profound influence on NaCl entry into gallbladder epithelial cells.

Electron-cytochemical localization of alkaline phosphatase to G cells of Necturus maculosus antrum.

Electron-cytochemical localization of alkaline phosphatase activity was performed on G cells of Necturus maculosus antral mucosa. Alkaline phosphatase activity was localized to the nuclear membrane, the Golgi/endoplasmic reticulum, and the limiting membranes of G cell peptide-secretion vesicles. There was no specific localization of alkaline phosphatase activity to the plasma membrane. Treatment of the tissues with levamisole (an alkaline phosphatase inhibitor) did not markedly reduce the specific alkaline phosphatase activity. Specific lead deposition was reduced by removal of the substrate from the reaction mixture. The results from this study on N. maculosus G cells demonstrate that alkaline phosphatase activity can be found in a non-mammalian gastric endocrine cell and that specific activity was localized primarily to those intracellular structures involved with protein biosynthesis.

Measurement of the effective thickness of the mucosal unstirred layer in Necturus gallbladder epithelium.

The effective thickness of the unstirred fluid layer (USL) adjacent to an epithelial barrier can be estimated from the time course for the accumulation or depletion of a solute at the membrane surface. In 1985 we reported an unstirred layer thickness of approximately 70 microns for Necturus gallbladder epithelium. In our earlier studies the delay caused by noninstantaneous bulk solution mixing was not taken into account and thus the USL thickness was systematically overestimated. In the present studies we describe an analysis of the time course of solute arrival at the membrane surface that takes into account noninstantaneous bulk solution mixing. We also describe a simple technique to monitor the accumulation or depletion of a solute at the membrane surface. The time course for the change in the concentration of either tetramethylammonium (TMA+) or tetrabutylammonium (TBA+) upon elevation of bulk solution concentration is sensed at the membrane surface with an ion-sensitive microelectrode. Because of the high selectivity of the ion-sensitive resin for TMA+ or TBA+ over other monovalent cations in the solution (Na+ and K+), a low concentration (1-2 mM) of the probe can be used. By measuring the time course of the arrival of first one probe and then the other, under identical superfusion conditions, sufficient information is obtained to eliminate multiple fits to the data, obtained when only one probe is used. Neglecting bulk solution mixing caused an error greater than 50% in estimated apparent USL thickness. The effective thickness of the USL depends critically upon chamber geometry, flow rate, and the position of superfusion and suction pipettes. Under our experimental conditions the effective USL at the mucosal surface of Necturus gallbladder epithelium was approximately 40 microns.

Ultrastructure of apical specializations of taste cells in the mudpuppy, Necturus maculosus.

The first interaction of taste stimuli with lingual chemoreceptors occurs on the apical membrane of taste cells, since only that portion is exposed to the oral cavity. To gain better insight into this interaction, we examined the pore region of taste buds in Necturus maculosus with scanning electron microscopy (SEM), transmission electron microscopy, and high-voltage electron microscopy. SEM of the pore reveals a patchwork distribution of three morphologically distinct types of apical specializations: long and branched (LB) microvilli, short and unbranched (SU) microvilli, and bundles of stereocilia. As demonstrated in thin and thick sections, LB microvilli are specializations of dark cells, SU microvilli are the apical specializations of light cells, and stereocilia arise from a cell that has the cytoplasmic markers characteristic of light cells. When left in place, the pore mucus completely covers the SU microvilli and partially covers the LB microvilli. However, stereocilia project above the surface and thus are highly exposed to taste stimuli in the oral cavity. These three morphologically distinct types of apical specializations may reveal functional differences among taste cells. The initial interaction between chemical stimulus and taste cell, and possibly chemoreceptor specificity itself, may be influenced by the morphology of the apical ending.

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)

Seasonal variations in the fine structure of the Necturus maculosus urinary bladder epithelium: low transporters and high transporters.

Although the urinary bladder of Necturus maculosus provides an important model system for studying the mechanisms of active Na absorption, little critical attention has been paid to the fine structure of its epithelium. Moreover, two distinct groups of urinary bladders, low and high Na transporters, have been described based on short-circuit current or transepithelial potential difference. In the present study, over an 11-month period, stable electrical parameters (short-circuit current, transepithelial potential difference, and resistance) were recorded from 63 chamber-mounted bladders. Analysis of these parameters revealed a highly significant difference between two groups (low transporters and high transporters) occurring at different times of the year. Consistent with these data, in urine collected from the bladders, the Na concentration in low transporters was significantly higher than that in high transporters. A subpopulation of these bladders was subsequently fixed and examined at the light and/or electron microscopic level. Low-transporting bladders were characterized unequivocally by a thin, stratified squamous epithelium only 6-15 micron thick. High-transporting bladders were composed predominantly of columnar-shaped granular cells up to 70 micron in height, with ciliated, mitochondria-rich, and basal cells present in small numbers. There is thus a correlation between transport activity, as measured by electrophysiological techniques and urine sodium analysis, and the structure of the tissue. Moreover, these parameters exhibit significant seasonal variation, the underlying mechanisms of which remain obscure.

Electron microscopic study of the innervation of the renal tubules and urinary bladder epithelium in Rana catesbeiana and Necturus maculosus.

The fine structure of the kidney and the bladder of the bullfrog (Rana catesbeiana), the bullfrog tadpole, and the mudpuppy (Necturus maculosus) were studied with special attention to the innervation of renal tubule cells and bladder epithelial cells. In the bullfrog kidney, nerve terminals and varicosities were frequently associated with the tubule cells, apparently in an increasing order from the proximal tubule to the connecting tubule. Although these terminals and varicosities did not directly contact the tubular cell membrane, an aggregation of synaptic vesicles on the side facing the tubule was considered as morphological evidence that neurotransmitter can be released here and can affect the transport activity of the tubule cells. The association of nerve varicosities with canaliculi cells in the connecting tubule was also demonstrated. In the bullfrog tadpoles, renal tubule cells were occasionally innervated. In the mudpuppy, renal tubule cells were only poorly innervated. The epithelium of the bullfrog bladder was commonly innervated. Nerve terminals with synaptic vesicles were located very near basal cells and even contacted them directly on rare occasions. In the mudpuppy, the innervation of the bladder epithelium was observed infrequently. The bullfrog tadpoles did not possess an apparent bladder. In all materials studied, renal arterioles and bladder smooth muscle cells were innervated.

Hydrostatic pressure changes related to paracellular shunt ultrastructure in proximal tubule.

We examined the effets of changes in hydrostatic pressures on the ultrastructural geometry of the lateral intercellular space and tight junctions in proximal tubules of contrtol (C) and volume-expanded (VE) Necturus kidney. The following groups of tubules were studied: (1) C, free-flow pressure, (2) C, stopped-flow, high-luminal pressure, (3) C, stopped-flow, low-luminal pressure, (4) VE, free-flow pressure, and (5) VE, stopped-flow, high-luminal pressure. Intratubular and peritubular capillary pressures were monitored before and during standardized perfusion-fixation for electron microscopy, and complete cross-sections of all sampled tubules were subjected to morphometric analysis. Average lateral intercellular space widths decreased significantly in C and VE stopped-flow tubules with high-luminal pressures but widened greatly in C stopped-flow tubules with low-luminal pressures. The length or width of the tight junctions did not change between the five experimental conditions. The ultrastructural changes correlate with the applied transepithelial pressure gradients rather than with transepithelial volume fluxes. The narrowing of lateral intercellular spaces in high pressure tubules correlate with the previously described increase in electrical resistance expressed per unit length tubule indicating that in these conditions part of the paracellular resistance is located in the free interspaces. The geometry of the lateral intercellular space in the proximal tubule of Necturus favors models of near-isotonic transport that do not depend on long and narrow interspaces.