Neural computations in the tiger salamander and mudpuppy outer retinae and an analysis of GABA action from horizontal cells.

A neural network architecture based on the neural anatomy and function of retinal neurons in tiger salamander and mudpuppy retinae is proposed to study basic aspects of early visual information processing. The model predictions for the main response characteristics of retinal neurons are found to be in agreement with neurophysiological data, including the antagonistic role of horizontal cells in the outer plexiform layer. The examination of possible gamma-aminobutyric acid (GABA) action from horizontal cells suggests that GABA(A) alone, GABA(B) alone, or their weighted combination can generate the response characteristics observed in bipolar cells.

Electrophysiology of Necturus taste cells.

Taste buds are sensory end organs that detect chemical substances occurring in foodstuffs and relay the relative information to the brain. The mechanisms by which the chemical stimuli are converted into biological signals represent a central issue in taste research. Our understanding of how taste buds accomplish this operation relies on the detailed knowledge of the biological properties of taste bud cells-the taste cells-and of the functional processes occurring in these cells during chemostimulation. The amphibian Necturus maculosus (mudpuppy) has proven to be a very useful model for studying basic cellular processes of vertebrate taste reception, some of which are still awaiting to be explored in mammals. The main advantages offered by Necturus are the large size of its taste cells and the relative accessibility of its taste buds, which can therefore be handled easily for experimental manipulations. In this review, I summarize the functional properties of Necturus taste cells studied with electrophysiological techniques (intracellular recordings and patch-clamp recordings). My focus is on ion channels in taste cells and on their role in signal transduction, as well as on the functional relationships among the cells inside Necturus taste buds. This information has revealed to be well suited to outline some of the general physiological processes occurring during taste reception in vertebrates, including mammals, and may represent a useful framework for understanding how taste buds work.

Structure and functional connections of presynaptic terminals in the vertebrate retina revealed by activity-dependent dyes and confocal microscopy.

The fluorescent dyes sulforhodamine 101 (SR 101) and FM1-43 were used as activity-dependent dyes (ADDs) to label presynaptic terminals in the retinas of a broad range of animals, including amphibians, mammals, fish, and turtles. The pattern of dye uptake was studied in live retinal preparations by using brightfield, fluorescence, and confocal microscopy. When bath-applied to the retina-eyecup, these dyes were avidly sequestered by the presynaptic terminals of virtually all rods, cones, and bipolar and amacrine cells; ganglion cell dendrites and horizontal cells lacked significant dye accumulation. Other structures stained with these dyes included pigment epithelial cells, cone outer segments, and Müller cell end-feet. Studies of dye uptake in dark- and light-adapted preparations showed significant differences in the dye accumulation pattern in the inner plexiform layer (IPL), suggesting a dynamic, light-modulated control of endocytotic activity. Presynaptic terminals in the IPL could be segregated on the basis of volume: bipolar varicosities in the IPL were typically larger than those of amacrine cells. The combination of retrograde labeling of ganglion cells and presynaptic terminal labeling with ADDs served as the experimental preparation for three-dimensional reconstruction of both structures, based on dual detector, confocal microscopy. Our results demonstrate a new approach for studying synaptic interactions in retinal function. These findings provide new insights into the likely number and position of functional connections from amacrine and bipolar cell terminals onto ganglion cell dendrites.

Effects of inhibitory neurotransmitters on the mudpuppy (Necturus maculatus) locomotor pattern in vitro.

Effects of inhibitory neurotransmitters on the locomotor rhythm and pattern generation were investigated using an in vitro preparation isolated from the mudpuppy (Necturus maculatus). The preparation consisted of the first five segments of the spinal cord and the right forelimb attached by the brachial nerves. During N-methyl-d-aspartate (NMDA)-induced locomotion, the rhythmic motor output (EMG) was recorded unilaterally from elbow flexor and extensor muscles. While neither glycine nor gamma-aminobutyric acid (GABA)-related substances induced locomotion in the absence of NMDA, they modulated NMDA-induced locomotion. Bath application of glycine and GABA suppressed the rhythmic motor pattern induced by NMDA. Addition of glycine receptor antagonist strychnine or GABA(A) receptor antagonist bicuculline disrupted the phase relationship between antagonistic motor pools during ongoing locomotion, thereby changing the normal alternating pattern into synchronous EMG bursts. Both the GABA(A) receptor agonist muscimol and GABA(B) receptor agonist baclofen mimicked the effects of GABA as they either slowed down or stopped locomotion. Nipecotic acid, a GABA uptake blocker, had a similar effect. This suggested that an endogenous release of GABA modulated the locomotor rhythm. The endogenous release was antagonized by the GABA(A) and GABA(B) receptor antagonists bicuculline and CGP-35348, respectively. Immunocytochemistry revealed that glycine and GABA-positive neurons and fibers were present in mudpuppy spinal cord. Although the GABAergic neurons were more numerous than glycinergic neurons, both cell types contributed processes directed towards the white matter and occasionally towards the ependymal lining of the central canal. Our results suggest that inhibitory neurotransmitters exert powerful actions upon the neuronal network governing forelimb locomotion in the mudpuppy. The effects we observed may be mediated by a network of segmentally distributed glycinergic and GABAergic spinal neurons.

Identification, localization, and modulation of neural networks for walking in the mudpuppy (Necturus maculatus) spinal cord.

We tested the hypothesis that the neural networks for walking in the mudpuppy can be divided into a flexor and an extensor center, each of which contains collections of interneurons localized in the vicinity of their motoneuron pools. Combining a battery of techniques, we identified and localized the elbow flexor center and its motoneuron pool in the C2 segment and the elbow extensor center and its motoneuron pool in the C3 segment. Rhythmic flexion or extension of the limb in isolation could be induced by continuous trains of current pulses of the C2 or C3 segments, respectively. Independent activation could also occur after application of glutamate receptor agonist NMDA. Part of segment C2 in isolation generated rhythmic elbow flexor bursts, whereas part of segment C3 in isolation generated rhythmic elbow extensor bursts. An isolated region spanning the C3 roots generated both flexor and extensor bursts. The step cycle was modulated in a phase-dependent manner by stimulation of the dorsal roots, the ventral roots, or either of the two centers. The effects of ventral root stimulation were removed by deafferentation to block reafferent input attributable to muscle contraction induced by the stimulation. We conclude that the neural networks for walking contain at least a flexor and an extensor generator that are localized in close apposition to the motoneuron pools, that the two centers can work independently despite the fact that there are reciprocal inhibitory interconnections between them, and that sensory input interacts with the spinal neural networks to reset the ongoing walking rhythm in a phase-dependent manner.

Serotonin modulates voltage-dependent calcium current in Necturus taste cells.

Necturus taste buds contain two primary cell types: taste receptor cells and basal cells. Merkel-like basal cells are a subset of basal cells that form chemical synapses with taste receptor cells and with innervating nerve fibers. Although Merkel-like basal cells cannot interact directly with taste stimuli, recent studies have shown that Merkel-like basal cells contain serotonin (5-HT), which may be released onto taste receptor cells in response to taste stimulation. With the use of whole cell voltage clamp, we examined whether focal applications of 5-HT to isolated taste receptor cells affected voltage-activated calcium current (I(Ca)). Two different effects were observed. 5-HT at 100 microM increased I(Ca) in 33% of taste receptor cells, whereas it decreased I(Ca) in 67%. Both responses used a 5-HT receptor subtype with a pharmacological profile similar to that of the 5-HT1A receptor, but the potentiation and inhibition of I(Ca) by 5-HT were mediated by two different second-messenger cascades. The results indicate that functional subtypes of taste receptor cells, earlier defined only by their sensitivity to taste stimuli, may also be defined by their response to the neurotransmitter 5-HT and suggest that 5-HT released by Merkel-like basal cells could modulate taste receptor function.

Development of voltage-dependent currents in taste receptor cells.

Taste buds, the specialized end organs of gustation, comprise a renewing sensory epithelium. Undifferentiated basal cells become taste receptor cells by elongating and extending processes apically toward the taste pore. Mature taste cells are electrically excitable and express voltage-dependent Na+ Ca2+, and K+ currents, whereas basal stem cells exhibit only slowly activating K+ currents. The question we have addressed in the present study is whether contact with the taste pore is required for expression of voltage-dependent inward currents in Necturus taste cells. Mature taste cells were distinguished from developing cells by labeling the apical surface of the cells with fluorescein-isothiocyanate-conjugated wheat germ agglutinin (FITC-WGA), while the tissue was still intact. Elongate cells lacking FITC-WGA staining were interpreted as developing taste cells that had not yet reached the taste pore. Giga-seal whole-cell recording revealed that most developing taste cells lacked inward currents. Although some developing cells expressed inward currents, they were much smaller than those of mature cells, and the activation kinetics of the K+ currents were slower than in mature cells. Electron microscopy confirmed the identity of labeled and unlabeled cells. All FITC-WGA-labeled cells exhibited the ultrastructural characteristics of mature taste receptor cells, whereas most unlabeled taste cells had a characteristic morphology that was markedly different from mature taste receptor cells or basal stem cells. These data suggest that contact with the taste pore is required for the development of inward currents in taste cells.

Electrophysiological and morphological properties of light and dark cells isolated from mudpuppy taste buds.

Isolated Necturus taste receptor cells were studied by giga-seal whole-cell recording and electron microscopy to correlate electrophysiological properties with taste cell structural features. Dark (type I) cells were identified by the presence of dense granular packets in the supranuclear and apical regions of the cytoplasm. In response to a series of depolarizing voltage commands from a holding potential of -80 mV, these cells exhibited a transient, TTX-sensitive inward Na+ current, a sustained outward K+ current, and a slowly inactivating inward Ca++ current. Light (type II) cells were identified by a lack of granular packets and by an abundance of smooth endoplasmic reticulum distributed throughout the cell. In addition, isolated light cells had clear vesicular inclusions in the cytoplasm and blebs on the plasma membrane. Light cells were divided into two functional populations based upon electrophysiological criteria: cells with inward and outward currents, and cells with outward currents only. Light cells with inward and outward currents had voltage-activated Na+, K+, and Ca++ currents with properties similar to those of dark cells. In contrast, the second group of light cells had only voltage-activated outward K+ currents in response to depolarizing voltage commands. These data suggest that dark cells and light cells with inward and outward currents are capable of generating action potentials and releasing neurotransmitters onto gustatory afferent neurons in response to taste stimulation. In contrast, light cells with outward currents only likely serve a different function in the taste bud.

Ca(2+)-dependent Cl- conductance in taste cells from Necturus.

1. Taste responses adapt to a constant chemical stimulus. The present study describes a new ionic conductance in taste cells--a Ca(2+)-dependent anion conductance that may explain taste adaptation. 2. Patch-clamp recordings were made on isolated Necturus taste cells or on taste cells in lingual slices. When Na+ and K+ currents were eliminated with tetrodotoxin (TTX) and tetraethyl-ammonium (TEA) in the bath and replacing K+ with N-methyl-D-glucamine (NMDG+) in the pipette, Ca2+ currents were followed by prolonged outward currents. Outward current was abolished when Ca2+ was substituted with Ba2+ or when Cl- was replaced with large organic anions (methanesulfonate, isethionate, or ascorbate). 3. The outward, Ca-dependent current was reduced by certain agents that block Cl- conductances in other tissues, namely 4-acet-amido-4-isothiocyanostilbene-2,2-disulfonic acid (SITS) and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS). However, other Cl- channel blockers--9-AC, furosemide and an antibody to Cl channels-had little or no specific effect on the Ca-dependent outward current in Necturus taste cells. 4. We postulate that the biological action of this Ca-dependent anion conductance in situ is to terminate depolarizing receptor potentials, even during maintained chemostimulation, thereby playing an important role in chemosensory adaptation and modulation of impulse discharge patterns in taste buds.

The activity of interneurons during locomotion in the in vitro necturus spinal cord.

1. Less than two segments of the cervical spinal cord of the mudpuppy (Necturus maculatus) is sufficient to generate a locomotor rhythm with application of N-methyl-D-aspartic acid (NMDA). We have recorded intracellularly from rhythmically active interneurons in these segments and classified them according to their phase of firing within the step cycle and their afferent input. 2. Four classes of interneurons were found: flexor, flexor-->extensor, extensor, and extensor-->flexor. Interneurons that burst during the transition from flexion to extension or vice versa are referred to as "transitional" interneurons and represent the majority (68%) of rhythmically active interneurons studied in the mudpuppy spinal cord. 3. All flexor interneurons received only inhibitory input from cutaneous and dorsal root afferents, whereas the flexor-->extensor interneurons that responded received only excitatory input from dorsal root and cutaneous afferents. All extensor interneurons and all but one extensor-->flexor interneuron received no afferent input from the cutaneous or dorsal root afferents we stimulated. 4. Other interneurons have been classified as "tonic" cells. They fire continuously when the mudpuppy is walking and are silent when the mudpuppy is not walking. These interneurons receive no afferent input from the sources tested and may be responsible for turning locomotion on and off. 5. In conclusion, the presence of many transitional interneurons with specific patterns of afferent input may be required for the phasing of legged locomotion. We believe the in vitro preparation of the mudpuppy spinal cord and forelimb is an excellent model for studying the firing properties of interneurons during legged locomotion.

Convective fluid flow through the paracellular system of Necturus gall-bladder epithelium as revealed by dextran probes.

1. Bidirectional paracellular fluxes using radioactive dextrans as inert molecular probes have been measured across Necturus gall-bladder epithelium during conditions of normal fluid absorption. There is a net flux at all radii analysed (0.4-2.2 nm) in the direction of fluid absorption. 2. The net flux is substantial at all radii within the range. The data extraplate to 2 x 10(-6) cm s-1 at zero probe radius, which is very close to the rate of epithelial fluid absorption. 3. The unstirred layers at the epithelial faces during transport have been determined; their contribution to the net fluxes is negligible. 4. Two possible mechanisms for the net flow of probes are considered: (i) that the probes diffuse across the junctions and are then entrained in a local osmotic flow along the interspaces and subepithelium; (ii) that the probes are entrained in volume flow across the junctions and the emergent solution subsequently passes through the interspaces and subepithelium. Model calculations clearly rule out mechanism (i) in which the maximum net flow obtainable is less than 10% of that observed. In addition the presence of leak paths shunting the junctions is not compatible with the observed fluxes. With mechanism (ii) the net flows are correctly predicted with all the fluid flow being transjunctional. The fluid absorption is therefore entirely paracellular. 5. The slope of the net flow curve shows no apparent change in magnitude over the range of the probe radii, indicating that effectively only one population of convective channels is present with parallel walls separated by about 7.7 nm. This agrees with the width previously determined by electron microscopy. 6. If the fluid absorption is junctional then the cellular route offers little if any relative contribution. The hydraulic conductivity of the junctions is not high enough, or the osmotic permeability of the membranes low enough, to accommodate this by osmosis and therefore the junctional fluid absorption must be non-osmotic.

Properties of synaptic transmission from photoreceptors to bipolar cells in the mudpuppy retina.

1. Simultaneous, whole-cell recordings were obtained from synaptically coupled photoreceptor/bipolar cell pairs, by the use of direct visualization in a superfused, mudpuppy retinal slice preparation. 2. OFF-bipolar cells (BPs) generated sign-conserving responses when extrinsic current was injected into rods and cones, whereas ON-BPs generated a sign-reversing response. OFF-BPs (n = 24) responded faster than ON-BPs (n = 12), in terms of response latency (27.8 vs. 80.6 ms) and peak response times (50.5 vs. 159.8 ms) when current was injected into photoreceptors. We did not detect any significant difference between rod- versus cone-mediated latency or peak response times in the ON- and OFF-BP subtypes. 3. Rod and cone inputs to OFF-BPs were blocked by kynurenic acid (Kyn), but the doses required were significantly higher for rod inputs: the IC50 (the concentration at which an antagonist blocks 50% of the responses) for Kyn was 0.3 mM for cone inputs and 1 mM for rod inputs. 4. Rod inputs to OFF-BPs showed the same Kyn sensitivity as rod inputs to horizontal cells (HCs). However, cone inputs to HCs (IC50 < 200 microM) were more sensitive to Kyn than those to OFF-BPs. 5. The pharmacological studies presented here, together with previous studies, suggest that the sign-conserving pathway in the outer plexiform layer of the mudpuppy retina involves at least three subtypes of glutamate receptors: 1) cone-activated receptors of HCs; 2) cone-activated receptors of OFF-BPs; and 3) rod-activated receptors found in HCs and BPs.(ABSTRACT TRUNCATED AT 250 WORDS)

An intensity-dependent biphasic neuron in mudpuppy retina.

Intracellular recordings in dark-adapted mudpuppy retinas have revealed a type of infrequently encountered cell with unusual response properties. These cells may be a subclass of horizontal cell since they are encountered at the same depth as horizontal cells and have large receptive fields and response amplitudes. However, they differ from typical horizontal cells in that they are depolarized by low intensity illumination and hyperpolarized by higher intensity illumination at all wavelengths. Both types of responses appear to be driven mainly by 572 nm cones. Both the depolarizing and hyperpolarizing responses were unaffected by APB, indicating that they are not mediated by on-center bipolar cells.

An in vitro preparation of the mudpuppy for simultaneous intracellular and electromyographic recording during locomotion.

In this report we describe the development of an in vitro preparation of the mudpuppy (Necturus maculatus) used to investigate locomotion in walking vertebrates. The preparation consists of the first 5 segments of the cervical spinal cord and the attached forelimb. The preparation is bathed in a cooled (15 degrees C) and oxygenated spinal cord Ringers solution and remains viable for 36-100 h. Locomotion can be elicited during the first 36-48 h by applying the excitatory amino acid N-methyl D-aspartate (NMDA) to the bath. Cutaneous and dorsal root reflexes remain unchanged for much longer periods of time (72-100 h). During locomotion, intracellular recordings can be made from interneurons and motoneurons while simultaneous electromyographic (EMG) recordings are made from forelimb muscles. Rhythmically active interneurons can be classified according to their phase of activity during the step cycle. Further classification of interneurons involves both monitoring the afferent input to these cells from dorsal root and cutaneous afferents as well as using their action potentials as a trigger for averaging the ongoing locomotor EMG activity. In this way some of the input and output characteristics of the interneurons can be monitored. The ability to record simultaneously from interneurons and muscles offers distinct advantages over current in vitro preparations.

Apical K+ channels in Necturus taste cells. Modulation by intracellular factors and taste stimuli.

The apically restricted, voltage-dependent K+ conductance of Necturus taste receptor cells was studied using cell-attached, inside-out and outside-out configurations of the patch-clamp recording technique. Patches from the apical membrane typically contained many channels with unitary conductances ranging from 30 to 175 pS in symmetrical K+ solutions. Channel density was so high that unitary currents could be resolved only at negative voltages; at positive voltages patch recordings resembled whole-cell recordings. These multi-channel patches had a small but significant resting conductance that was strongly activated by depolarization. Patch current was highly K+ selective, with a PK/PNa ratio of 28. Patches containing single K+ channels were obtained by allowing the apical membrane to redistribute into the basolateral membrane with time. Two types of K+ channels were observed in isolation. Ca(2+)-dependent channels of large conductance (135-175 pS) were activated in cell-attached patches by strong depolarization, with a half-activation voltage of approximately -10 mV. An ATP-blocked K+ channel of 100 pS was activated in cell-attached patches by weak depolarization, with a half-activation voltage of approximately -47 mV. All apical K+ channels were blocked by the sour taste stimulus citric acid directly applied to outside-out and perfused cell-attached patches. The bitter stimulus quinine also blocked all channels when applied directly by altering channel gating to reduce the open probability. When quinine was applied extracellularly only to the membrane outside the patch pipette and also to inside-out patches, it produced a flickery block. Thus, sour and bitter taste stimuli appear to block the same apical K+ channels via different mechanisms to produce depolarizing receptor potentials.

A peptide from the Drosophila Shaker K+ channel inhibits a voltage-gated K+ channel in basolateral membranes of Necturus enterocytes.

A synthetic peptide composed of the first 22 amino acid residues of the Drosophila Shaker K+ channel inhibits a voltage-gated K+ channel in basolateral membrane vesicles from Necturus enterocytes reconstituted in planar phospholipid bilayers when added to the solution bathing the inner surface of this channel but not when added to the solution bathing its outer surface. A modified peptide in which the leucine in the 7 position is replaced with phenylalanine is also an effective inhibitor, but replacement of the leucine-7 with lysine or glutamate, or digestion with trypsin, renders the peptide ineffective; replacement of the leucine-7 with glycine markedly reduces but does not abolish the effectiveness of the peptide as an inhibitor. These results are analogous to those reported for the Shaker K+ channel +ADHoshi, T., Zagotta, W.N. & Aldrich, R.W. (1990) Science 250, 533-538; and Zagotta, W.N., Hoshi, T. & Aldrich, R.W. (1990) Science 250, 568-571.+BD and suggest that the molecular anatomy of the receptor at the inner face of the Necturus K+ channel with which the peptide interacts to bring about inhibition of that channel may be similar to that of the Shaker K+ channel.

Intracellular pH in isolated Necturus duodenal mucosa exposed to luminal acid.

Regulation of intracellular pH (pHi) and its maintenance within physiological ranges during exposure to luminal acid was studied in isolated Necturus duodenal mucosa using liquid sensor microelectrodes. Exposure of the mucosa to luminal pH 2.7 caused significant intraepithelial acidification. Subsequent removal of HCO3-/CO2 (HEPES/O2 substitution) from the serosal perfusate caused a further decrease of pHi. Blocking of HCO3- transport across the basolateral cell membrane by addition of 4-acetamido-4,isothiosyanostilbene-2,2-disulfonic acid (SITS) to serosal perfusate also caused a slight but significant decrease of pHi. Removal of Na+ (choline substitution) from the serosal perfusate during acid exposure likewise caused a significant decrease in pHi, as did serosal addition of an inhibitor of Na+/H+ antiport, 1 mmol/L amiloride. When Na+ was removed from the serosal perfusate after HCO3- removal, pHi first rapidly acidified; this was followed after an initial 5-minute steady state by an uncontrolled progressive acidification at a rate of 0.33 pH unit/15 min without any further steady state. A similar but weaker effect could also be shown with amiloride addition. The epithelial surface pH was 7.13 +/- 0.08 at the apex of mucosal villus and 7.42 +/- 0.11 (n = 5) in the cryptal area between the villi, i.e., greater than 1 pH unit higher than that of the luminal bulk solution (pH 6), thus suggesting active alkalization of the epithelial surface. Removal of serosal HCO3-/CO2 decreased surface pH significantly both at the villus apex and at the cryptal area, suggesting that the surface alkalization is mediated by transport of serosal HCO3- to the epithelial surface. The data suggest that pHi in acid-exposed duodenal mucosa is primarily maintained within physiological range by an HCO3(-)-dependent mechanism, which, at least in part, exerts its action extracellularly by forming an alkaline buffer layer at the epithelial surface. If adequate serosal (or systemic) HCO3- is not available, a second-line Na(+)-dependent and amiloride-sensitive pHi-regulatory mechanism, presumably an Na+/H+ antiport, becomes the main regulator of pHi.

Electrophysiological effects of basolateral [Na+] in Necturus gallbladder epithelium.

In Necturus gallbladder epithelium, lowering serosal [Na+] ([Na+]s) reversibly hyperpolarized the basolateral cell membrane voltage (Vcs) and reduced the fractional resistance of the apical membrane (fRa). Previous results have suggested that there is no sizable basolateral Na+ conductance and that there are apical Ca(2+)-activated K+ channels. Here, we studied the mechanisms of the electrophysiological effects of lowering [Na+]s, in particular the possibility that an elevation in intracellular free [Ca2+] hyperpolarizes Vcs by increasing gK+. When [Na+]s was reduced from 100.5 to 10.5 mM (tetramethylammonium substitution), Vcs hyperpolarized from -68 +/- 2 to a peak value of -82 +/- 2 mV (P less than 0.001), and fRa decreased from 0.84 +/- 0.02 to 0.62 +/- 0.02 (P less than 0.001). Addition of 5 mM tetraethylammonium (TEA+) to the mucosal solution reduced both the hyperpolarization of Vcs and the change in fRa, whereas serosal addition of TEA+ had no effect. Ouabain (10(-4) M, serosal side) produced a small depolarization of Vcs and reduced the hyperpolarization upon lowering [Na+]s, without affecting the decrease in fRa. The effects of mucosal TEA+ and serosal ouabain were additive. Neither amiloride (10(-5) or 10(-3) M) nor tetrodotoxin (10(-6) M) had any effects on Vcs or fRa or on their responses to lowering [Na+]s, suggesting that basolateral Na+ channels do not contribute to the control membrane voltage or to the hyperpolarization upon lowering [Na+]s. The basolateral membrane depolarization upon elevating [K+]s was increased transiently during the hyperpolarization of Vcs upon lowering [Na+]s. Since cable analysis experiments show that basolateral membrane resistance increased, a decrease in basolateral Cl- conductance (gCl-) is the main cause of the increased K+ selectivity. Lowering [Na+]s increases intracellular free [Ca2+], which may be responsible for the increase in the apical membrane TEA(+)-sensitive gK+. We conclude that the decrease in fRa by lowering [Na+]s is mainly caused by an increase in intracellular free [Ca2+], which activates TEA(+)-sensitive maxi K+ channels at the apical membrane and decreases apical membrane resistance. The hyperpolarization of Vcs is due to increase in: (a) apical membrane gK+, (b) the contribution of the Na+ pump to Vcs, (c) basolateral membrane K+ selectivity (decreased gCl-), and (d) intraepithelial current flow brought about by a paracellular diffusion potential.

A comparison of intact and in-vitro locomotion in an adult amphibian.

Locomotion was compared in an intact and in-vitro preparation of the adult mudpuppy (Necturus maculatus). The intact animals walked on an aquatic treadmill while in-vitro preparations were made to walk with a bath application of the excitatory amino acid NMA (N-methyl DL-aspartate). EMG recordings of shoulder muscles (pectoralis, latissimus dorsi, dorsalis scapulae, and procoracohumeralis) and elbow muscles (brachialis and extensor ulnae) were obtained from intact animals while recordings were made from only the elbow muscles in-vitro. The in-vitro preparation required magnesium in the bath to initiate and maintain locomotion, consistent with an NMDA mediated response. Also consistent with an NMDA response was the finding that glycine potentiated the NMA induced locomotion in-vitro. The range of cycle durations seen in-vitro was well within the range seen in the intact animal, while gait analysis demonstrated the similarity of intact and in-vitro locomotor cycles. In spite of these very similar locomotor patterns there are interesting differences in the patterned output seen in-vitro, such as the absence of a second burst in the brachialis muscle in-vitro.

Reconstitution of an epithelial chloride channel. Conservation of the channel from mudpuppy to man.

We have previously shown that monoclonal antibody E12 (MAb E12), one of several such antibodies raised against theophylline-treated Necturus gallbladder (NGB) epithelial cells, inhibits the chloride conductance in the apical membrane of that tissue. Since chloride channels are critical to the secretory function of epithelia in many different animals, we have used this antibody to determine whether the channels are conserved, and in an immunoaffinity column to isolate the channel protein. We now demonstrate that MAb E12 cross-reacts with detergent-solubilized extracts of different tissues from various species by enzyme-linked immunosorbent assay (ELISA). Western blot analysis shows that this monoclonal antibody recognizes proteins of Mr 219,000 in NGB, toad gallbladder, urinary bladder, and small intestine, A6 cells, rat colon, rabbit gastric mucosa, human lymphocytes, and human nasal epithelial cells, and inhibits the chloride conductance in toad gallbladder, rat colon, and human nasal epithelium. Detergent-solubilized protein eluted from an immunoaffinity column and then further purified via FPLC yields a fraction (Mr 200,000-220,000) which has been reconstituted into a planar lipid bilayer. There it behaves as a chloride-selective channel (PCl/PNa = 20.2 in a 150/50 mM trans-bilayer NaCl gradient) whose unit conductance is 62.4 +/- 4.6 pS, and which is blocked in the bilayer by the antibody. The gating characteristics of this channel indicate that it can exist as aggregates or as independent single channels, and that the antibody interferes with gating of the aggregates, leaving the unit channels unchanged. From these data we conclude that the protein of Mr 219,000 recognized by this monoclonal antibody is an important component of an epithelial chloride channel, and that this channel is conserved across a wide range of animal species.