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PubMed1985

Water permeability of Necturus gallbladder epithelial cell membranes measured by nuclear magnetic resonance.

In order to assess the contribution of transcellular water flow to isosmotic fluid transport across Necturus gallbladder epithelium, we have measured the water permeability of the epithelial cell membranes using a nuclear magnetic resonance method. Spin-lattice (T1) relaxation of water protons in samples of gallbladder tissue where the extracellular fluid contained 10 to 20 mM Mn2+ showed two exponential components. The fraction of the total water population responsible for the slower of the two was 24 +/- 2%. Both the size of the slow component, and the fact that it disappeared when the epithelial layer was removed from the tissue, suggest that it was due to water efflux from the epithelial cells. The rate constant of efflux was estimated to be 15.6 +/- 1.0 sec-1 which would be consistent with a diffusive membrane water permeability Pd of 1.6 X 10(-3) cm sec-1 and an osmotic permeability Pos of between 0.3 X 10(-4) and 1.4 X 10(-4) cm sec-1 osmolar-1. Using these data and a modified version of the standing-gradient model, we have reassessed the adequacy of a fluid transport theory based purely on transcellular osmotic water flow. We find that the model accounts satisfactorily for near-isosmotic fluid transport by the unilateral gallbladder preparation, but a substantial serosal diffusion barrier has to be included in order to account for the transport of fluid against opposing osmotic gradients.

Animals↗

PubMed1985

Protamine reversibly decreases paracellular cation permeability in Necturus gallbladder.

Protamine, a naturally occurring arginine-rich polycationic protein (pI 9.7 to 12), was tested in Necturus gallbladder using a transepithelial AC-impedance technique. Protamine sulfate or hydrochloride (100 micrograms/ml = 20 microM), dissolved in the mucosal bath, increased transepithelial resistance by 89% without affecting the resistance of subepithelial layers. At the same time, transepithelial voltage (psi ms) turned from slightly mucosa-positive values to mucosa-negative values of approximately +1 to -5 mV. The effect of protamine on transepithelial resistance was minimal at concentrations below 5 micrograms/ml but a maximum response was achieved between 10 and 20 micrograms/ml. Resistance started to increase within 1 min and was maximal after 10 min. These effects were not inhibited by serosal ouabain (5 X 10(-4) M) but could be readily reversed by mucosal heparin. The sequence of protamine effect and heparin reversal could be repeated several times in the same gallbladder. Mucosal heparin, a strong negatively charged mucopolysaccharide, or serosal protamine were without effect. Mucosal protamine reversibly decreased the partial ionic conductance of K and Na by a factor of 3, but did not affect Cl conductance. Net water transport from mucosa to serosa was reversibly increased by 60% by protamine. We conclude that protamine reversibly decreases the conductance of the cation-selective pathway through the tight junction. Although this effect is similar to that reported for 2,4,6-triamino-pyrimidinium (TAP), the mechanism of action may differ. We propose that protamine binds to the apical cell membrane and induces a series of intracellular events which leads to a conformational alteration of the tight junction structure resulting in decreased cationic permeability.

Animals↗

PubMed1983

Effects of Na-coupled alanine transport on intracellular K activities and the K conductance of the basolateral membranes of Necturus small intestine.

Intracellular electrical potentials and K activity, (K)c, were determined simultaneously in Necturus small intestine before and after the addition of alanine to the mucosal solution. As noted previously (Gunter-Smith, Grasset & Schultz, 1982), the addition of alanine to the mucosal solution resulted in a prompt depolarization of the electrical potential difference across the apical membrane (psi mc) and a decrease in the slope resistance of that barrier (rm). This initial response was followed by a slower repolarization of psi mc associated with a decrease in the slope resistance of the basolateral membrane (rs) so that when the steady state was achieved (rm/rs) did not differ significantly from control values in the absence of alanine. In the absence of alanine, psi mc averaged -32 mV and (K)c averaged 67 mM. When a steady state was achieved in the presence of alanine these values averaged -24 mV and 50 mM, respectively. The steady-state electrochemical potential differences for K across the basolateral membrane in the absence and presence of alanine did not differ significantly. Inasmuch as the rate of transcellular active Na transport or "pump activity" was increased two- to threefold in the presence of alanine, it follows that, if active Na extrusion across the basolateral membrane is coupled to active K uptake across that barrier with a fixed stoichiometry then, the decrease in rs must be due to an increase in the conductance of the basolateral membrane to K that parallels the increase in "pump activity". This "homocellular" regulatory mechanism serves to (i) prevent an increase in (K)c due to an increase in pump activity; and, (ii) repolarize psi mc and thus restore the electrical driving force for the rheogenic Na-coupled entry processes.

Alanine↗

PubMed1988

Voltage- and time dependence of apical membrane conductance during current clamp in Necturus gallbladder epithelium.

The effects of short (1 sec) and long (1 min) transepithelial current clamps on membrane voltages and resistances of Necturus gallbladder were investigated. Transepithelial and cell membrane current-voltage relationships determined from 1-sec clamps revealed that: a) depolarization of the apical membrane voltage (Vmc) results in a marked decrease in apical membrane fractional resistance (fRa), whereas hyperpolarization of Vmc results in either no change in fRa or a small increase, and b) the voltage-dependent changes in fRa are essentially complete within 500 msec. Exposure of the tissue to 5 mM TEA+ on the mucosal side caused no significant change in baseline Vmc (-69 +/- 2 mV) and yet virtually abolished the voltage dependence of fRa. A possible interpretation of these results is that two types of K+ channels exist in the apical membrane, with different voltage dependencies and TEA+ sensitivities. Acidification or Ba2+ addition to the mucosal solution also reduced the voltage-dependent changes in fRa. The time courses of the changes in fRa and in the cable properties of the epithelium were assessed during 1-min transepithelial current clamps (+/- 200 microA/cm2). No secondary change in fRa was observed with mucosa-to-serosa currents, but a slow TEA+-sensitive decrease in fRa (half-time of seconds) was evident with serosa-to-mucosa currents. Cable analysis experiments demonstrated that the initial (less than 500 msec) voltage-dependent decrease in fRa is due to a fall in apical membrane resistance. The later decrease in fRa is due to changes in both cell membrane resistances attributable to the increase in transcellular current flow resulting from a fall in paracellular conductance. The voltage dependence of the apical membrane conductance is a more significant problem in estimating fRa than the current-induced effects on the lateral intercellular spaces. In principle, TEA+ can be used to prevent the nonlinear behavior of Ra during measurements of the voltage divider or membrane resistance ratio.

Animals↗

PubMed1984

Electrophysiology of Necturus urinary bladder: II. Time-dependent current-voltage relations of the basolateral membranes.

As reported previously (S.R. Thomas et al., J. Membrane Biol. 73:157-175, 1983) the current-voltage (I-V) relations of the Na-entry step across the apical membrane of short-circuited Necturus urinary bladder in the presence of varying mucosal Na concentrations are (i) time-independent between 20-90 msec and (ii) conform to the Goldman-Hodgkin-Katz constant field flux equation for a single cation over a wide range of voltages. In contrast, the I-V relations of the basolateral membrane under these conditions are (i) essentially linear between the steady-state, short-circuited condition and the reversal potential (Es); and (ii) are decidedly time-dependent with Es increasing and the slope conductance, gs, decreasing between 20 and 90 msec after displacing the transepithelial electrical potential difference. Evidence is presented that this time-dependence cannot be attributed entirely to the electrical capacitance of the tissue. The values of gs determined at 20 msec are linear functions of the short-circuit current, Isc, confirming the relations reported previously, which were obtained using a more indirect approach. The values of Es determined at 20 msec are significantly lower than any reasonable estimate of the electromotive force for K across the basolateral membrane, indicating that this barrier possesses a significant conductance to other ions which may exceed that to K. In addition, these values increase linearly with decreasing Isc and approach the value of the electrical potential difference across the basolateral membrane observed when Na entry across the apical membrane is blocked with amiloride or when Na is removed from the mucosal solution. A possible explanation for the time-dependence of Es and gs is offered and the implications of these findings regarding the interpretation of previous microelectrophysiologic studies of epithelia are discussed.

Animals↗

PubMed1986

Current-voltage relations of sodium-coupled sugar transport across the apical membrane of Necturus small intestine.

The current-voltage (I-V) relations of the rheogenic Na-sugar cotransport mechanism at the apical membrane of Necturus small intestine were determined from the relations between the electrical potential difference across the apical membrane, psi mc, and that across the entire epithelium, psi ms, when the latter was varied over the range +/- 200 mV, under steady conditions in the presence of galactose and after the current across the apical membrane carried by the cotransporter, ImSNa, is blocked by the addition of phloridzin to the mucosal solution. ImSNa was found to be strongly dependent upon psi mc over the range -50 mV less than psi mc less than EmSNa where EmSNa is the "zero current" or "reversal" potential. Over the range of values of psi mc encountered under physiological conditions the cotransporter may be modeled as a conductance in series with an electromotive force so that ImSNa = gmSNa (EmSNa - psi mc) where gmSNa is the contribution of this mechanism to the conductance of the apical membrane and is "near constant." In several instances ImSNa "saturated" at large hyperpolarizing or depolarizing values of psi mc. The values of EmSNa determined in the presence of 1, 5, and 15 mM galactose strongly suggest that if the Na-galactose cotransporters are kinetically homogeneous, the stoichiometry of this coupled process is unity. Finally, the shapes of the observed I-V relations are consistent with the predictions of a simple kinetic model which conforms with current notions regarding the mechanico-kinetic properties of this cotransport process.

Animals↗

PubMed1982

Occluding junctions of the Necturus gallbladder.

The paracellular conducting pathway of the Necturus gallbladder was studied with electrophysiological and electromicroscopic methods. The first one consists of the passage of short (5 msec) and small (32 microA cm-2) current pulses associated with a voltage scanning of the plane of the epithelium at the apical surface with a microelectrode to detect the regions where current flows. The procedure shows that (a) the conductance is evenly distributed along the intercellular regions along the intercellular spaces of the cells where occluding junctions are located; (b) the field above the occluding junctions has the shape of a bell, so that the junction can be sensed at 1-2 micron from the region where the intercellular space is visualized by light microscopy; (c) the intersections between three cells, in spite of having 3 half-junctions contributing (instead of two), do not have a higher conductance than the rest of the occluding junction. Scanning electron microscopy shows that (a) cells are densely covered by microvilli which interdigitate above the region of the occluding junctions, and (b) are covered by a surface coat. With transmission electron microscopy, (a) the opening of the occluding junctions at the apical border appears irregular, and most of them oblique; (b) in the last microns the actual mouth of the junction may deviate from the course of the interspace. Freeze-fracture replicas indicate that (a) the occluding junction has a uniform width and little variations in the number of strands around the cell, except (b) at intersections between 3 cells where both, its width and the number of strands, increase toward the basal region.

Animals↗

PubMed1982

Structure of Necturus gallbladder epithelium during transport at low external osmolarities.

Gallbladders transport isotonically over a wide range of osmolarities. This ability has been assumed to depend on the geometry of the lateral intercellular spaces. We report that this geometry in the Necturus gallbladder varies extensively with the external osmolarity and depends in vitro on the integrity of the subepithelial tissues. The structure of the living epithelium was studied by Nomarski light microscopy while ultrastructural effects were revealed by electron microscopy. The short-term effects (less than 60 min) of low external osmolarities were: 1) the cells became bell-shaped with an increased cell height measured centrally, 2) lateral intercellular spaces lost their convoluted character; and 3) numerous membrane-bound cavities appeared in the cells. Furthermore, long-term exposure to the low external osmolarities caused an uneven density of epithelial cells. With subepithelial tissues intact, blistering of the epithelium cell layer was evident. Qualitative electron-microscopic data indicate that the membrane of the cavities was recruited from the basolateral cell membrane. This agrees well with light-microscopic observation that the cavities were initiated as invaginations of this cell membrane.

Animals↗

PubMed1982

Steady states and the effects of ouabain in the Necturus gallbladder epithelium: a model analysis.

A simple numerical model for the Necturus gallbladder epithelium is presented. K+, Na+ and Cl- cross the mucosal and serosal membranes as well as the junctions by means of electrodiffusion; furthermore the mucosal membrane contains a neutral entry mechanism for NaCl and the serosal membrane contains an active pump for K+ and Na+. The values which have been used for the model are taken from the literature. The model can only attain steady states if the resistance of the serosal membrane is lower than 1000 omega cm2. Values reported in the literature for the resistance of this membrane vary from about 3000 to about 100 omega cm2. We shall argue, however, that the higher estimates are in error because they are derived from a model of the tissue in which each membrane and the junction are modeled by a resistor; this procedure is invalid because the resistance of the lateral intercellular space relative to the resistance of the tight junctions is neglected and consequently the resistance of the serosal membrane is overestimated by a factor of about four. Apart from predicting a realistic steady state at normal external concentrations the model can predict quantitatively several experimental results obtained from the living epithelium. We have focused on the experiments which test the permeabilities of the serosal membrane and the properties of the pump: i) Replacement of serosal Cl- by an impermeant ion. ii) Replacement of serosal K+ by Na+. iii) Inhibiting the (Na+, K+)-pump. The best correspondence between model and experiments is obtained when the pump is assumed to be electrogenic (or rheogenic) with a ratio of coupling between Na+ and K+ of 3:2. In this case both model and direct experiments (also presented in this paper) show an initial abrupt depolarization of 6 to 7 mV. The model also shows that it cannot be concluded from i and ii that the Cl- permeability of the serosal membrane is low. The model explains, even with high passive Cl- permeabilities, why the intracellular Cl- concentration is relatively unaffected by paracellular currents, a fact which in other epithelia has been taken as an implication of a low Cl- permeability of the serosal membranes.

Animals↗

PubMed1982

Cell pH and luminal acidification in Necturus proximal tubule.

Cellular potential and pH measurements (pHi) were carried out in the perfused kidney of Necturus on proximal tubules with standard and recessed-tip glass microelectrodes under control conditions and after stimulation of tubular bicarbonate reabsorption. Luminal pH and net bicarbonate reabsorption were measured in parallel experiments with recessed-tip glass or antimony electrodes, both during stationary microperfusions as well as under conditions of isosmotic fluid transport. A mean cell pH of 7.15 was obtained in control conditions. When the luminal bicarbonate concentration was raised to 25 and 50 mM, pH, rose to 7.44 and 7.56, respectively. These changes in pHi were fully reversible. Under all conditions intracellular H+ was below electrochemical equilibrium. Thus the maintenance of intracellular pH requires "active" H+ extrusion across one or both of the cell membranes. The observed rise in pHi and the peritubular depolarization after stimulation of bicarbonate reabsorption are consistent with enhanced luminal hydrogen ion secretion and augmentation of peritubular bicarbonate exit via an anion-conductive transport pathway.

Animals↗

PubMed1981

Mechanisms of cation permeation across apical cell membrane of Necturus gallbladder: effects of luminal pH and divalent cations on K+ and Na+ permeability.

Conventional microelectrode techniques were combined with unilateral mucosal ionic substitutions to determine the effects of luminal pH and luminal alkali-earth cation concentrations on apical membrane cation permeability in Necturus gallbladder epithelium. Acidification of the mucosal solution caused reversible depolarization of both cell membranes and increase of transepithelial resistance. Low pH media also caused: (a) reduction of the apical membrane depolarization induced by high K, and (b) increase of the apical membrane hyperpolarization produced by Na replacement with Li or N-Methyl-D-glucamine. These results, in conjunction with estimates of cell membrane conductances, indicate that acidification of the luminal solution produces a reduction of apical membrane K permeability (PK). Addition of alkali earth cations (Mg2+, Ca2+, Sr2+, or Ba2+) produced cell membrane depolarization, increase of relative resistance of the luminal membrane and reduction of the apical membrane potential change produced by a high-K mucosal medium. These results, as those produced by low pH, can be explained by a reduction of apical membrane PK. The effects of Ba2+ on membrane potential and relative apical membrane PK were larger than those of all other four cations at all concentrations tested (1-10 mM). The effect of Sr2+ was significantly larger than those of Mg2+ and Ca2+ at 10 mM, but not different at 5 mM. The reduction of PK produced by mucosal acidification appears to be mediated by: (a) nonspecific titration of membrane fixed negative charges, and (b) an effect of luminal proton activity on the apical K channel. Divalent cations reduce apical membrane PK probably by screening negative surface charges. The larger magnitude of the effects of Ba2+ and Sr2+ can be explained by binding to membrane sites, in the surface or in the K channel, in addition to their screening effect. We suggest that the action of luminal pH on K secretion in some segments of the renal tubule could be mediated in part by this pH-dependent K permeability of the luminal membrane.

Animals↗

PubMed1983

Chloride transport across the basolateral cell membrane of the Necturus proximal tubule: dependence on bicarbonate and sodium.

The transport of chloride across the Necturus proximal tubule cell was studied in the doubly-perfused kidney using conventional, chloride-sensitive and pH-sensitive microelectrodes. Lowering chloride activity in the basolateral solution results in a reduction in intracellular Cl- activity (aiCl). This reduction in aiCl is inhibited by removing either HCO-3 or Na+ from the perfusion solution, indicating that both HCO-3 and Na+ are required for Cl- movement across the basolateral cell membrane. Reducing either HCO-3 or Na+ in the basolateral solution causes an increase in aiCl. Thus changes in either Na+ or HCO-3 chemical gradients across the basolateral cell membrane significantly affect chloride movement. Changing intracellular pH by means of NH4Cl exposure results in an increase in aiCl followed by a sharp decrease when NH4Cl is removed. These changes in intracellular chloride do not occur in the absence of HCO-3. Likewise, the decrease in aiCl following NH4Cl treatment requires the presence of Na+ in the basolateral solution. We conclude that chloride is transported across the basolateral cell membrane in exchange for both Na+ and HCO-3. Our results also support the presence of a Na+/Cl- cotransport mechanism on the apical cell membrane.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed1988

Base induced hyperpolarization of the cell potential in HCO3- free perfused Necturus renal proximal tubules.

Short-term peritubular alkalinization from 7.5 to 8.5 hyperpolarized (-8.8 mV) the basolateral membrane potential (V1) in HCO3- free Herpes buffered Necturus renal proximal tubule cells. This sustained base induced hyperpolarization (BIH) was associated with an increase in the peritubular apparent transference number for potassium (tK+). The apparent transference number for potassium (tK+) was estimated at pH 7.5 and 8.5 by raising peritubular K+ from 2.5 to 10 mmol/l. tK+ increased linearly as V1 hyperpolarized, whereas tK+ measured in the presence of peritubular Ba2+ at pH 7.5 and 8.5 was nearly zero. However, the BIH persisted in the presence of barium at the peritubular, luminal or both sides of the epithelium. Moreover this BIH was also accompanied by a small hyperpolarization (-0.4 mV) of the transepithelial membrane potential (V3) in the absence or presence of peritubular and/or luminal Ba2+. Therefore we conclude that BIH must originate from additional mechanisms other than an increase in peritubular or luminal potassium conductance.

Animals↗

PubMed1984

Intracellular activities during volume regulation by Necturus gallbladder.

Necturus gallbladder epithelial cells regulate their volume after a change in solution osmolality. We determined the intracellular activities of Na, K and Cl when the mucosal bathing solution osmolality was increased 18% by the addition of mannitol. The gallbladder was mounted in a rapid flow chamber and punctured simultaneously with two single-barrelled microelectrodes. One electrode sensed membrane potential and the other was sensitive to the activity of Na, K or Cl. Cell volume measurements, made in previous studies utilizing quantitative light microscopy, indicated that hypertonicity of the mucosal bath first caused a cell shrinkage of 15% followed by volume readjustment. Some loss of Na, K and Cl was observed during shrinkage; subsequently during volume regulation, the intracellular quantities of all three ions increased. The loss of Na during the initial cell shrinkage could be blocked by ouabain and was therefore due to increased transport. K and Cl losses were probably related to the increase in their concentrations during shrinkage. The gain of Na, K and Cl during volume regulation was similar in magnitude to the loss of these solutes during cell shrinkage. The increase of Na, K and Cl during volume regulation accounted for about 60% of the increase of cell solutes during this period indicating that other solutes also contributed to the volume regulation response.

Animals↗

PubMed1987

pH-dependent electrical properties and buffer permeability of the Necturus renal proximal tubule cell.

Necturus kidneys were perfused with Tris-buffered solutions at three different pH values, i.e. 7.5, 6.0 and 9.0. A significant drop in fluid absorption occurred at pH 6.0, whereas pH 9.0 did not increase volume flow significantly. When acute unilateral, i.e. either in the lumen or the peritubular capillaries, and bilateral pH changes were elicited in both directions from 7.5 to 9.0 at a constant Tris-butyrate buffer concentration, both peritubular membrane potential difference V1 and transepithelial potential difference V3 hyperpolarized, independently of the side where the change in pH was brought about. Acid perfusions at pH 6.0 caused a similar response but of opposite sign. Analysis of the potential changes shows that pH influences not only the electromotive force and resistance of the homolateral membrane, but also the electrical properties of the paracellular path. Interference of pH with Na, Cl or K conductance was assessed. Any appreciable role for sodium or chloride was excluded, whereas the potassium transference number (tK) of the peritubular membrane increased 16% in alkaline pH. However, this increase accounts only for 19 to 36% of the observed hyperpolarization. Since changes in Tris-butyrate buffer concentration at constant pH do not affect V1 or V3 considerably, the hyperpolarization in pH 9 cannot be explained by an elevation in internal pH only, or by a Tris-H+ ion diffusion potential only. The role of the permeability of the buffers: bicarbonate, butyrate and phosphate, in determining electrical membrane parameters was evaluated. Transport numbers of the buffer anions ranked as follows: tHCO3 greater than tbutyrate greater than tphosphate. It is concluded that modulation of membrane potential by extracellular pH is mediated primarily by a change in peritubular cell membrane tK and additionally by membrane currents carried by buffer anions.

Animals↗

PubMed1990

Barium blocks cell membrane and tight junction conductances in Necturus gallbladder epithelium. Experiments with an extended impedance analysis technique.

The site and concentration dependence of the blocking effect of Ba2+ on Necturus gallbladder epithelium has been investigated. A new approach was used which combines time-dependent electrical cell coupling analysis with intermittently performed measurements of transepithelial and apparent intracellular impedance. From the coupling pulse data the sum of apical and basolateral membrane conductances is obtained, which is then held constant during fitting of the impedance data. This combination technique yields more reliable estimates of apical and basolateral membranes resistances (Ra, Rbl) and of tight junction resistance (Rj) than our previous impedance analysis technique. Using the new approach we have found that luminal Ba2+ concentrations between 0.5 and 1.0 mmol/l increase Ra with saturation-type kinetics without affecting Rbl and Rj, while higher luminal Ba2+ concentrations progressively increase Rj. Corresponding effects were observed under serosal Ba2+. The results validate the new impedance analysis approach and demonstrate that millimolar concentrations of Ba2+ block tight junction conductances. Accordingly, Ba2+ can no longer be considered a tool to exclusively alter cell membrane resistances in epithelia.

Animals↗

PubMed2002

Effects of cholinergic and noradrenergic agents on locomotion in the mudpuppy (Necturus maculatus).

Some neurotransmitters act consistently on the central pattern generator (CPG) for locomotion in a wide range of vertebrates. In contrast, acetylcholine (ACh) and noradrenaline (NA) have various effects on locomotion in different preparations. The roles of ACh and NA have not been studied in amphibian walking, so we examined their effects in an isolated spinal cord preparation of the mudpuppy ( Necturus maculatus). This preparation contains a CPG that produces locomotor activity when N-methyl- D-aspartic acid (NMDA), an excitatory amino acid agonist, is added to the bath. The addition of carbachol, a long acting ACh agonist, to the bath disrupted the walking rhythm induced by NMDA, while not changing the level of activity in flexor and extensor motoneurons. Adding clonidine, an alpha(2)-noradrenergic agonist, had no effect on the NMDA-induced walking rhythm. Physostigmine, an ACh-esterase inhibitor, disrupted the walking rhythm, presumably by potentiating the effects of endogenously released ACh. Atropine, an ACh antagonist that binds to muscarinic ACh receptors, blocked the effects of carbachol, indicating that the action is mediated, at least in part, by muscarinic receptors. In the absence of carbachol, atropine had no effect. Locomotion was not induced by carbachol, atropine or clonidine in a resting spinal cord preparation. Cholinergic actions do not seem to be essential to the CPG for walking in the mudpuppy, but ACh may convert a rhythmic walking state to a more tonic state with occasional bursts of EMG activity for postural adjustments.

Acetylcholine↗

PubMed1999

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.

Animals↗