Calcium ion levels in resting and depolarized goldfish retinal ganglion cell somata and growth cones.

1. We have estimated free, intracellular calcium ion concentrations ([Ca]i) in isolated retinal ganglion cells of adult goldfish by ratio-imaging fura-2 emission intensity at two excitation wavelengths. Here we describe [Ca]i in these cells, both at rest and during depolarization by elevated levels of extracellular potassium ions ([K]o). 2. [K]o was varied between 5 and 60 mM in sodium-free, tetrodotoxin-containing salines. Ganglion cell membrane potential, measured with patch electrodes, fell with each increment of [K]o used, from approximately -70 mV in 5 mM K+ to approximately -20 mV in 60 mM K+. 3. In control saline, [Ca]i was roughly 120 nM in cell somata and at least twofold higher in their growth cones. [Ca]i increased in both somata and growth cones to as high as 1.5 microM in salines containing 60 mM K+. [Ca]i exceeded 1.5 microM in some cells in high-K+ salines, although these levels could not be quantified accurately with fura-2. 4. Increases in [Ca]i elicited by elevated [K]o persisted for the duration of the exposure to high-K+ saline and were blocked by replacement of most of the bath Ca2+ by Co2+. These increases in [Ca]i were also sensitive to dihydropyridine calcium-channel ligands, viz., enhanced by BAY K 8644 (3 microM) and antagonized by nifedipine (10 microM). 5. Partial recovery of control [Ca]i occurred when [K]o was reduced to 5 mM after exposure to high-K+ saline and in high-K+ saline when nifedipine was included. These results show that goldfish retinal ganglion cells can partially buffer intracellular Ca2+ in the absence of extracellular Na+ ions. 6. These results provide measurements of the changes in [Ca]i brought about by depolarization of goldfish retinal ganglion cells in Na(+)-free salines. In these salines, at least part of the increase in [Ca]i appears to result from Ca2+ influx through a voltage-activated, noninactivating calcium conductance in the somata and growth cones of these cells. These measurements complement whole-cell patch-clamp and vibrating microprobe recordings from the somata and neurites of these cells and also immunocytochemical studies and patch-clamp measurements in amphibian, reptilian, and mammalian retinal ganglion cells.     

The ionic basis of the resting potential and a slow depolarizing response in Rohon-Beard neurones of Xenopus tadpoles.

1. Rohon-Beard cells in the spinal cord of Xenopus laevis tadpoles have been studied in animals 4-days to 2-weeks-old (Nieuwkoop & Faber, 1956, stages 45-49). These neurones have an unusually large resting membrane potential of -88 mV, in Ringer solution containing 3-0 mM K+. 2. Their resting potential (R..) depends on the concentration gradient of K+ across the cell membrane. These cells follow the prediction of the Nernst equation for a K+-selective electrode, down to external K+ concentrations as low as 1-0 mM (R.P. -118 mV). 3. The resting potentials of muscle cells in these animals exhibit the same dependence on external [K+], as has been shown previously. 4. Rohon-Beard cells can be driven antidromically, bu stimulation of the anterior end of the spinal cord with brief current pulses through a suction electrode. Antidromic action potentials fail to invade the cell body with repeated stimulation at 1Hz. 5. Even when impulses fail to invade Rohon-Beard somata, slow depolarizations can be produced by single shocks or trains of shocks which cause impulse activity in other neurones. The response can be observed to a single stimulus or to a train of stimuli. The magnitude of the depolarization is graded, depending on the number of stimuli and the frequency of stimulation. 6. Support is presented for the hypothesis that the slow depolarization in Rohon-Beard cells is mediated by the release of K+ into their environment by the impulse activity of neighbouring neurones. The slow depolarization increases in solutions containing 1-5 mM-K+, and decreases in solutions containing 6-0 mM-K+. The changes are in quantitative agreement with those anticipated by theory. 7. The slow depolarization is unlikely to be due to a conductance change produced by a synaptic transmitter, since hyperpolarization and depolarization of the Rohon-Beard cell with injected current do not change the amplitude of the response. Further, low Ca-high Mg solutions which block neuromuscular transmission do not block the response. 8. The possible role of the slow depolarizing response in the physiological activity of these neurones is discussed.     

The action potential of chick dorsal root ganglion neurones maintained in cell culture.

1. The directly evoked action potential of dissociated, embryonic, chick, dorsal root ganglion (DRG) neurones maintained in cell culture is prolonged compared to spinal cord cell spikes and the re-polarization phase is marked by a plateau. 2. Evidence was obtained that both Ca2+ and Na+ carry inward current across the active soma membrane. Ca2+ because: overshooting spikes persist in tetrodotoxin (TTX) or Na+-free media; in the presence of TTX (or absence of Na+) spike size varies directly with extracellular Ca2+ and spikes are eliminated by Co2+. Na+ because: spikes persist in the presence of Co2+ or Ca2+-free media; in the presence of Co2+ (or absence of Ca2+) spike varies directly with extracellular Na+ and spikes are blocked by TTX. 3. On the other hand, Ca2+ plays less if any role in action potentials conducted along sensory nerve cell processes. Conducted spikes could not be evoked in TTX containing or Na+-free media. 4. A long-lasting depolarization follows the action potential in some neurones. This depolarization is associated with an increase in membrane conductance and appears to drive the membrane potential to ca. -30mV. It persists when conducted impulses are blocked so it is probably not a recurrent synaptic potential. 5. It is suggested that combined Ca2+-Na+ spikes observed in isolated sensory neurones in vitro reflect the action potential of adult sensory cells but the possibility that they represent an early stage in development is also discussed.     

Three pharmacologically distinct potassium channels in molluscan neurones.

1. Potassium currents were studied under voltage-clamp conditions in nerve cell bodies of the nudibranch Tritonia diomedia. 2. Potassium currents could be separated into three distinct components on the basis of their sensitivity to 4-aminopyridine (4-AP), tetraethyl-ammonium (TEA) and to Co2+ and Mn2+ ions. 3. A transient potassium current, similar to the fast outward current described by Connor & Stevens (1971b) and Neher (1971), was blocked by externally applied 4-AP but was much less sensitive to TEA or to Co2+ or Mn2+. A single 4-AP ion binds each receptor with an apparent dissociation constant of 1-5 X 10(-3) M. 4-AP decreases the rates of activation and inactivation and reduces the maximum conductance of transient current channels. 4. Delayed outward current was not effected by 4-AP at concentrations which blocked the transient current, but it could be divided into two components by external application of TEA and Co2+ or Mn2+. 5. A voltage-dependent component of delayed current, termed K-current, was blocked by TEA. Each K-current receptor binds a single TEA ion with an apparent dissociation constant of 8 X 10(-3) M. Co2+ and Mn2+ have little or no effect on K-current. 6. A second component of delayed outward current, termed C-current, depends on Ca2+ entry for its activation. It is similar to the Ca2+ dependent potassium current reported by Meech & Stranden (1975) in Helix cells. C-current is essentially blocked by 30 mM external Co2+ or Mn2+. It is little affected by TEA, however, being reduced by about 20% at a TEA concentration of 100 mM. 7. It is concluded that three sets of potassium selective channels contribute to the outward current and that these channels can be separated pharmacologically.     

Effects of the calcium ionophore A23187 on pancreatic acinar cell membrane potentials and amylase release.

1. The effects of the Ca2+-ionophore A23187 and the non-metabolizable cholinergic agonist bethanechol on acinar cell membrane potentials and amylase release from the superfused mouse pancreas were studied. 2. In the presence of extracellular Ca2+ (2.56 mM), A23187 (10(-5)M) and bethanechol (3 X 10(-5)M) caused an equal increase in the release of amylase. Both stimulants depolarized theacinar cells, A23187 by 6-0 mV and bethanechol by 12-3 mV. 3. When Ca2+ and Mg2+ were removed from the superfusate, the ability of A23187 to increase the rate of amylase release was virtually abolished, while the effect of bethanechol remained unaltered. Similarly, in the absence of these divalent cations, A23187 did not cause depolarization of the acinar cells, while depolarization in response to bethanechol was largely normal. Consequently it is unlikely that cholinergic agonists initiate secretion by activating a Ca2+-ionophore-like mechanism in the cell membrane. 4. When the concentration of Ca2+ in the medium was raised to 10 mM was the only extracellular divalent cation present, the depolarization in response to A23187 was increased to 11-8 mV. When Mg2+ in a concentration of 10 mM was the only extracellular divalent cation, the depolarization was only 2-1 mV. 5. The Ca2+ dependent, A23187-induced depolarization was abolished in the absence of Na+ (Tris substitution). Addition of Na+ to the superfusate caused an immediate depolarization. 6. It is concluded that the Ca2+ dependent depolarization of pancreatic acinar cells induced by A23187 is not directly due to an increased divalent cation conductance. Our findings are consistent with the view that the depolarization is due to an increased influx of Na+ resulting from a Ca2+ mediated increase in Na+ permeability.     

Roles of Ca2+ influx through ATP-activated channels in catecholamine release from pheochromocytoma PC12 cells.

1. Extracellular ATP evokes catecholamine release concomitant with depolarization in pheochromocytoma PC12 cells. Roles of Ca2+ influx through ATP-activated channels during the catecholamine release were investigated. 2. Norepinephrine or dopamine release induced by > or = 100-microM concentrations of ATP was insensitive to 300 microM Cd2+, whereas the release induced by increasing extracellular KCl (50-150 mM) was completely blocked by this concentration of Cd2+. 3. ATP (100 microM) increased the intracellular free Ca2+ concentration measured with fura-2. The increase was not affected by 300 microM Cd2+ or 100 microM nicardipine, suggesting that Ca2+ influx through ATP-activated channels but not through voltage-gated Ca2+ channels contributes to the ATP-evoked catecholamine release. 4. Inward currents permeating through voltage-gated Ca2+ channels were measured using the whole-cell voltage clamp. In the presence of 10 microM ATP, a concentration that induces an ATP-activated channel-mediated current equivalent to that induced by 100 microM ATP during the depolarization in "non-voltage clamped" cells, the Ca2+ current activated by a voltage step to +10 mV was reduced. The reduction in the Ca2+ channel-mediated current was not observed when the extracellular Ca2+ was replaced with Ba2+. 5. The ATP (100 microM)-evoked dopamine release was inhibited by 300 microM Cd2+ when measured with extracellular Ba2+ instead of Ca2+. This effect of Ba2+ may not be related to K+ channel-blocking activity, because the ATP-evoked dopamine release obtained with 5 mM tetraethylammonium (TEA) was not inhibited by Cd2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro associative conditioning of Hermissenda: cumulative depolarization of type B photoreceptors and short-term associative behavioral changes

Cumulative depolarization of Hermissenda type B photoreceptors, a short-term neural correlate of associative learning, was produced by simulating associative training in the isolated nervous system (in vitro conditioning). This simulation entailed stimulation and recording from three classes of neurons normally affected by the associative training procedure: a type B photoreceptor, the silent/excitatory (S/E) optic ganglion cell, and a statocyst caudal hair cell. Exposure of the isolated nervous system to five simultaneous pairings of light and current-induced impulse activity of the caudal hair cell resulted in an average 10-mV depolarization of type B cells. Cumulative depolarization was found to be pairing specific, to occur with a minimal number of training trials, and was paralleled by short-term pairing-specific changes in phototactic behavior for the intact animal. Two important determinants of cumulative depolarization were found to be the magnitude and duration of the long-lasting depolarization (LLD) response of type B cells to light, and a pairing-specific synaptic facilitation of the LLD response. The synaptic facilitation arose from two distinct sources: increased excitatory postsynaptic potential (EPSP) feedback on B cells following light and caudal hair cell stimulation pairings, and disinhibition of the type B photoreceptor following pairings. The S/E optic ganglion cell was found to be a potent regulator of B cell EPSPs. Cumulative depolarization was substantially reduced when the S/E cell was hyperpolarized throughout the course of pairings. Conversely, pairings of light with depolarizing current stimulation of the S/E cell were sufficient to produce cumulative depolarization of B cells. Precluding disinhibition of the B cell from the caudal hair cell was also found to attenuate cumulative depolarization. Additional constraints, inherent to the neural organization of the visual and statocyst neural systems were found to further limit the degree of cumulative depolarization. Among the most important of these were the interpairing interval and light intensity. Exposure of intact animals of five pairings of light and rotation resulted in short-term suppression of phototactic behavior. Like the cumulative depolarization of B cells with in vitro conditioning procedures, these changes were relatively pairing specific and persisted for comparable durations of time. Cumulative depolarization of B cells appears to be an important initial step in the production of long-term associative neural and behavioral changes in Hermissenda.     

Whole-cell K+ currents in isolated rabbit corneal epithelial cells.

Membrane currents were recorded from enzymatically isolated cells from basal layers of rabbit corneal epithelium by the whole-cell clamp technique. Pipettes contained 140.4 mM KCl and extracellular K+ concentration was varied. The membrane currents on step voltage changes were rectangular currents with some fluctuations. The fluctuations disappeared near the zero-current potential. The reversal potential in normal Tyrode's solution with 5.4 mM K+ was -57.8 +/- 6.2 mV (mean +/- S.D., n = 10). Increasing [K+]o from 5.4 to 140.4 mM shifted the reversal potentials in the positive direction with a slope of 41.0 mV/decade. Concomitant depolarization of the resting potential was observed on increasing [K+]o. The whole-cell currents were blocked by Cs+ or Ba2+. These suggest that the major current component in the corneal epithelial cells in K+.     

Cation effects on acid secretion in rabbit gastric glands

The ability of isolated gastric glands to produce acid as a function of intra- and extracellular concentrations of K+ and Na+ was investigated. Ouabain inhibited acid formation as measured by the aminopyrine (AP) accumulation technique in a dose-dependent manner with an ED50 of 2 X 10(-6) M at 5.4 mM extracellular K+. This inhibition was counteracted by increasing extracellular K+ or decreasing extracellular Na+. In K+-free solutions with regular Na+, the AP accumulation was almost totally abolished; the readdition of K+ rapidly restored the response with a K0.5 of 1 mM extracellular K+. In the absence of Na+, with or without ouabain, there was always a significant residual AP accumulation, but the K0.5 of extracellular K+ required for acid formation increased to about 20 mM. Despite repeated washings in Na+-K+-free solutions, the intracellular K+ content could not be decreased below 24 mM above the apparent K0.5 for K+. It was found that the intracellular K+ could be depleted without disturbance of the acid secretory function if the glands were treated with the neutral ionophore amphotericin B and ouabain in K+-Na+-free solutions. Complete dose-response curves of H+ secretion as a function of K+ concentration could now be obtained. Thus the K0.5 for K+ activation of AP accumulation was 16.5 +/- 0.9 mM; upon histamine stimulation of the glands, the K0.5 decreased to 10.4 +/- 0.7 mM. Intracellular K+ concentrations above approximately 60 mM inhibited AP accumulation. Na+ dose dependently inhibited the K+-induced response. At low extracellular K+ concentrations, a ouabain-insensitive K+ accumulation mechanism was unmasked. This K+ uptake was partly inhibited y p-chloromercuribenzene sulfonic acid and by inhibitors of the gastric H+-K+-ATPase and amplified by inhibitors of K+ conductance channels such as Ba2+. The K+ content of gastric glands is mainly regulated by the Na+-K+-ATPase, but additional K+-uptake pathways are present. The affinity for K+ is increased in histamine-stimulated glands, which might indicate one mechanism of stimulating the secretory response. Na+ inhibits secretion possibly by binding to the cytosolic cation site of the gastric ATPase. The cation relationship to acid secretion in the rabbit parietal cell is similar to that described for vesicles isolated from hog gastric mucosa.     

Stimulus-secretion coupling of parathyroid hormone release: studies of 45Ca and 86Rb fluxes

Pieces of rat parathyroid glands were used to study fluxes of 45Ca and 86Rb. The uptake of 45Ca increased with the extracellular Ca2+ concentration up to at least 5 mM. A rise of extracellular Ca2+ had dual effects on 45Ca efflux in terms of an initial stimulation and a subsequent inhibition. However, K+ depolarization neither affected the uptake nor the efflux of 45Ca indicating a lack of voltage-dependent Ca2+ channels. The depolarization obtained with exposure to Ca2+ cannot be attributed to a decreased K+ permeability, since the 86Rb concentrating ability diminished and the efflux of the isotope increased when parathyroid pieces were exposed to a raised Ca2+ concentration. A stimulation of 86Rb efflux by the Ca2+ ionophore A-23187 indicated that the parathyroid cells possess a K+ permeability activated by cytoplasmic Ca2+. It is suggested that Ca2+ fluxes through channels sensitive to activation by Ca2+ are important both for the membrane potential and the cytoplasmic Ca2+ activity.     

Voltage-sensitive calcium flux into bovine chromaffin cells occurs through dihydropyridine-sensitive and dihydropyridine- and omega-conotoxin-insensitive pathways

The fluorescent Ca2+ indicator FURA-2 was used to characterize the depolarization-related intracellular Ca2+ signalling process in bovine adrenal chromaffin cells. Depolarization with high K+ (10-65 mM) gave rise to a very rapid increase in intracellular free Ca2+ concentration, which subsequently decayed slowly towards a "plateau". The size of this initial increase varied sigmoidally with the calculated membrane potential, the relationship being described well by a Boltzmann distribution function for a transition between two states (transition potential, -23 mV). A dihydropyridine calcium channel agonist [(+)202-791, 1 microM] raised intracellular free Ca2+ concentration further in the presence of 30 mM K+, and it enhanced the initial intracellular Ca2+ response to depolarization. Voltage-sensitive calcium channels in chromaffin cells are believed to include the L-type. Several dihydropyridine calcium channel antagonists [(-)202-791, nifedipine, nitrendipine; 1-5 microM], known to be active on L-type channels, caused only modest inhibition of K+ -induced increase in intracellular free Ca2+ concentration: c. 50% (at 30 mM K+) and 25% (at 40-70 mM K+). In addition, omega-conotoxin GVIA (1-10 microM), a blocker of neuronal N- and L-type calcium channels, reduced the initial increase in intracellular free Ca2+ concentration only slightly at 55 mM K+. Further, the dihydropyridine-insensitive component of the intracellular Ca2+ signal was also insensitive to omega-conotoxin, which was otherwise quite active in a central nervous rat in vivo preparation Gd3+ (40 microM), a potent calcium antagonist in the chromaffin cell, blocked the intracellular Ca2+ response to depolarization. When added at different times after K+ stimulation, however, Gd3+ reduced intracellular free Ca2+ concentration to control levels along a slow time course of several minutes. Similar results were obtained when EGTA was added to reduce extracellular Ca2+ concentration to sub-nanomolar levels, in the presence of high K+. We conclude that bovine chromaffin cells are equipped with at least two different classes of voltage-dependent calcium channels, only one of which is likely to be the L-type channel. We also propose that depolarization, in addition to stimulating Ca2+ influx, may also lead to enhancement of Ca2+ release from an intracellular store.     

Extracellular pH dynamics of retinal horizontal cells examined using electrochemical and fluorometric methods

Extracellular H(+) has been hypothesized to mediate feedback inhibition from horizontal cells onto vertebrate photoreceptors. According to this hypothesis, depolarization of horizontal cells should induce extracellular acidification adjacent to the cell membrane. Experiments testing this hypothesis have produced conflicting results. Studies examining carp and goldfish horizontal cells loaded with the pH-sensitive dye 5-hexadecanoylaminofluorescein (HAF) reported an extracellular acidification on depolarization by glutamate or potassium. However, investigations using H(+)-selective microelectrodes report an extracellular alkalinization on depolarization of skate and catfish horizontal cells. These studies differed in the species and extracellular pH buffer used and the presence or absence of cobalt. We used both techniques to examine H(+) changes from isolated catfish horizontal cells under identical experimental conditions (1 mM HEPES, no cobalt). HAF fluorescence indicated an acidification response to high extracellular potassium or glutamate. However, a clear extracellular alkalinization was found using H(+)-selective microelectrodes under the same conditions. Confocal microscopy revealed that HAF was not localized exclusively to the extracellular surface, but rather was detected throughout the intracellular compartment. A high degree of colocalization between HAF and the mitochondrion-specific dye MitoTracker was observed. When HAF fluorescence was monitored from optical sections from the center of a cell, glutamate produced an intracellular acidification. These results are consistent with a model in which depolarization allows calcium influx, followed by activation of a Ca(2+)/H(+) plasma membrane ATPase. Our results suggest that HAF is reporting intracellular pH changes and that depolarization of horizontal cells induces an extracellular alkalinization, which may relieve H(+)-mediated inhibition of photoreceptor synaptic transmission.     

Rat hippocampal neurons in culture: Ca2+ and Ca2+-dependent K+ conductances

Rat hippocampal neurons grown in dissociated cell culture were studied in a medium containing 1 microM tetrodotoxin (TTX) and 25 mM tetraethylammonium (TEA), which eliminated the Na+ and K+ conductances normally activated by depolarizing current injections. In this medium depolarizing current pulses evoked depolarizing regenerative potentials and afterhyperpolarizations in most cells. Both of these events were blocked by close application of Co2+ or Cd2+. These events resemble Ca2+ spikes reported previously in hippocampal pyramidal cells. The membrane potential at which these Ca2+ spikes could be triggered and the rheobase current necessary were dependent on the potential at which the cell was conditioned: the more depolarized the holding potential, the more negative the absolute potential at which a spike could be triggered and the less rheobase current required. The duration of these Ca2+ spikes was also sensitive to the holding potential: the more depolarized the holding level, the longer the duration of the triggered spikes. The amplitude and duration of the Ca2+ spikes were enhanced in a reversible manner by 0.5-1.0 mM 4-aminopyridine (4-AP) delivered in the vicinity of the cell. Two-electrode voltage-clamp analysis of cells studied in TTX, TEA-containing medium revealed an inward current response that peaked in 25-50 ms during depolarizing commands. This response first became detectable during commands to -30 mV. It peaked in amplitude during commands to -10 mV and was enhanced in medium containing elevated [Ca2+]0. It was blocked by either 20 mM Mg2+, 0.2 mM Cd2+, 5 mM Co2+, or 5 mM Mn2+. These results have led us to identify this inward current response as ICa2+. 4-AP enhanced the magnitude and duration of ICa2+ independent of the drug's depressant effects on a transient K+ current also observed under these same experimental conditions. In many but not all cells the Ca2+ spike was followed by a long-lasting hyperpolarization associated with an increase in membrane conductance. This was blocked by Co2+. Under voltage clamp ICa2+ was followed by a slowly developing outward current response that was attenuated by Co2+ or Cd2+. These properties observed under current- and voltage-clamp recording conditions are superficially similar to those previously reported for Ca2+-dependent K+ conductance mechanisms (IC) recorded in these and other membranes. Long-lasting tail currents following activation of IC inverted in the membrane potential range for the K+ equilibrium potential found in these cells.     

Serotonergic stretch receptors induce plateau properties in a crustacean motor neuron by a dual-conductance mechanism.

1. The mechanisms for induction of bistable plateau potential properties by a set of serotonergic/cholinergic peripheral stretch receptor cells [gastropyloric receptor (GPR) cells] were examined in the crab stomatogastric ganglion (STG) with the use of intracellular recording techniques. 2. GPR cell stimulation evoked nicotinic excitatory postsynaptic potentials (EPSPs) and induced plateau potential capability in the dorsal gastric (DG) motor neuron. The plateau potential could be triggered during a GPR train either by the summating nicotinic EPSPs or by brief intracellular current injection. After pharmacological blockade of nicotinic and muscarinic receptors, a slow depolarization in response to GPR stimulation was revealed. Prolonged plateau potentials could still be evoked after this treatment. Local application of serotonin (5-HT; 10 microM to 1 mM) mimicked the noncholinergic plateau inducing effects of GPR stimulation in the DG motor neuron. 3. The synergistic action of acetylcholine (ACh) and 5-HT was examined by stimulating the GPR cells at different frequencies (1-20 Hz). The plateau induction was present down to 2 Hz. The time to onset for triggering a plateau during a GPR train was determined by the co-released ACh. 4. The 5-HT-evoked slow depolarization persisted in tetrodotoxin (TTX; 0.1-1 microM), and the DG motor neuron could still produce a plateau potential on brief depolarization in the absence of the spike-generating mechanism. 5. In normal TTX-containing saline, the 5-HT-evoked depolarization was accompanied by a weak and variable decrease in apparent input conductance. After substituting one-half of the extracellular sodium with either Trisma-HCl or choline, the decrease in apparent input conductance became more pronounced. This decrease was converted to an increase in apparent input conductance when extracellular Ca2+ was replaced with Mg2+. 6. Under voltage-clamp conditions, local application of 5-HT caused a slow inward current of prolonged duration in DG. The current versus voltage relationship had an inverted U-shape with no apparent reversal potential in the entire voltage range investigated (-90 to -5 mV). The 5-HT-induced changes in input conductance showed a complex voltage dependence, with a conductance decrease from moderately depolarized voltages. 7. Extracellular Cs+ (2-4 mM) caused the DG to hyperpolarize 2-4 mV from rest, whereas lowering extracellular Ca2+ caused it to depolarize 7-15 mV. The combined action of low extracellular Ca2+ and 2-4 mM Cs+ caused an almost complete block of the slow 5-HT-evoked depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of mitochondrial dysfunction in the Ca2+-induced decline of transmitter release at K+-depolarized motor neuron terminals

The present study tested whether a Ca2+-induced disruption of mitochondrial function was responsible for the decline in miniature endplate current (MEPC) frequency that occurs with nerve-muscle preparations maintained in a 35 mM potassium propionate (35 mM KP) solution containing elevated calcium. When the 35 mM KP contained control Ca2+ (1 mM), the MEPC frequency increased and remained elevated for many hours, and the mitochondria within twitch motor neuron terminals were similar in appearance to those in unstimulated terminals. All nerve terminals accumulated FM1-43 when the dye was present for the final 6 min of a 300-min exposure to 35 mM KP with control Ca2+. In contrast, when Ca2+ was increased to 3.6 mM in the 35 mM KP solution, the MEPC frequency initially reached frequencies >350 s-1 but then gradually fell approaching frequencies <50 s-1. A progressive swelling and eventual distortion of mitochondria within the twitch motor neuron terminals occurred during prolonged exposure to 35 mM KP with elevated Ca2+. After approximately 300 min in 35 mM KP with elevated Ca2+, only 58% of the twitch terminals accumulated FM1-43. The decline in MEPC frequency in 35 mM KP with elevated Ca2+ was less when 15 mM glucose was present or when preparations were pretreated with 10 microM oligomycin and then bathed in the 35 mM KP with glucose. When glucose was present, with or without oligomycin pretreatment, a greater percentage of twitch terminals accumulated FM1-43. However, the mitochondria in these preparations were still greatly swollen and distorted. We propose that prolonged depolarization of twitch motor neuron terminals by 35 mM KP with elevated Ca2+ produced a Ca2+-induced decrease in mitochondrial ATP production. Under these conditions, the cytosolic ATP/ADP ratio was decreased thereby compromising both transmitter release and refilling of recycled synaptic vesicles. The addition of glucose stimulated glycolysis which contributed to the maintenance of required ATP levels.     

Photoresponse from the statocyst hair cell in Lymnaea stagnalis

Sensory cells for associative learning of light and turbulence were studied in Lymnaea. Intracellular recordings with Lucifer Yellow filled electrodes were made from photoreceptors and statocyst hair cells. Photoreceptors had a long latency, graded depolarizing response to a flash of light; they extended their axon to the cerebral ganglion. The caudal hair cell, one of 12 cells in the statocyst, responded to brief light with a depolarization and superimposed impulse activity. It formed its terminal arborization close to the photoreceptor endings in the cerebral ganglion. Ca(2+)-free saline reversibly abolished the photoresponse in the hair cell, suggesting the information was conveyed via a chemical synapse. These findings demonstrated that sensory information for associative learning was convergent at the statocyst hair cell.     

Endogenous Na(+)-K+ (or NH4+)-2Cl- cotransport in Rana oocytes; anomalous effect of external NH4+ on pHi.

1. In Rana oocytes, measurements with chloride-sensitive microelectrodes show that the mean intracellular chloride activity (34.8 +/- 6.3 mM, n = 79) is three times higher than that expected for the passive distribution of chloride ions across the outer membrane (12.4 mM, mean membrane potential -43 +/- 8.8 mV, n = 79). 2. Reuptake of chloride into oocytes depleted by prolonged exposure to chloride-free saline takes place against the electrochemical gradient. 3. Chloride reuptake does not take place in sodium-free solution or in a sodium-substituted potassium-free solution. It is inhibited by bumetanide (10(-5) M) in the bathing medium. 4. The overall stoichiometry of the transport mechanism deduced from simultaneous measurements of intracellular sodium and chloride using ion-selective electrodes is 1Na+:1K+:2Cl-. 5. Ammonium ions substitute for potassium on the cotransporter. 6. In oocytes smaller than 0.9 mm in diameter, exposure to external ammonium causes an alkaline shift in intracellular pH as the NH3 enters and takes up H+ to form NH4+. We propose that chloride-dependent NH4+ transport contributes to the accumulation of NH4+ and causes the 'postexposure' acidification as the intracellular NH4+ releases H+ to form NH3 which is then lost from the cell. 7. In larger oocytes ammonium exposure produces a rapid reduction in pHi which may be explained in part by cotransport-mediated uptake of NH4+. Evidence is also provided for a second chloride-dependent NH4+ transport mechanism and a chloride-independent process.     

Energetics of sugar transport by isolated intestinal epithelial cells: effects of cytochalasin B.

The capability of isolated intestinal epithelial cells to establish concentration gradients of 3-O-methylglucose (3-OMG) by a Na+-dependent transport system is limited by concomitant function of a Na+-independent, facilitated diffusion transport system. Monosaccharides accumulated by the active system are continuously lost via the passive system, which acts to lower steady-state sugar gradients maintained by the cell. Cytochalasin B is a potent inhibitor of the passive system and allows the cells to establish a sugar gradient that is much higher than normal. When extracellular [3-OMG] is 1 mM, cytochalasin induces sugar accumulation ratios of 30-fold (+/- phlorizin) in contrast to control ratios of approximately 10-fold. When [3-OMG] is 0.1 mM, cytochalasin (0.1 mM) induces 40-fold accumulation ratios. When changes in extracellular sugar concentration are considered, steady-state concentration gradients observed are 70-fold. For a Na:sugar coupling stoichiometry of 1:1, gradients of this magnitude represent the approximate theoretical maximum for a transport system driven exclusively by the transmembrane electrochemical potential for Na+.     

86Rubidium release from cultured primary astrocytes: effects of excitatory and inhibitory amino acids

The effects of high K+, glutamate and its analogue, kainate, on K+ release were studied in primary astrocyte cultures prepared from newborn rat brains using 86Rb+ as a tracer for K+. An increase in 86Rb+ release was observed when the extracellular K+ concentration was elevated (10-40 mM). Glutamate and kainate stimulated the release in a dose-dependent manner, 100 microM concentrations being about as equally effective as high K+ (40 mM). Both compounds also caused an increase in the absorbance of the cyanine dye, 3,3'-diethylthiadicarbocyanine, indicating depolarization of the membrane. No significant Na+-dependent uptake of [3H]kainate occurred in the cells, thus excluding the possibility that depolarization was due to electrogenic uptake of amino acid into the cells. GABA and taurine significantly depressed the high K+- and glutamate-induced 86Rb+ release. Taurine itself caused a small increase in 86Rb+ release and the membrane was depolarized, judging from the increase in the absorbance of the cyanine dye, 3,3'-diethylthiadicarbocyanine. No effect of taurine was observed when the Cl- concentration was reduced in the experimental medium. The results suggest that cultured astrocytes respond by membrane depolarization to high external K+ and to glutamate and kainate. The degree of this depolarization can be modified by the inhibitory amino acids GABA, taurine and glycine, the effect of taurine probably being mediated by an increase in Cl- conductance across the cell membrane. The role of functional receptors for amino acid transmitters and the effects observed are discussed.