Suramin affects capsaicin responses and capsaicin-noxious heat interactions in rat dorsal root ganglia neurones.

The effect of suramin, an inhibitor of G protein regulated signalling, was studied on the membrane currents induced by noxious heat and by capsaicin in cultured dorsal root ganglia neurones isolated from neonatal rats. Whole-cell responses induced by a heat ramp (24-52 degrees C) were little affected by suramin. The noxious heat-activated currents were synergistically facilitated in the presence of 0.3 microM capsaicin 13.2-fold and 6.3-fold at 40 degrees C and 50 degrees C, respectively. In 65% of neurones, the capsaicin-induced facilitation was inhibited by 10 microM suramin to 35 +/- 6% and 53 +/- 6% of control at 40 degrees C and 50 degrees C (S.E.M., n = 15). Suramin 30 microM caused a significant increase in the membrane current produced by a nearly maximal dose (1 microM) of capsaicin over the whole recorded temperature range (2.4-fold at 25 degrees C and 1.2-fold at 48 degrees C). The results demonstrate that suramin differentially affects the interaction between capsaicin and noxious heat in DRG neurones and thus suggest that distinct transduction pathways may participate in vanilloid receptor activation mechanisms.

The effects of capsaicin and acidity on currents generated by noxious heat in cultured neonatal rat dorsal root ganglion neurones.

1. The effects of capsaicin, acidic pH, ATP, kainate and GABA on currents generated by noxious heat were studied in cultured dorsal root ganglion (DRG) neurones (< 20 microm in diameter) isolated from neonatal rats. The patch clamp technique was used to record membrane currents or changes of membrane potential. 2. In agreement with previous results, inward membrane currents (I(heat)) induced by a 3 s ramp of increasing temperature from room temperature (approximately 23 degrees C) to over 42 degrees C varied greatly between cells (-100 pA to -2.4 nA at 48 degrees C) and had a temperature coefficient (Q(10)) > 10 over the range of 43-52 degrees C. 3. Capsaicin potentiated the heat-induced current even when capsaicin, at room temperature, had little or no effect on its own. In cells in which capsaicin induced no or very small membrane current at room temperature (< 50 pA), I(heat) exhibited detectable activation above 40 degrees C and increased 5.1 +/- 1.1 (n = 37) and 6.3 +/- 2.0 (n = 18) times at 0.3 and 1 microM capsaicin, respectively. 4. A rapid decrease in extracellular pH from 7.3 to 6.8, 6.3 or 6.1 produced an inward current which inactivated in ~5 s either completely (pH 6.8 or 6.3) or leaving a small current (approximately 50 pA) for more than 2 min (pH 6.1). After inactivation of the initial low pH-induced current, I(heat) at 48 degrees C increased 2.3 +/- 0.4 times at pH 6.8, 4.0 +/- 0.6 times at pH 6.3 and 4.8 +/- 0.8 times at pH 6.1 with a Q(10) > 10 (n = 16). 5. ATP (n = 22), kainate (n = 7) and GABA (n = 8) at 100 microM, produced an inactivating inward current in all heat-sensitive DRG neurones tested. During inactivation and in the presence of the drug, I(heat) was increased slightly with ATP and unaffected with kainate and GABA. These agents apparently do not directly affect the noxious heat receptor. 6. The results indicate a novel class of capsaicin-sensitive cells, in which capsaicin evokes no or very small inward current but nevertheless increases sensitivity to noxious heat.

Membrane currents and morphological properties of neurons and glial cells in the spinal cord and filum terminale of the frog.

Using the patch-clamp technique in the whole-cell configuration combined with intracellular dialysis of the fluorescent dye Lucifer yellow (LY), the membrane properties of cells in slices of the lumbar portion of the frog spinal cord (n=64) and the filum terminale (FT, n=48) have been characterized and correlated with their morphology. Four types of cells were found in lumbar spinal cord and FT with membrane and morphological properties similar to those of cells that were previously identified in the rat spinal cord (Chvátal, A., Pastor, A., Mauch, M., Syková, E., Kettenmann, H., 1995. Distinct populations of identified glial cells in the developing rat spinal cord: Ion channel properties and cell morphology. Eur. J. Neurosci. 7, 129-142). Neurons, in response to a series of symmetrical voltage steps, displayed large repetitive voltage-dependent Na(+) inward currents and K(+) delayed rectifying outward currents. Three distinct types of non-neuronal cells were found. First, cells that exhibited passive symmetrical non-decaying currents were identified as astrocytes. These cells immunostained for GFAP and typically had at least one thick process and a number of fine processes. Second, cells with the characteristic properties of rat spinal cord oligodendrocytes, with passive symmetrical decaying currents and large tail currents after the end of the voltage step. These cells exhibited either long parallel or short hairy processes. Third, cells that expressed small brief inward currents in response to depolarizing steps, delayed rectifier outward currents and small sustained inward currents identical to rat glial precursor cells. Morphologically, they were characterized by round cell bodies with a number of finely branched processes. LY dye-coupling in the frog spinal cord gray matter and FT was observed in neurons and in all glial populations. All four cell types were found in both the spinal cord gray matter and FT. The glia/neuron ratio in the spinal cord was 0.78, while in FT it was 2.0. Moreover, the overall cell density was less in the FT than in the spinal cord. The present study shows that the membrane and morphological properties of glial cells in the frog and rat spinal cords are similar. Such striking phylogenetic similarity suggests a significant contribution from distinct glial cell populations to various spinal cord functions, particularly ionic and volume homeostasis in both mammals and amphibians.

Müller glial cells in anuran retina.

Whereas in the brain, the activity of the neurons is supported by several types of glial cells such as astrocytes, oligodendrocytes, and ependymal cells, the retina (evolving from the brain during ontogenesis) contains only one type of macroglial cell, the Müller (radial glial) cells, in most vertebrates including the anurans. These cells span the entire thickness of the tissue, and thereby contact and ensheath virtually every type of neuronal cell body and process. This intimate topographical relationship is reflected by a multitude of functional interactions between retinal neurons and Müller glial cells. Müller cells are the principal stores of retinal glycogen, and are thought to fuel retinal neurons with substrate (lactate/pyruvate) for their oxidative metabolism. Furthermore, Müller cells are involved in the control and homeostasis of many constituents of the extracellular space, such as potassium and perhaps other ions, signaling molecules, and of the extracellular pH. They also seem to play important roles in recycling mechanisms of photopigment molecules and neurotransmitter molecules such as glutamate and GABA. By containing the main retinal stores of glutathione, Müller cells may protect retinal neurons against free radicals. Moreover, Müller cells express receptors for many neuroactive substances, and may also release such substances to their neighbouring neurons. Thus, Müller cells exert many functions crucial for signal processing in the normal retina. Moreover, Müller cells change their properties in cases of retinal disease and injury, and may either support the survival of neuronal cells or accelerate the progress of neuronal degeneration.

Spatial distribution of spermine/spermidine content and K(+)-current rectification in frog retinal glial (Müller) cells.

Previous studies in retinal glial (Müller) cells have suggested that (1) the dominant membrane currents are mediated by K(+) inward-rectifier (Kir) channels (Newman and Reichenbach, Trends Neurosci 19:307-312, 1996), and (2) rectification of these Kir channels is due largely to a block of outward currents by endogenous polyamines such as spermine/spermidine (SPM/SPD) (Lopatin et al., Nature 372:366-369, 1994). In frog Müller cells, the degree of rectification of Kir-mediated currents is significantly higher in the endfoot than in the somatic membrane (Skatchkov et al., Glia 27:171-181, 1999). This article shows that in these cells there is a topographical correlation between the local cytoplasmic SPM/SPD immunoreactivity and the ratio of inward to outward K(+) currents through the surrounding membrane area. Throughout the retina, Müller cell endfeet display a high SPM/SPD immunolabel (assessed by densitometry) and a large inward rectification of K(+) currents, as measured by the ratio of inward to outward current produced by step changes in [K(+)](o). In the retinal periphery, Müller cell somata are characterized by roughly one-half of the SPM/SPD immunoreactivity and K(+)-current rectification as the corresponding endfeet. In the retinal center, Müller cell somata are virtually devoid of both SPM/SPD immunolabel and K(+)-current inward rectification. Comparing one region of the retina with another, we find an exponential correlation between the local K(+) rectification and the local SPM/SPD content. This finding suggests that the degree of inward rectification in a given membrane area is determined by the local cytoplasmic polyamine concentration.

Potassium buffering by Müller cells isolated from the center and periphery of the frog retina.

Müller (radial glial) cells span the retina from the outer to the inner limiting membranes. They are the only glial cells found in the amphibian retina. The thickness of the frog (Rana pipiens) retina decreases by a factor of about four from the center to the periphery. Thus, Müller cells were isolated, by enzymatic dissociation, with stalk lengths from 20 to 140 microm. Their ability to transfer K(+) via the stalk between soma and endfoot was studied. Membrane currents were recorded using the whole-cell voltage-clamp technique with the pipette sealed to either the endfoot or the soma. Inward (I(KIN)) or outward (I(KO)) currents were elicited by rapid increases (3 to 10 mM) or decreases (3 to 1 mM) of the extracellular K(+) concentration ([K(+)](o)) either by local application (close or distant to the recording pipette) or around the entire cell (whole cell perfusion). For the long central cells, the ratio I(KIN)/I(KO) was 4.6 +/- 0.6 SE (n = 9) at the endfoot and 1.7 +/- 0.1 SE (n = 8) at the soma. In cells from the retinal periphery, the ratio I(KIN)/I(KO) was higher, 7.0 +/- 0.27 (n = 8) at the endfoot and 3.2 +/- 0.1 (n = 10) at the soma. The results suggest that there is less inward rectification in the somatic than in the endfoot membrane. As expected from previous studies, the sensitivity of the cells to K(+) was higher at the endfoot than at the soma. The amplitude of I(KIN) at the endfoot compared to the soma was about 8-fold for the long central cells but only about 1.5-fold for the short peripheral cells. Currents spread readily from endfoot to soma in the peripheral cells. In the long central Müller cells the soma and endfoot appeared electrotonically isolated. The "functional length constant", lambda, of cell stalk processes was about 70 microm. The relative decrement of large inward currents was stronger than that of smaller outward currents; this difference ("artificial rectification") is explained by a simple model, where larger currents (inward) are attenuated more than smaller (outward) currents. The data support the hypothesis that in the retinal periphery, Müller cells provide extensive spatial K(+) buffering from both plexiform layers into the vitreous body. In the central retina, however, such currents are limited within a short (interlaminar) range.

Temperature coefficient of membrane currents induced by noxious heat in sensory neurones in the rat.

1. Membrane currents induced by noxious heat (Iheat) were studied in cultured dorsal root ganglion (DRG) neurones from newborn rats using ramps of increasing temperature of superfusing solutions. 2. Iheat was observed in about 70 % of small (< 25 microm) DRG neurones. At -60 mV, Iheat exhibited a threshold at about 43 C and reached its maximum, sometimes exceeding 1 nA, at 52 C (716 +/- 121 pA; n = 39). 3. Iheat exhibited a strong temperature sensitivity (temperature coefficient over a 10 C temperature range (Q10) = 17.8 +/- 2.1, mean +/- s.d., in the range 47-51 C; n = 41), distinguishing it from the currents induced by capsaicin (1 microM), bradykinin (5 microM) and weak acid (pH 6.1 or 6.3), which exhibited Q10 values of 1.6-2.8 over the whole temperature range (23-52 C). Repeated heat ramps resulted in a decrease of the maximum Iheat and the current was evoked at lower temperatures. 4. A single ramp exceeding 57 C resulted in an irreversible change in Iheat. In a subsequent trial, maximum Iheat was decreased to less than 50 %, its threshold was lowered to a temperature just above that in the bath and its maximum Q10 was markedly lower (5.6 +/- 0.8; n = 8). 5. DRG neurones that exhibited Iheat were sensitive to capsaicin. However, four capsaicin-sensitive neurones out of 41 were insensitive to noxious heat. There was no correlation between the amplitude of capsaicin-induced responses and Iheat. 6. In the absence of extracellular Ca2+, Q10 for Iheat was lowered from 25.3 +/- 7.5 to 4. 2 +/- 0.4 (n = 7) in the range 41-50 C. The tachyphylaxis, however, was still observed. 7. A high Q10 of Iheat suggests a profound, rapid and reversible change in a protein structure in the plasma membrane of heat-sensitive nociceptors. It is hypothesized that this protein complex possesses a high net free energy of stabilization (possibly due to ionic bonds) and undergoes disassembly when exposed to noxious heat. The liberated components activate distinct cationic channels to generate Iheat. Their affinity to form the complex at low temperatures irreversibly decreases after one exposure to excessive heat.

Procaine excites nociceptors in cultures from dorsal root ganglion of the rat.

Procaine, a classical local anesthetic, produces, at low concentration (2-200 microM), excitation in a distinct population of small sensory neurons isolated from newborn rats (2D) and cultured for 1-5 days. The excitation or inward current (>50 pA) induced by procaine was observed in 59 out of 78 neurons. Nearly all these procaine-sensitive neurons (56 of 59) were also sensitive to capsaicin while 8 procaine-insensitive neurons responded to capsaicin (1 microM). In procaine-sensitive neurons tested for responsiveness to noxious heat, a 10 s temperature ramp from 24 to 48 degrees C induced an inward current of 413 +/- 47 pA (SEM, n = 27) and this current was enhanced, in the presence of procaine, about 3-fold (2.8 +/- 0.4, SEM, n = 27). The responses to procaine were concentration dependent and underwent pronounced tachyphylaxis after repeated applications. The voltage-current relationship exhibited outward rectification and the apparent reversal at 25 +/- 4.2 mV (SEM, n = 9) suggesting that the current is carried by cations including Ca2+. This procaine effect may offer an explanation for toxic consequence of the clinical use of local anesthetics.

K+ Channel density increases selectively in the endfoot of retinal glial cells during development of Rana catesbiana.

The radial glial cells that span the retina, described by Müller in 1851, have a remarkable distribution of ion channels in adult amphibia that mediate extracellular K+ spatial buffering. 94% of the total membrane conductance of these cells resides in inward rectifier K+ channels in the endfoot processes apposed to the vitreous humour. We now report that this regional specialization is found in Müller cells isolated from adult (>120 day old) bullfrogs but to a far less extent in those from 10-20 day old tadpoles (stages 34-36). Using the cell attached configuration of the patch-clamp technique, we found, in agreement with previous studies in salamanders, that the endfoot of adult cells had 19.2+/-2.4 (mean +/- S.E., n = 81) channels/patch, whereas the soma had 1.81+/-0.28 (n = 21) channels/patch. In the tadpole, the respective values were 4.29+/-0.26 (n = 79) for the endfoot and 2.26+/-0.24 (n = 27) for the soma. The slope conductance of the inward rectifier K+ channel in 115 mM K+, 19.2+/-0.25 pS (n = 205), channel kinetics and the resting membrane potential (-69+/-2.7 mV, n = 224) were similar at both the endfoot and soma of both adults and embryos. We conclude that during development, the K+ conductance of the Müller cell endfoot, but not of the soma, increases due to a selective clustering of inwardly rectifying K+ channels in that specific region of the cell membrane. The properties of the channels change little during the transformation from tadpole to adult bullfrog.

Glial calcium: homeostasis and signaling function.

Glial cells respond to various electrical, mechanical, and chemical stimuli, including neurotransmitters, neuromodulators, and hormones, with an increase in intracellular Ca2+ concentration ([Ca2+]i). The increases exhibit a variety of temporal and spatial patterns. These [Ca2+]i responses result from the coordinated activity of a number of molecular cascades responsible for Ca2+ movement into or out of the cytoplasm either by way of the extracellular space or intracellular stores. Transplasmalemmal Ca2+ movements may be controlled by several types of voltage- and ligand-gated Ca(2+)-permeable channels as well as Ca2+ pumps and a Na+/Ca2+ exchanger. In addition, glial cells express various metabotropic receptors coupled to intracellular Ca2+ stores through the intracellular messenger inositol 1,4,5-triphosphate. The interplay of different molecular cascades enables the development of agonist-specific patterns of Ca2+ responses. Such agonist specificity may provide a means for intracellular and intercellular information coding. Calcium signals can traverse gap junctions between glial cells without decrement. These waves can serve as a substrate for integration of glial activity. By controlling gap junction conductance, Ca2+ waves may define the limits of functional glial networks. Neuronal activity can trigger [Ca2+]i signals in apposed glial cells, and moreover, there is some evidence that glial [Ca2+]i waves can affect neurons. Glial Ca2+ signaling can be regarded as a form of glial excitability.

Changes in glial K+ currents with decreased extracellular volume in developing rat white matter.

Whole cell patch-clamp recordings of K+ currents from oligodendrocyte precursors in 10-day-old rats (P10) and, following myelination, in mature oligodendrocytes from 20-day-old rats (P20) were correlated with extracellular space (ECS) diffusion parameters measured by the local diffusion of iontophoretically injected tetramethylammonium ions (TMA+). The aim of this study was to find an explanation for the changes in glial currents that occur with myelination. Oligodendrocyte precursors (P10) in slices from corpus callosum were characterized by the presence of A-type K+ currents, delayed and inward rectifier currents, and lack of tail currents after the offset of a voltage jump. Mature oligodendrocytes in corpus callosum slices from P20 rats were characterized by passive, decaying currents and large tail currents after the offset of a voltage jump. Measurements of the reversal potential for the tail currents indicate that they result from increases in [K+]e by an average of 32 mM during a 20 msec 100 mV voltage step. Concomitant with the change in oligodendrocyte electrophysiological behavior after myelination there is a decrease in the ECS of the corpus callosum. ECS volume decreases from 36% (P9-10) to 25% (P20-21) of total tissue volume. ECS tortuosity lambda = (D/ADC)0.5, where D is the free diffusion coefficient and ADC is the apparent diffusion coefficient of TMA+ in the brain, increases as measured perpendicular to the axons from 1.53 +/- 0.02 (n = 6, mean +/- SEM) to 1.70 +/- 0.02 (n = 6). TMA+ non-specific uptake (k') was significantly larger at P20 (5.2 +/- 0.6 x 10(-3) s(-1), n = 6) than at P10 (3.5 +/- 0.4 x 10(-3) s(-1), n = 6). It can be concluded that membrane potential changes in mature oligodendrocytes are accompanied by rapid changes in the K+ gradient resulting from K+ fluxes across the glial membrane. As a result of the reduced extracellular volume and increased tortuosity, the membrane fluxes produce larger changes in [K+]e in the more mature myelinated corpus callosum than before myelination. These conclusions also account for differences between membrane currents in cells in slices compared to those in tissue culture where the ECS is essentially infinite. The size and geometry of the ECS influence the membrane current patterns of glial cells and may have consequences for the role of glial cells in spatial buffering.

Glial-neuronal interactions in non-synaptic areas of the brain: studies in the optic nerve.

Optic nerves, like other CNS tracts, consist of axons closely apposed across narrow extracellular clefts to the cell bodies and processes of glial cells. Despite the anatomical simplicity of these pathways and the absence of synapses, a surprising range of interactions occurs between axons and glial cells mediated by changes in the chemical composition of the extracellular fluid produced by glial or neuronal stimulation. Some of the interactions are relatively brief, resulting from alterations in extracellular ions such as K+ or H+, or alterations of small molecules like glutamate or ATP. Other interactions involve much longer time periods and presumably larger signaling molecules, like peptides or proteins. These play a role not only in the development of axonal pathways but also in the processes of degeneration and regeneration that follow brain injury or disease.

Potassium currents in cultured glia of the frog optic nerve.

The processes that participate in clearing increases in [K+]o produced by active neurons include KCl uptake, Na pump stimulation, and spatial buffering. The latter process requires glial cells to carry: 1) inward K+ currents in regions where K+ is elevated at a glial membrane potential more negative than EK; and 2) outward K+ currents at normal K+ and glial membrane potential more positive than EK (Orkand et al: J Neurophysiol 29:788, 1966). Techniques for isolation and culturing glial cells brought new possibilities for studying ionic channels involved in spatial buffering. However, they raised the question of the extent to which the properties of ionic channels are changed due to the process of culturing when glial cells are exposed to an artificial environment and deprived of direct interaction with neurons. We studied potassium currents in glial cells from the frog optic nerve that were cultured for 1-8 days. At 24-48 h, cells exhibited an inwardly rectifying Cs+ blocked current (IK(IN)) that increased in amplitude and shifted its threshold of activation to EK when [K+]o was increased from 3 to 6 or 10 mM. IK(IN), diminished after 3 days in culture and virtually disappeared after 5 days. At 24-48 h, a potassium delayed rectifier current (IKD) was relatively small but became large at 3 days, and was practically the only current present after 5 days. IKD was activated at -8.5 +/- 0.58 mV(SE, n = 48) and 58 +/- 2.2% (SE, n = 48) blocked by 20 mM tetraethylammonium. The results of this study support the idea that the inward rectifying potassium channels (Kir) are responsible for carrying K+ into glial cells whenever [K+]o increases. However, the delayed rectifier potassium channels (KD) cannot provide the pathway for outward K+ current during spatial buffering, and another mechanism must be involved in this process. Our study provides further evidence that culture conditions can greatly influence functional expression of ionic channels in glial cells.

Effect of cutting the optic nerve on K+ currents in endfeet of Muller cells isolated from frog retina.

Membrane currents were recorded from Muller cells isolated from normal retinas and from retinas whose ganglion cell axons had been cut in the optic nerve 30-60 days previously. The surgical procedure did not block the retinal blood supply and did not allow the axons to regenerate. The principal finding was that after severing the optic nerve there was less inward rectification in response to voltage commands. That is, the maintained inward current (I K(IN)) produced in response to a hyperpolarizing voltage command decreased leading to a decrease in the ratio I K(IN)/I K(OUT) In 98 mM [K+]O, this ratio was 2.86 +/- 0.21 (mean +/- SE; n = 24) in controls and 1.13 +/- 0.13 (n = 21) in Muller cells from denervated retinae. Barium, a blocker of the potassium inward rectifier (I (KIR)), eliminated this difference. Moreover, severing the optic nerve also decreased the resting potentials of Muller cells in 2.5 mM [K+]O from -83 +/- 7 mV to -63 +/- 9 mV. The results suggest that the voltage-dependent behavior and selectivity of K+ inward rectifying channels (K (ir)) in the endfeet depends on the integrity of the closely apposed ganglion cells.

Nerve impulses increase glial intercellular permeability.

Coordinating the activity of neurons and their satellite glial cells requires mechanisms by which glial cells detect neuronal activity and change their properties as a result. This study monitors the intercellular diffusion of the fluorescent dye Lucifer Yellow (LY), following its injection into glial cells of the frog optic nerve, and demonstrates that nerve impulses increase the permeability of interglial gap junctions. Consequently, the spatial buffer capacity of the neuroglial cell syncytium for potassium, other ions, and small molecules will be enhanced; this may facilitate glial function in maintaining homeostasis of the neuronal microenvironment.

Effects of low doses of caffeine on [Ca2+]i in voltage-clamped snail (Helix aspersa) neurones.

1. We have measured cytosolic free Ca2+ concentrations ([Ca2+]i) in voltage-clamped snail neurones using fura-2. Transient increases in [Ca2+]i were induced by depolarizing voltage steps of 20-60 mV for 0.1-10 s from a holding potential of -50 or -60 mV. 2. Low doses of caffeine, 0.2-1 mM, increased the size of the [Ca2+]i transients by both increasing the peak and producing an undershoot. 3. Ryanodine, an inhibitor of Ca2+ release from the intracellular Ca2+ stores, and cyclopiazonic acid (CPA), an inhibitor of the Ca(2+)-ATPase of the intracellular Ca2+ stores, both reduced the size of the [Ca2+]i transients and blocked the effects of caffeine on the transients. 4. The effects of caffeine and CPA were greater on transients produced by long, small, rather than short, large depolarizations. This suggests that calcium-induced calcium release (CICR) played a greater role in the [Ca2+]i increase resulting from longer, smaller depolarizations. 5. Increasing the extracellular pH from 7.5 to over 9, which inhibits the plasmalemmal Ca(2+)-H(+)-ATPase, increased the resting [Ca2+]i level. Depolarization-induced [Ca2+]i transients became much larger while the two effects of caffeine remained. CPA was ineffective at high pH. 6. In some experiments the increase in basal [Ca2+]i caused by alkaline pH was reduced by 0.2 or 0.5 mM caffeine. The increase in basal [Ca2+]i caused by maintained depolarization was reduced, after a transient increase, by 0.5 mM caffeine. Both reduction and increase were blocked by CPA. 7. We conclude that low doses of caffeine can increase uptake by intracellular Ca2+ stores. Caffeine could also release Ca2+ from ryanodine-insensitive Ca(2+)-ATPase-dependent stores as well as facilitating normal ryanodine-sensitive CICR.

Potassium currents in endfeet of isolated Müller cells from the frog retina.

Voltage dependent potassium currents were recorded using the whole-cell mode of the patch-clamp technique for the first time from endfeet of Müller cells dissociated from the frog retina. Recordings from intact cells and isolated endfeet indicate that the inward rectifier potassium channel is the dominant ion channel in these cells and that the density of these channels is highest in the endfoot as has been previously reported for several other species. The present study uses rapid changes in [K+]o to understand the behavior of these channels in buffering [K+]o in the retina. With rapid changes in [K+]o, it was found that, at a membrane potential of -90mV, which is close to EK, increasing [K+]o from 3 to 10 mM produced an inward K+ current 5.48 +/- 0.89 SD (n = 9) times larger than outward current induced by decreasing [K+]o from 3 to 1 mM. The outward current was maximal at a holding potential of about -80mV and exhibited inactivation at more positive potentials. At -40 mV both the inward and outward currents are markedly reduced. The current voltage curve for the inward current was linear at holding potentials from -50 mV to -140 mV. Using 20 mV voltage steps, it was found that the voltage dependent K+ currents were unaffected by the addition of 2 mM Cd2+, a blocker of Ca(2+)-activated potassium currents, decreasing [Cl-]o from 120 mM to 5 mM or the substitution of 30 mM Na+ by TEA. The addition of 5 mM [Cs+]o blocked only the inward current. Both the outward and the inward currents disappeared in the absence of intracellular and extracellular K+; 0.3 mM [Ba2+]o blocked the inward current completely and strongly inhibited the outward current in a time and voltage dependent manner. We conclude that at physiological [K+]o and membrane potential, the K+ channels in the Müller cell endfoot are well suited to carry K+ both inward and outward across the membrane as required for spatial buffering.

Serotonin- and proton-induced and modified ionic currents in frog sensory neurons.

Using the whole-cell patch-clamp technique, the effects of serotonin (5-HT) and increased acidity to produce membrane currents and to modify high threshold voltage-dependent calcium currents were studied in isolated dorsal root ganglion (DRG) cells of the frog maintained in short-term culture. DRG cells were classified by morphology into two types: (1) cells with a large number of dark rusty brown granules, and (2) cells devoid of these granules or with few scattered pale granules. Fast application of 5-HT (10-30 microM) induced a rapidly desensitizing inward current with a reversal potential at about 0 mV in 38 of 50 granule-containing neurons (76%) which was never observed (0/35) in "clear" neurons. This current was blocked by 10 nM (+)-tubocurarine. In addition, a small noninactivating outward current was also observed in most DRG neurons during 5-HT superfusion. A sudden decrease of pH from 7.4 to 6 or 5.8 induced a fast inactivating inward current of 100-300 pA in 74% of the "clear" neurons and only 24% of the granule-containing neurons. Small noninactivating membrane currents induced by lowering pH were observed in all neurons. Both 5-HT and increased extracellular H+ reduced the magnitude of high threshold calcium currents in all DRG neurons. It is suggested that the 5-HT receptors are expressed on a morphologically distinct population of neurons while the cells with channels responsible for the fast inactivating proton-induced current cannot be related to any distinct morphological cell type.

NMDA receptors on adult frog spinal motoneurons in culture.

Whole-cell membrane currents induced by superfusion of NMDA were examined in cultured motoneurons from the spinal cord of the adult frog in Mg(2+)-free Ringer solution containing 10 microM glycine. The amplitude of the response to 100 microM NMDA was 280 +/- 37 pA (mean +/- S.D.; n = 24) with a reversal potential +6.1 +/- 3.0 mV (mean +/- S.D.; n = 6). At a membrane potential of -60 mV, the response to 100 microM NMDA was blocked by 0.1 mM Mg2+ or 100 microM AP5. From the dose-response curve, the estimated EC50 was 77 microM and the calculated Hill coefficient was 1.6. NMDA receptors on frog motoneurons appear to have properties similar to those of mammals but may be expressed at lower density.

Facilitation of sodium currents in frog neuroglia by nerve impulses: dependence on external calcium.

Facilitation of voltage-gated sodium currents in glial membranes by nerve impulses has been studied by using both the whole cell and loose-patch clamp techniques in the isolated intact optic nerve of the frog. During facilitation there is a shift in the voltage dependence of glial Na+ channels such that a given depolarization produces a larger inward Na+ current. Decreasing external calcium from 4 times normal to 0.2 times normal produced a similar shift in the current-voltage relation. Increasing the external calcium concentration to 4-5 times normal blocks facilitation. In reduced calcium, 0.1-0.2 times normal, the peak of facilitation was unaffected, but its decay was slowed. The addition of 1 mM nickel and 2 mM cobalt or 2 mM cadmium, to prevent depletion of extracellular calcium that might result from voltage-dependent entry of calcium into the axons, did not block the facilitation. The results suggest that, even though facilitation is blocked by high extracellular calcium, a decrease in extracellular calcium produced by axon impulses is not the cause of the facilitation.