Non-NMDA receptor-mediated modulation of voltage-activated outward currents in chick neurones

The effect of kainate on the voltage-activated current of cultured embryonic chick telencephalic neurones was studied by whole-cell voltage clamp recording. In addition to opening non-NMDA receptor coupled ion channels, kainate produced additional effects, with a slower time course: it modulated voltage-activated currents. These effects were blocked by the non-NMDA receptor antagonists CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) and GYKI 53784 ((-)-1-(4-aminophenyl)-3- methylcarbamoyl-4-methyl-7,8-methylenedioxy-3,4- dihydro-5H-2,3-benzodiazepine), indicating that non NMDA receptors are involved. It has been shown that the reversible inhibition of outward potassium currents was partly due to a decrease of current amplitude and partly to a marked shift of the inactivation curve towards more negative potentials.     

Modulation of voltage-activated calcium currents by mechanical stimulation in rat sensory neurons

We examined the effects of mechanical stress, induced by a stream of bath solution, on evoked action potentials, electrical excitability, and Ca2+ currents in rat dorsal root ganglion neurons in culture with the use of the whole cell patch-clamp technique. Action-potential duration was altered reversibly by flow in 39% of the 51 neurons tested, but membrane potential and excitability were unaffected. The flow-induced increases and decreases in action-potential duration were consistent with the different effects of flow on two types of Ca2+ channel, determined by voltage-clamp recordings of Ba2+ currents. Current through omega-conotoxin-sensitive (N-type) Ca2+ channels increased by an estimated 74% with flow, corresponding to 23% increase in the total high voltage-activated current, whereas current through low-threshold voltage-activated (T-type) channels decreased by 14%. We conclude that modulation of voltage-activated Ca2+ currents constitutes a route by which mechanical events can regulate Ca2+ influx in sensory neurons.     

Calcium currents and calcium signaling in rod bipolar cells of rat retinal slices

Combined electrophysiological and imaging techniques were used to study calcium currents (ICa) and their sites of origin at rod bipolar cells in rat retinal slices. We report here for the first time the successful whole-cell patch-clamp recording from presynaptic boutons that were compared with somatic recordings. TTX-resistant inward currents were elicited in response to depolarization. The kinetic and pharmacological properties of ICa were very similar for recordings obtained from the soma and the presynaptic terminals. ICa activated maximally between -30 and -20 mV was enhanced by Bay K 8644 and was blocked by isradipine and nifedipine. Peak amplitude and time to peak were -31.3 +/- 1.2 pA and 3.2 +/- 0.2 msec with somatic recordings (n = 54), whereas the corresponding values were -31.6 +/- 6.1 pA and 3.2 +/- 0.7 msec in recordings obtained directly from terminals (n = 6). ICa showed little inactivation during sustained depolarizations. No T-type ICa was observed with depolarizations from -90 mV. Concomitant with Ca2+ entry, depolarization induced the appearance of transient outward currents that resembled IPSCs and were blocked by GABA and glycine receptor antagonists, suggesting that they arise from activation of amacrine feedback synapses. Upon depolarization, intracellular Ca2+ ([Ca2+]i) rises were restricted to the presynaptic terminals with no somatic or axonal changes and were linearly dependent on pulse duration when using a low-affinity Ca2+ indicator. In cone bipolar cells, ICa inactivated markedly, and [Ca2+]i rises occurred in the axon, as well as in the presynaptic terminals.     

Evidence for calcium/calmodulin dependence of spinule retraction in retinal horizontal cells

Horizontal cells of the carp retina alter their synaptic connections with cones during dark and light adaptation. At light onset, dendrites of horizontal cells, which are positioned laterally at the ribbon synapse, form "spinules," little processes with membrane densities. Spinules are retracted again during dark adaptation. Spinule retraction is also elicited upon glutamate application to the retina. In the present study, we address the question whether calcium/calmodulin-dependent pathways are involved in dark- and glutamate-evoked spinule retraction. Light-adapted retinas were isolated and subsequently dark adapted during incubation in media of different calcium concentrations. Spinule retraction was clearly blocked in low-calcium solutions (5 microM and 50 nM CaCl2). Incubation in medium containing cobalt chloride (2 mM) had the same effect. Both treatments blocked the glutamate-induced spinule retraction as well. These results indicate that spinule retraction is induced by a calcium influx into horizontal cells. To investigate whether calmodulin, the primary calcium receptor in eukaryotic cells, is present at the site of spinule formation, light- and dark-adapted retinas, embedded in LR White resin, were labelled with an antibody against calmodulin and gold-conjugated secondary antibodies. Horizontal cell dendrites at the ribbon synapse revealed strong calmodulin immunoreactivity, which was more than twice as high in light- as in dark-adapted retinas. The incubation of isolated retinas with the calmodulin antagonists W5 and W13 inhibited spinule retraction. In summary, these results suggest that spinule retraction may be regulated by calcium influx into horizontal cells and subsequent calcium/calmodulin-dependent pathways.     

Activation and inactivation properties of voltage-gated calcium currents in developing cat retinal ganglion cells

The correlated activity of developing retinal ganglion cells is essential for the reorganization and refinement of retinogeniculate projections. Previous studies have uncovered marked changes in the spiking properties of retinal ganglion cells during this period of reorganization; however, a full understanding of the changes in the underlying ionic conductances has yet to be obtained. To this end, the whole-cell configuration of the patch-clamp technique was used to record currents conducted by voltage-gated calcium channels in 83 dissociated cat retinal ganglion cells obtained from animals aged between embryonic day 34 and postnatal day 105. Calcium currents, magnified by using barium as the major charge carrier, were isolated by substituting choline for Na+ in the bathing solution and Cs+ for K+ in the electrode solution. Three voltage-gated Ca2+ conductances were identified based on their voltage dependence and kinetics of activation and inactivation: a transient low-voltage-activated conductance, a transient high-voltage-activated conductance and a sustained high-voltage-activated conductance. During the developmental period examined there were significant increases in the densities of all three conductances, as well as significant changes in some of their activation and inactivation properties. These findings, together with those reported previously for the voltage-gated Na+ and K+ conductances, are related to the generation of excitability in developing retinal ganglion cells during a period critical to the normal development of the visual system. Furthermore, while the sustained high-voltage-activated conductance was present in all of the retinal ganglion cells observed, only about 72% expressed the transient high-voltage-activated current. During the developmental period examined, there was also an increase in the proportion of cells expressing the transient low-voltage-activated conductance. This, along with our previous finding that retinal ganglion cells heterogeneously express different types of voltage-gated K+ channels, strongly suggests that the spiking patterns observed in different classes of retinal ganglion cell may be due, in part, to their intrinsic membrane properties.     

Characterization of spontaneous inhibitory synaptic currents in salamander retinal ganglion cells

Spontaneous and light-evoked postsynaptic currents (sPSCs and lePSCs, respectively) in retinal ganglion cells of the larval tiger salamander were recorded under voltage-clamp conditions from living retinal slices. The focus of this study is to characterize the spontaneous inhibitory PSCs (sIPSCs) and their contribution to the light-evoked inhibitory PSCs (leIPSCs) in ON-OFF ganglion cells. sIPSCs were isolated from spontaneous excitatory PSCs (sEPSCs) by application of 10 microM 6,7-dinitroquinoxaline-2,3-dione (DNQX) + 50 microM 2-amino-5-phosphonopentanoic acid (AP5). In approximately 70% of ON-OFF ganglion cells, bicuculline (or picrotoxin) completely blocks sIPSCs, suggesting all sIPSCs in these cells are mediated by GABAergic synaptic vesicles and gamma-aminobutyric acid-A (GABAA) receptors (GABAergic sIPSCs, or GABAsIPSCs). In the remaining 30% of - ganglion cells, bicuculline (or picrotoxin) blocks 70-98% of the sIPSCs, and the remaining 2-30% are blocked by strychnine (glycinergic sIPSCs, or GLYsIPSCs). GABAsIPSCs occur randomly with an exponentially distributed interval probability density function, and they persist without noticeable rundown over time. The GABAsIPSC frequency is greatly reduced by cobalt, consistent with the idea that they are largely mediated by calcium-dependent vesicular release. GABAsIPSCs in DNQX + AP5 are tetrodotoxin (TTX) insensitive, suggesting that amacrine cells that release GABA under these conditions do not generate spontaneous action potentials. The average GABAsIPSCs exhibited linear current-voltage relation with a reversal potential near the chloride equilibrium potential, and an average peak conductance of 319.67 +/- 252.83 (SD) pS. For GLYsIPSCs, the average peak conductance increase is 301.68 +/- 94.34 pS. These parameters are of the same order of magnitude as those measured in inhibitory miniature postsynaptic currents (mIPSCs) associated with single synaptic vesicles in the CNS. The amplitude histograms of GABAsIPSCs did not exhibit multiple peaks, suggesting that the larger events are not discrete multiples of elementary events (or quanta). We propose that each GABAsIPSC or GLYsIPSC in retinal ganglion cells is mediated by a single or synchronized multiple of synaptic vesicles with variable neurotransmitter contents. In a sample of 16 ON-OFF ganglion cells, the average peak leIPSC (held at 0 mV) at the light onset is 509.0 +/- 233.85 pA and that at the light offset is 529.0 +/- 339.88 pA. The approximate number of GABAsIPSCs and GLYsIPSCs required to generate the average light responses, calculated by the ratio of the charge (area under current traces) of the leIPSCs to that of the average single sIPSCs, is 118 +/- 52 for the light onset, and 132 +/- 76 for the light offset.     

G-Protein-coupled modulation of presynaptic calcium currents and transmitter release by a GABAB receptor

Presynaptic GABAB receptors play a regulatory role in central synaptic transmission. To elucidate their underlying mechanism of action, we have made whole-cell recordings of calcium and potassium currents from a giant presynaptic terminal, the calyx of Held, and EPSCs from its postsynaptic target in the medial nucleus of the trapezoid body of rat brainstem slices. The GABAB receptor agonist baclofen suppressed EPSCs and presynaptic calcium currents but had no effect on voltage-dependent potassium currents. The calcium current-EPSC relationship measured during baclofen application was similar to that observed on reducing [Ca2+]o, suggesting that the presynaptic inhibition generated by baclofen is caused largely by the suppression of presynaptic calcium influx. Presynaptic loading of the GDP analog guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS) abolished the effect of baclofen on both presynaptic calcium currents and EPSCs. The nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) suppressed presynaptic calcium currents and occluded the effect of baclofen on presynaptic calcium currents and EPSCs. Photoactivation of GTPgammaS induced an inward rectifying potassium current at the calyx of Held, whereas baclofen had no such effect. We conclude that presynaptic GABAB receptors suppress transmitter release through G-protein-coupled inhibition of calcium currents.     

Effects of caffeine and 3-isobutyl-1-methylxanthine on voltage-activated potassium currents in vertebrate neurones and secretory cells

1. The effects of caffeine and 3-isobutyl-l-methylxanthine (IBMX) on voltage-activated K+ currents were examined by use of patch clamp recording techniques in dissociated chick autonomic ganglion neurones, chick pineal cells and rat anterior pituitary cells. 2. In chick ciliary ganglion neurones, caffeine (0.1-10 mM) produced a robust blockade of delayed rectifier K+ currents (IDR). Blockade was rapid in onset and concentration- and voltage-dependent. Caffeine produced greater inhibition with larger depolarizing voltage pulses. Similar inhibition of IDR was observed in excised outside-out 'maxi-patches' indicating a direct effect on the K+ channels. Caffeine also inhibited IDR in chick sympathetic neurones, chick pineal cells and rat anterior pituitary cells. 3. Application of 10 mM caffeine caused inhibition of transient A-currents (IA) in chick ciliary ganglion neurones. Inhibition of IA was voltage-dependent with greater inhibition observed at more positive command potentials. Application of 1 mM caffeine did not cause inhibition of IA. 4. Application of 1 mM IBMX, a structural analogue of caffeine, caused inhibition of IDR and IA in chick ciliary ganglion neurones. The voltage-dependence of the inhibition of both currents was qualitatively different from that observed with caffeine. The inhibitory effects of 1 mM IBMX and 10 mM caffeine on IDR and IA were additive. 5. Direct inhibition of voltage-activated K+ currents can potentially produce significant secondary effects on intracellular free Ca2+. These results indicate that caution must be used in the design and interpretation of experiments in which millimolar concentrations of caffeine or IBMX are used in pharmacological studies of intracellular Ca2+ dynamics or other second messenger mechanisms.     

The mechanism by which NBQX enhances NMDA currents in retinal ganglion cells

When the quinoxaline NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo (F) quinoxaline), a KA/AMPA antagonist, is bath applied to the tiger salamander retina, a paradoxical action is evident in the light-evoked synaptic responses of ganglion cells: NBQX enhances excitatory synaptic currents at light onset observed under whole-cell voltage-clamp conditions in a perfused retinal slice preparation. This observation was surprising because synaptic inputs into ganglion cells that are mediated by KA/AMPA receptors are entirely blocked by NBQX. Thus, the NBQX-enhanced current is entirely mediated by NMDA receptors. The purpose of this study was to determine the mechanism(s) by which blocking KA/AMPA receptors appears to enhance NMDA currents. Using hyperosmotic sucrose stimulation to activate neurotransmitter release from the inner retina, we observed that NBQX augmented the sucrose-evoked response, suggesting that at least a component of this enhancement may reside in the inner retina. NBQX does not enhance NMDA currents activated by bath applied NMDA, demonstrating that the NBQX-induced enhancement does not result from modulation of NMDA receptors. Voltage-clamp studies, carried out at the appropriate holding potential, indicate that NBQX enhances glutamatergic transmission and reduces inhibitory inputs onto ganglion cells. In the presence of strychnine and picrotoxin, the NBQX-induced enhancement of NMDA currents is eliminated, suggesting that NBQX facilitates the expression of NMDA currents by a selective and partial reduction of inhibitory mechanisms. Additional studies suggest that part of the NMDA enhancement by NBQX is evident at the postsynaptic level, but a presynaptic component probably also participates, perhaps at the level of bipolar cell terminals. One way to account for this observation is to assume that a subpopulation of inhibitory amacrine cells requires KA/AMPA receptors exclusively for their synaptic activation: previous studies of sustained amacrine cells support this interpretation. Thus the NBQX-induced enhancement phenomenon may reflect a network-selective distribution of NMDA and KA/AMPA receptors among third-order neurons.     

Intracellular calcium, currents, and stimulus-response coupling in endothelial cells

Vascular endothelium appears to be a unique organ. It not only responds to numerous hormonal and chemical signals but also senses changes in physical parameters such as shear stress, producing mediators that modulate the responses of numerous cells, including vascular smooth muscle, platelets, and leukocytes. In many cases, the initial response of endothelial cells to these diverse signals involves elevation of cytosolic Ca2+ and activation of Ca(2+)-dependent enzymes, including nitric oxide synthase and phospholipase A2. Both the release of Ca2+ from intracellular stores, most likely the endoplasmic reticulum, and the influx of Ca2+ from the extracellular space contribute to the [Ca2+]i increase. The most important trigger for Ca2+ release is inositol 1,4,5-trisphosphate, which is generated by the action of phospholipase C, a plasmalemmal enzyme activated in many cases by the receptor-G protein cascade. Ca2+ influx appears to be related to the activity of receptor-G protein-enzyme complex and to the degree of fullness of the endoplasmic reticulum but does not involve voltage-gated Ca2+ channels. The magnitude of the Ca2+ influx depends on the electrochemical gradient, which is modulated by the membrane potential, Vm. Under basal conditions, Vm is dominated by a large inward rectifier K+ current. Some stimuli, e.g., acetylcholine, have been shown to hyperpolarize Vm, thus increasing the electrochemical gradient for Ca2+, which appears to be modulated by activation of Ca(2+)-dependent K+ and Cl- currents. However, the lack of potent and specific blockers for many of the described or postulated channels (e.g., nonselective cation channel, Ca(2+)-activated Cl- channel) makes an estimation of their effect on endothelial cell function rather difficult. Possible future directions of research and clinical implications are discussed.     

Inwardly rectifying K+ currents in isolated human retinal pigment epithelial cells

PURPOSE: K+ channels in the retinal pigment epithelium (RPE) play a number of important roles, including the establishment of membrane potential, the transport of K+ between the subretinal space and choroid, and the generation of the c-wave of the electroretinogram. Previous studies on amphibian RPE demonstrated that these functions are likely served by an inwardly rectifying K+ channel. The aim of this study was to characterize inwardly rectifying K+ channels in cultured and freshly isolated adult human RPE (hRPE) cells. METHODS: Single cells were dispersed enzymatically from primary cultures of adult hRPE or from fresh adult hRPE-choroid. Ionic currents were recorded using either the perforated-patch or whole-cell configuration of the patch-clamp technique. RESULTS: In 5 mM external K+, roughly 20% of cultured hRPE cells exhibited a strong inwardly rectifying K+ conductance that passed inward but little outward current. This conductance increased when [K+]o was increased and exhibited a voltage-dependent block by external Na+ at negative potentials. In contrast, all freshly isolated hRPE cells exhibited a mild inwardly rectifying K+ conductance that mediated substantial outward current at physiological voltages. This conductance decreased when [K+]o was increased and showed no voltage-dependent block by external Na+. CONCLUSION: The authors conclude that fresh hRPE cells express a mild inwardly rectifying K+ conductance. The operation of this conductance at physiological voltages makes it a likely candidate for the resting K+ conductances of the apical and basolateral membranes. Cultured hRPE cells express a functionally different channel type that may reflect a change in phenotype.     

Riluzole interacts with voltage-activated sodium and potassium currents in cultured rat cortical neurons

Inhaled nitric oxide is a selective pulmonary vasodilator used for the treatment of pulmonary hypertension. The potential adverse effects of inhaled nitric oxide are unknown and represent the focus of the present studies. Whereas inhalation of nitric oxide (10 to 100 ppm, 5 h) by Balb/c mice had no effect on the number or type of cells recovered from the lung, a dose-related increase in bronchoalveolar lavage protein was observed, suggesting that nitric oxide induces alveolar epithelial injury. To determine if this was associated with altered alveolar macrophage activity, we quantified production of reactive oxygen and nitrogen intermediates by these cells. Interferon-gamma, alone or in combination with lipopolysaccharide (LPS), induced expression of inducible nitric oxide synthase (iNOS) protein and nitric oxide production by alveolar macrophages. Cells from mice exposed to 20 to 100 ppm nitric oxide produced significantly more nitric oxide and expressed greater quantities of iNOS than cells from control animals. Superoxide anion production and peroxynitrite generation by alveolar macrophages were also increased after exposure of mice to nitric oxide. This was correlated with increased antinitrotyrosine antibody binding to macrophages in histologic sections. Taken together, these data demonstrate that inhaled nitric oxide primes lung macrophages to release reactive oxygen and nitrogen intermediates. Increased production of these mediators by macrophages following inhalation of nitric oxide may contribute to tissue injury.     

Dopaminergic modulation of NMDA-induced whole cell currents in neostriatal neurons in slices: contribution of calcium conductances

The present experiments were designed to examine dopamine (DA) modulation of whole cell currents mediated by activation of N-methyl-D-aspartate (NMDA) receptors in visualized neostriatal neurons in slices. First, we assessed the ability of DA, D1 and D2 receptor agonists to modulate membrane currents induced by activation of NMDA receptors. The results of these experiments demonstrated that DA potentiated NMDA-induced currents in medium-sized neostriatal neurons. Potentiation of NMDA currents occurred at three different holding potentials, although it was more pronounced at -30 mV. It was mediated by D1 receptors, because it was mimicked by D1 agonists and blocked by exposure to a D1 antagonist. Activation of D2 receptors produced inconsistent effects on NMDA-induced membrane currents. Either decreases, increases, or no effects on NMDA currents occurred. Second, we examined the contributions of intrinsic, voltage-dependent conductances to DA potentiation of NMDA currents. Blockade of K+ conductances did not prevent DA enhancement of NMDA currents. However, voltage-activated Ca2+ conductances provided a major contribution to DA modulation. The dihydropyridine L-type Ca2+ channel blockers, nifedipine, and methoxyverapamil (D-600), markedly reduced but did not totally eliminate the ability of DA to modulate NMDA currents. The D1 receptor agonist SKF 38393 also enhanced Ba2+ currents in neostriatal neurons. Together, these findings provide evidence for a complex interplay between DA, NMDA receptor activation and dihydropyridine-sensitive Ca2+ conductances in controlling responsiveness of neostriatal medium-sized neurons.     

Estimating the contributions of NMDA and non-NMDA currents to EPSPs in retinal ganglion cells

Whole-cell recordings were obtained from retinal ganglion cells of the tiger salamander (Ambystoma tigrinum) in a superfused slice preparation to evaluate contributions of NMDA (N-methyl-D-aspartate) and KA/AMPA (kainate/alpha-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid) receptors to excitatory postsynaptic potentials (EPSPs) of retinal ganglion cells. Synaptic activation of retinal ganglion cells was achieved through the use of a brief pressure pulse of hyperosmotic Ringer (Ringer + sucrose) delivered through a microelectrode visually placed in the inner plexiform layer while whole-cell recordings were obtained from adjacent cells in the ganglion cell layer. Separation of NMDA and KA/AMPA excitatory postsynaptic currents (EPSCs) was achieved through the application of the antagonists NBQX and D-AP7, while inhibitory currents were blocked by strychnine and picrotoxin. Simple addition of the two independent EPSCs showed, most often, that the sum of the KA/AMPA and NMDA currents was less than the control response, but in some cases the sum of the two currents exceeded the magnitude of the control response. Neither result was consistent with expectations based on voltage-clamp principles and the assumption that the two currents were independent; for this reason, we considered the possibility of nonlinear interactions between KA/AMPA and NMDA receptors. Computer simulations were carried out to evaluate the summation experiments. We used both an equivalent cylinder model and a more realistic, compartmental model of a ganglion cell constrained by a passive leakage conductance, a linear KA/AMPA synaptic current, and a nonlinear NMDA current based on the well-known, voltage-sensitive Mg2+ block. Computer simulation studies suggest that the hypo- and hyper-summation of NMDA and KA/AMPA currents, observed physiologically, can be accounted for by a failure to adequately space clamp the neuron. Clamp failure leads to enhanced NMDA currents as the ion channels are relieved of the Mg2+ block; their contribution is thus exaggerated depending on the magnitude of the conductance change and the spatial location of the synaptic input.     

Altered calcium channel currents in Purkinje cells of the neurological mutant mouse leaner

Mutations of the alpha1A calcium channel subunit have been shown to cause such human neurological diseases as familial hemiplegic migraine, episodic ataxia-2, and spinocerebellar ataxia 6 and also to cause the murine neurological phenotypes of tottering and leaner. The leaner phenotype is recessive and characterized by ataxia with cortical spike and wave discharges (similar to absence epilepsy in humans) and a gradual degeneration of cerebellar Purkinje and granule cells. The mutation responsible is a single-base substitution that produces truncation of the normal open reading frame beyond repeat IV and expression of a novel C-terminal sequence. Here, we have used whole-cell recordings to determine whether the leaner mutation alters calcium channel currents in cerebellar Purkinje cells, both because these cells are profoundly affected in leaner mice and because they normally express high levels of alpha1A. In Purkinje cells from normal mice, 82% of the whole-cell current was blocked by 100 nM omega-agatoxin-IVA. In Purkinje cells from homozygous leaner mice, this omega-agatoxin-IVA-sensitive current was 65% smaller than in control cells. Although attenuated, the omega-agatoxin-IVA-sensitive current in homozygous leaner cells had properties indistinguishable from that of normal Purkinje neurons. Additionally, the omega-agatoxin-IVA-insensitive current was unaffected in homozygous leaner mice. Thus, the leaner mutation selectively reduces P-type currents in Purkinje cells, and the alpha1A subunit and P-type current appear to be essential for normal cerebellar function.     

Interactions of inhibition and excitation in the light-evoked currents of X type retinal ganglion cells

The excitatory and inhibitory conductances driving the light-evoked currents (LECs) of cat and ferret ON- and OFF-center X ganglion cells were examined in sliced and isolated retina preparations using center spot stimulation in tetrodotoxin (TTX)-containing Ringer. ON-center X ganglion cells showed an increase in an excitatory conductance reversed positive to +20 mV during the spot stimulus. At spot offset, a transient inhibitory conductance was activated on many cells that reversed near ECl. OFF-center X ganglion cells showed increases in a sustained inhibitory conductance that reversed near ECl during spot stimulation. At spot offset, an excitatory conductance was activated that reversed positive to +20 mV. The light-evoked current kinetics of ON- and OFF-center X cells to spot stimulation did not significantly differ in form from their Y cell counterparts in TTX Ringer. When inhibition was blocked, current-voltage relations of the light-evoked excitatory postsynaptic currents (EPSCs) of both ON- and OFF-X cells were L-shaped and reversed near 0 mV. The EPSCs averaged between 300 and 500 pA at -80 mV. The metabotropic glutamate receptor agonist 2-amino-4-phosphonobutyric acid (APB), was used to block ON-center bipolar cell function. The LECs of ON-X ganglion cells were totally blocked in APB at all holding potentials. APB caused prominent reductions in the dark holding current and synaptic noise of ON-X cells. In contrast, the LECs of OFF-X ganglion cells remained in APB. An increase in the dark holding current was observed. The excitatory amino acid receptor antagonist combination of D-amino-5-phosphono-pentanoic acid (D-AP5) and 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(F)-quinoxalinedione (NBQX) was used to block ionotropic glutamate receptor retinal neurotransmission. The LECs of all ON-X ganglion cells were totally blocked, and their holding currents were reduced similar to the actions of APB. For OFF-X ganglion cells, the antagonist combination always blocked the excitatory current at light-OFF; however, in many cells, the inhibitory current at light-ON remained. ON-center X ganglion cells receive active excitation during center illumination, and a transient inhibition at light-OFF. In contrast OFF-center X ganglion cells experience a sustained active inhibition during center illumination, and a shorter increase in excitation at light-offset. Cone bipolar cells provide a resting level of glutamate release on X ganglion cells on which their light-evoked currents are superimposed [corrected].     

LTP in CA1 of the adult rat hippocampus and voltage-activated calcium channels

It is difficult to induce long-term potentiation (LTP) in CA1 of hippocampal slices from 120-day-old rats when a single 100 Hz, 1 s tetanus is administered in extracellular solution containing 2.0 mM calcium and 2.0 mM magnesium. However, in the presence of 2.5 mM calcium and 1.3 mM magnesium LTP is reliably induced by this same stimulus. Although the amplitude of LTP is similar to that observed in slices from 30-day-old rats, LTP in slices from mature rats is not inhibited by MK-801 but is blocked by nifedipine. These results suggest that factors contributing to LTP in slices from mature rats require careful consideration under different experimental paradigms.     

Optical measurement of presynaptic calcium currents

Measurements of presynaptic calcium currents are vital to understanding the control of transmitter release. However, most presynaptic boutons in the vertebrate central nervous system are too small to allow electrical recordings of presynaptic calcium currents (I(Ca)pre). We therefore tested the possibility of measuring I(Ca)pre optically in boutons loaded with calcium-sensitive fluorophores. From a theoretical treatment of a system containing an endogenous buffer and an indicator, we determined the conditions necessary for the derivative of the stimulus-evoked change in indicator fluorescence to report I(Ca)pre accurately. Matching the calcium dissociation rates of the endogenous buffer and indicator allows the most precise optical measurements of I(Ca)pre. We tested our ability to measure I(Ca)pre in granule cells in rat cerebellar slices. The derivatives of stimulus-evoked fluorescence transients from slices loaded with the low-affinity calcium indicators magnesium green and mag-fura-5 had the same time courses and were unaffected by changes in calcium influx or indicator concentration. Thus both of these indicators were well suited to measuring I(Ca)pre. In contrast, the high-affinity indicator fura-2 distorted I(Ca)pre. The optically determined I(Ca)pre was well approximated by a Gaussian with a half-width of 650 micros at 24 degrees C and 340 micros at 34 degrees C.     

Antagonists-resistant calcium currents in rat embryo motoneurons

Ca2+ channels diversity of cultured rat embryo motoneurons was investigated with whole-cell current recordings. In 5-20 mM Ba2+, the whole-cell currents were separated in low- (LVA) and high-voltage-activated (HVA) current. The LVA current was evident since the first day in culture, while the HVA component was small and increased with time. Recordings after 4 days revealed approximately 20% L-, approximately 45% N- and approximately 35% P- and R-type currents. P-type currents were revealed only in 40% of motoneurons, in which 20-200 nM omega-Aga-IVA caused 20% irreversible block of total current. The remaining 60% of cells were insensitive even to higher doses of the toxin (500 nM in 5 mM Ba2+), suggesting weak expression and heterogeneous distribution of P-type channels compensated by high densities of HVA Ca2+ channels resistant to all the antagonists (R-type). A significant residual current could also be resolved after prolonged applications of 5 microM omega-CTx-MVIIC, which allowed separation of N- and P-type currents by the distinct onset of toxin block. The antagonists-resistant current reveals biophysical characteristics typical of HVA channels, but distinct from the alphaE channel. The current activates around -20 mV in 20 mM Ba2+; inactivates slowly and independently of Ca2+; is blocked by low [Cd2+] and high [Ni2+]; and is larger with Ba2+ than Ca2+. The uncovered R-type calcium current can account for part of the presynaptic Ca2+ current controlling neurotransmitter release at the mammalian neuromuscular junction whose activity is resistant to DHP-and omega-CTx-GVIA, and displays anomalous sensitivity to omega-Aga-IVA and omega-CTx-MVIIC.