Analyses of bipolar cell responses elicited by polarization of horizontal cells

Simultaneous intracellular recordings were made from a bipolar cell and a horizontal cell in the carp retina. The properties of the bipolar cell were studied while injecting current into the horizontal cell. Hyperpolarization of horizontal cells, irrespective of their type, elicited a hyperpolarizing response in on-center bipolar cells and a depolarizing response in off-center bipolar cells. Analyses of the ionic mechanisms of bipolar cell responses revealed that depolarization of horizontal cells simulated and hyperpolarization opposed the effect of central illumination. The effect of polarization was exerted in such a manner that each type of horizontal cells modified the transmission from those photoreceptors from which they receive main inputs. In on- center bipolar cells, for example, the L-type horizontal cells receiving inputs mainly from red cones modified the cone-bipolar transmission accompanied by a conductance change of K+ and/or Cl- channels, and the intermediate horizontal cells receiving inputs from rods modified the rod-bipolar transmission accompanied by a conductance change of Na+ channels. In off-center bipolar cells, the effect of polarization of any type of horizontal cells was mediated mainly by conductance changes of Na+ channels. Feedback mechanisms from horizontal cells to photoreceptors could explain these results reasonably well.     

Application of transretinal current stimulation for the study of bipolar-amacrine transmission

Transretinal current flowing from the receptor side to the vitreous side depolarizes the axon terminals of retinal cells and facilitates the release of transmitter. Such current elicited a depolarizing response in off-center bipolar cells and a hyperpolarizing response in on-center bipolar cells. It also elicited a response of relatively complex waveform in amacrine cells. The responses elicited in bipolar cells were suppressed in the presence of 5-10 mM glutamate in the perfusing Ringer solution, while the responses of amacrine cells persisted, although their waveform changed to a simple one that showed monotonic depolarization irrespective of the type of amacrine cell and were accompanied by a decrease in the membrane resistance. The results indicate excitatory synaptic transmission from bipolar cells to amacrine cells. Since the response elicited by current in ON-OFF cells was almost identical to those elicited in ON or OFF amacrine cells, the transient nature of their light response cannot be due to their membrane properties. ON-OFF cells responded to transretinal current flowing in the opposite direction with a small hyperpolarization accompanied by a resistance increase. The hyperpolarizing response was suppressed by the addition of GABA in glutamate Ringer solution. The results suggest an activation by the current of GABA-ergic feedback pathways from amacrine cells to bipolar cells.     

Active calcium responses recorded optically from nerve terminals of the frog neurohypophysis

Voltage-sensitive dyes were used to record by optical means membrane potential changes from nerve terminals in the isolated frog neurohypophysis. Following the block of voltage-sensitive Na+ channels by tetrodotoxin (TTX) and K+ channels by tetraethylammonium (TEA), direct electric field stimulation of the nerve terminals still evoked large active responses. These responses were reversibly blocked by the addition of 0.5 mM CdCl2. At both normal and low [Na+]o, the regenerative response appeared to increase with increasing [Ca++]o (0.1-10 mM). There was a marked decrease in the size of the response, as well as in its rate of rise, at low [Ca++]o (0.2 mM) when [Na+]o was reduced from 120 to 8 mM (replaced by sucrose), but little if any effect of this reduction of [Na+]o at normal [Ca++]o. In normal [Ca++]o, these local responses most probably arise from an inward Ca++ current associated with hormone release from these nerve terminals. At low [Ca++]o, Na+ appears to contribute to the TTX-insensitive inward current.     

Calcium entry in squid axons during voltage clamp pulses

Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with sodium ion sensitive, current and voltage electrodes. The axons were usually bathed in a solution of varying Ca2+ concentration ([Ca2+]o) containing 150mM each of Na+, K+ and an inert cation such as Li+, Tris or N-methylglucamine and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic Ca2+ level, [Ca2+]i. The effect of membrane voltage on [Ca2+]i was found to depend on the concentration of internal Na+ ([Na+]i). Voltage clamp hyperpolarizing pulses were found to cause a reduction of [Ca2+]i. For depolarizing pulses a relationship between [Ca2+]i gain and [Na+]i indicates that Ca2+ entry is sigmoid with a half maximal response at 22 mM Na+. This Ca2+ entry is a steep function of [Na+]i suggesting that 4 Na+ ions are required to promote the influx of 1 Ca2+. There was little change in Ca2+ entry with depolarizing pulses when [Ca2+]o is varied from 1 to 10mM, while at 50mM [Ca2+]o calcium entry clearly increases suggesting an alternate pathway from that of Na+/Ca2+ exchange. This entry of Ca2+ at high [Ca2+]o, however, was not blocked by Cs+o. The results obtained lend further support to the notion that Na+/Ca2+ exchange in squid giant axon is sensitive to membrane voltage no matter whether this is applied as a constant change in membrane potential or as an intermittent one.     

GnRH-induced cytosolic calcium oscillations in pituitary gonadotrophs: phase resetting by membrane depolarization.

Cultured rat pituitary gonadotrophs under whole-cell voltage clamp conditions respond to the hypothalamic hormone GnRH with synchronized oscillatory changes in both cytosolic Ca2+ concentration ([Ca2+]i) and [Ca2+]i-activated, apamin-sensitive K+ current (IK(Ca)). We found, and report here for the first time, that in GnRH-stimulated cells a brief depolarizing pulse can elicit a transient [Ca2+]i rise similar to the endogenous cycle. Furthermore, Ca2+ entry during a single depolarizing pulse was found to shift the phase of subsequent endogenous [Ca2+]i oscillations, which thereafter continue to occur at their previous frequency before the pulse. Application of two consecutive depolarizing pulses showed that the size of the [Ca2+]i rise evoked by the second pulse depended on the time lapsed between two consecutive pulses, indicating that each endogenous or evoked [Ca2+]i rise cycle leaves the Ca2+ release mechanism of the gonadotroph in a refractory state. Recovery from this condition can be described by an exponential function of the time lapsed between the pulses (time constant of ca. 1 s). We propose that the underlying mechanism in both refractoriness after endogenous cycles and phase resetting by a brief pulse of Ca2+ entry involves the InsP3 receptor-channel molecule presumed to be located on the cytosolic aspect of the endoplasmic reticulum membrane.     

Membrane potential responses of paramecium caudatum to external Na+

The membrane potential responses of Paramecium caudatum to Na+ ions were examined to understand the mechanisms underlying the sensation of external inorganic ions in the ciliate by comparing the responses of the wild type and the behavioral mutant. Wild-type cells exhibited initial continuous backward swimming followed by repeated transient backward swimming in the Na+-containing test solution. A wild-type cell impaled by a microelectrode produced initial action potentials and a sustained depolarization to an application of the test solution. The prolonged depolarization, the depolarizing afterpotential, took place subsequently after stimulation. The ciliary reversal of the cell was closely associated with the depolarizing responses. When the application of the test solution was prolonged, the wild-type cell produced sustained depolarization overlapped by repeated transient depolarization. A behavioral mutant defective in the Ca2+ channel, CNR (caudatum non reversal), produced a sustained depolarization but no action potential or depolarizing afterpotential. The mutant cell responded to prolonged stimulation with sustained depolarization overlapped by transient depolarization, although it did not show backward swimming. The results suggest that Paramecium shows at least two kinds of membrane potential responses to Na+ ions: a depolarizing afterpotential mediating initial backward swimming and repeated transient depolarization responsible for the repeated transient backward swimming.     

P2X2 receptors are essential for [Ca2+]i increases in response to ATP in cultured rat myenteric neurons

We characterized ATP-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and membrane current in cultured rat myenteric neurons using ratiometric Ca2+ imaging with fura-2 and the whole cell patch-clamp technique, respectively. Neuronal cells were functionally identified by [Ca2+]i responses to high K+ and nicotine, which occurred only in cells positive for neuron-specific protein gene product 9.5 immunoreactivity. ATP evoked a dose-dependent increase of [Ca2+]i that was greatly decreased by the removal of extracellular Ca2+ concentration ([Ca2+]o). The amplitude of the [Ca2+]i response to ATP was reduced by half in the presence of voltage-dependent Ca2+ channel blockers. In [Ca2+]o-free solution, ATP produced a small transient rise in [Ca2+]i similar to that induced by P2Y agonists. At -60 mV, ATP evoked a slowly inactivating inward current that was suppressed by the removal of extracellular Na+ concentration. The current-voltage relation for ATP showed an inward rectification with the reversal potential of about 0 mV. The apparent rank order of potency for the purinoceptor agonist-induced increases of [Ca2+]i was ATP > or = adenosine 5'-O-3-triphosphate > or = CTP > or = 2-methylthio-ATP > benzoylbenzoyl-ATP. A similar potency order was obtained with current responses to these agonists. P2 antagonists inhibited inward currents induced by ATP. Ca2+ and Mg2+ suppressed the ATP-induced current, and Zn2+, Cu2+, and protons potentiated it. RT-PCR and immunocytochemical studies showed the expression of P2X2 receptors in cultured rat myenteric neurons. These results suggest that ATP mainly activates ionotropic P2X2 receptors, resulting in a [Ca2+]i increase dependent on [Ca2+]o in rat myenteric neurons. A small part of the ATP-induced [Ca2+]i increase may be also mediated via a P2Y receptor-related mechanism.     

Presynaptic facilitation at the crayfish neuromuscular junction. Role of calcium-activated potassium conductance

Membrane potential was recorded intracellularly near presynaptic terminals of the excitor axon of the crayfish opener neuromuscular junction (NMJ), while transmitter release was recorded postsynaptically. This study focused on the effects of a presynaptic calcium-activated potassium conductance, gK(Ca), on the transmitter release evoked by single and paired depolarizing current pulses. Blocking gK(Ca) by adding tetraethylammonium ion (TEA; 5-20 mM) to a solution containing tetrodotoxin and aminopyridines caused the relation between presynaptic potential and transmitter release to steepen and shift to less depolarized potentials. When two depolarizing current pulses were applied at 20-ms intervals with gK(Ca) not blocked, the presynaptic voltage change to the second (test) pulse was inversely related to the amplitude of the first (conditioning) pulse. This effect of the conditioning prepulse on the response to the test pulse was eliminated by 20 mM TEA and by solutions containing 0 mM Ca2+/1 mM EGTA, suggesting that the reduction in the amplitude of the test pulse was due to activation of gK(Ca) by calcium remaining from the conditioning pulse. In the absence of TEA, facilitation of transmitter release evoked by a test pulse increased as the conditioning pulse grew from -40 to -20 mV, but then decreased with further increase in the conditioning depolarization. A similar nonmonotonic relationship between facilitation and the amplitude of the conditioning depolarization was reported in previous studies using extracellular recording, and interpreted as supporting an additional voltage-dependent step in the activation of transmitter release. We suggest that this result was due instead to activation of a gK(Ca) by the conditioning depolarization, since facilitation of transmitter release increased monotonically with the amplitude of the conditioning depolarization, and the early time course of the decay of facilitation was prolonged when gK(Ca) was blocked. The different time courses for decay of the presynaptic potential (20 ms) and facilitation (greater than 50 ms) suggest either that residual free calcium does not account for facilitation at the crayfish NMJ or that the transmitter release mechanism has a markedly higher affinity or stoichiometry for internal free calcium than does gK(Ca). Finally, our data suggest that the calcium channels responsible for transmitter release at the crayfish NMJ are not of the L, N, or T type.     

Deprivation of Na+, Ca2+ and Mg2+ from the extracellular solution increases cytosolic Ca2+ and stimulates catecholamine secretion from cultured bovine adrenal chromaffin cells

We report here that exposing cultured chromaffin cells to a low ionic strength medium (with sucrose in place of NaCl to maintain osmolarity) can induce a marked elevation in cytosolic Ca2+ concentration ([Ca2+]i) and catecholamine (CA) release. To determine the underlying mechanism, we first studied the effects of low [Na+]o on single cell [Ca2+]i (using fluo-3 as Ca2+ indicator) and CA release from many cells. In a Mg2+ and Ca2+-deficient medium, lowering the external concentration of Na2+ ([Na+]o) evoked CA secretion preceded by a transitory [Ca2+]i rise, the amplitude of which was inversely related to [Na+]o. By contrast, in the presence of either [Ca2+]o (2 mM) and [Mg2+]o (1.4 mM) or [Mg2+]o alone (3.4 mM), lowering the ionic strength was without effect. Furthermore, in a physiologic [Na+]o, [Ca2+]o and [Mg2+]o medium, two or three consecutive applications of the cholinergic agonist oxotremorine-M (oxo-M) consistently evoked a substantial [Ca2+]i rise. By contrast, consecutive applications of oxo-M in a Ca2+-deficient medium failed to evoke a rise in [Ca2+]i after the first exposure to the agonist. To clarify the underlying mechanism, we measured and compared the effects of low [Na+]o and the cholinergic agonists nicotine and oxo-M on changes in [Ca2+]i; we studied the effects of these agonists on both membrane potential, Vm (under current clamp conditions), and [Ca2+]i by single cell microfluorimetry (indo-1 as Ca2+ indicator). We observed that, in the presence of [Ca2+]o and [Mg2+]o, lowering [Na+]o had no effect on Vm. In a Ca2+-deficient medium, lowering [Na+]o depolarized the membrane from ca. –60 to –10 mV. As expected, we found that nicotine (10 M) depolarized the membrane (from ca. –60 to –20 mV) and simultaneously evoked a substantial [Ca2+]i rise that was [Ca2+]o-dependent. However, contrary to our expectations, we found that the muscarinic agonist oxo-M (50 M) also depolarized the membrane and induced an elevation in [Ca2+]i. Furthermore, both signals were blocked by D-tubocurarine, insinuating the nicotinic character of oxo-M in adrenal chromaffin cells from bovine. These results suggest that both nicotine and oxo-M stimulate Ca2+ entry, probably through voltage-gated Ca2+-channels. We also show here that oxo-M (and not low [Na+]o) stimulates phosphoinositide turnover.     

Comparison of changes evoked by GABA (gamma-aminobutyric acid) and anoxia in [K+]o, [Cl-]o, and [Na+]o in stratum pyramidale and stratum radiatum of the guinea pig hippocampus

Ion-selective microelectrode recordings were made to assess a possible contribution of extracellular gamma-aminobutyric acid (GABA) accumulation to early responses evoked in the brain by anoxia and ischemia. Changes evoked by GABA or N2 in [K+]o, [Cl-]o, [Na+]o, and [TMA+]o were recorded in the cell body and dendritic regions of the stratum pyramidale (SP) and stratum radiatum (SR), respectively, of pyramidal neurons in CA1 of guinea pig hippocampal slices. Bath application of GABA (1-10 mM) for approximately 5 min evoked changes in [K+]o and [Cl-]o with respective EC50 levels of 3.8 and 4.1 mM in SP, and 4.7 and 5.6 mM in SR. In SP 5 mM GABA reversibly increased [K+]o and [Cl-]o and decreased [Na+]o; replacement of 95% O2 -5% CO2 by 95% N2 -5% CO2 for a similar period of time evoked changes which were for each ion in the same direction as those with GABA. In SR both GABA and N2 caused increases in [K+]o and decreases in [Cl-]o and [Na+]. The reduction of extracellular space, estimated from levels of [TMA+]o during exposures to GABA and N2, was 5-6% and insufficient to cause the observed changes in ion concentration. Ion changes induced by GABA and N2 were reversibly attenuated by the GABA(A) receptor antagonist bicuculline methiodide (BMI, 100 microM). GABA-evoked changes in [K+]o in SP and SR and [Cl-]o in SP were depressed by > or =90%, and of [Cl-]o in SR by 50%; N2-evoked changes in [K+]o in SP and SR were decreased by 70% and those of [Cl-]o by 50%. BMI blocked delta [Na+]o with both GABA and N2 by 20-30%. It is concluded that during early anoxia: (i) accumulation of GABA and activation of GABA(A) receptors may contribute to the ion changes and play a significant role, and (ii) responses in the dendritic (SR) regions are greater than and (or) differ from those in the somal (SP) layers. A large component of the [K+]o increase may involve a GABA-evoked Ca2+-activated gk, secondary to [Ca2+]i increase. A major part of [Cl-]o changes may arise from GABA-induced g(Cl) and glial efflux, with strong stimulation of active outward transport and anion exchange at SP, and inward Na+/K+/2Cl- co-transport at SR. Na+ influx is attributable mainly to Na+-dependent transmitter uptake, with only a small amount related to GABA(A) receptor activation. Although the release and (or) accumulation of GABA during anoxia might be viewed as potentially protectant, the ultimate role may more likely be an important contribution to toxicity and delayed neuronal death.     

Voltage dependent activation of tonic contraction in cardiac myocytes.

Contractions of isolated single myocytes of guinea pig heart stimulated by rectangular depolarizing pulses consist of a phasic component and a voltage dependent tonic component. In this study we analyzed the mechanism of activation of the graded, sustained contractions elicited by slow ramp depolarization and their relation to the components of contractions elicited by rectangular depolarizing pulses. Experiments were performed at 37 degrees C in ventricular myocytes of guinea pig heart. Voltage-clamped myocytes were stimulated by the pulses from the holding potential of -40 to +5 mV or by ramp depolarization shifting voltage within this range within 6 s. [Ca2+]i was monitored as fluorescence of Indo 1-AM and contractions were recorded with the TV edge-tracking system. Myocytes responded to the ramp depolarization between -25 and -6 mV by the slow, sustained increase in [Ca2+]i and shortening, the maximal amplitude of which was in each cell similar to that of the tonic component of Ca2+ transient and contraction. The contractile responses to ramp depolarization were blocked by 200 microM ryanodine and Ca2+-free solution, but were not blocked by 20 microM nifedipine or 100-200 microM Cd2+ and potentiated by 5 mM Ni2+. The responses to ramp depolarization were with this respect similar to the tonic but not to the phasic component of contraction: both components were blocked by 200 microM ryanodine, and were not blocked by Cd2+ or Ni2+ despite complete inhibition of the phasic Ca2+ current. However, the phasic component but not the tonic component of contraction in cells superfused with Ni2+ was inhibited by nifedipine. Both components of contraction were inhibited by Ca2+-free solution superfused 15 s prior to stimulation. CONCLUSIONS: In myocytes of guinea pig heart the contractile response to ramp depolarization is equivalent to the tonic component of contraction. It is activated by Ca2+ released from the sarcoplasmic reticulum by the ryanodine receptors. Their activation and inactivation is voltage dependent and it does not depend on the Ca2+ influx by the Ca2+ channels or reverse mode Na+/Ca2+ exchange, however, it may depend on Ca2+ influx by some other, not yet defined route.     

Ionic mechanisms underlying the responses of off-center bipolar cells in the carp retina. I. Studies on responses evoked by light

Off-center bipolar cells show hyperpolarizing responses to spot illumination in the receptive field center and depolarization responses to an annulus in the surround. To understand the ionic mechanisms underlying these responses, we examined the current-voltage relationship of these bipolar cells, input resistance changes during their light-evoked responses, and the reversal potentials of these responses. Off-center bipolar cells generally showed inward rectification when they were hyperpolarized and outward rectification when they were strongly depolarized. The membrane potential at which the I-V relationship deviated from linearity varied in individual cells. Hyperpolarizing center responses were generally accompanied by a resistance increase, irrespective of signal inputs either from red- sensitive cones or from rods, and the response polarities reversed at greater than +50 mV. Depolarizing surround responses were accompanied by a resistance decrease with a reversal potential at about +28 mV (one case). From the above observations, it is suggested that the center responses are generated by a decrease in sodium conductance (gNa) and the surround response is generated by an increase in gNa.     

Caffeine slows turn-off of calcium release in voltage clamped skeletal muscle fibers.

Myoplasmic free calcium transients delta [Ca2+] were monitored with the calcium indicators antipyrylazo III and fura-2 in voltage clamped cut frog skeletal muscle fibers, in the presence and absence of 0.5 mM caffeine. Without caffeine delta [Ca2+] began to decline within a few milliseconds of fiber repolarization for pulses of all durations. In caffeine delta [Ca2+] continued to rise for 10-60 ms after 10 or 20 ms depolarizing pulses, indicating that the release of calcium from the sarcoplasmic reticulum (SR) continued well after repolarization of transverse tubular (TT) membranes in the presence of caffeine. Caffeine also increased the peak amplitude of delta [Ca2+] for all pulses and slowed the decline of delta [Ca2+] after pulses of all durations. The rate of calcium release from the SR calculated from delta [Ca2+] showed that for 10 ms pulses in caffeine release did not turn off abruptly on repolarization but instead declined to zero with a time constant essentially the same as the time constant for inactivation of SR calcium release during depolarizing pulses in the presence or absence of caffeine. The observed loss of TT membrane potential control of SR calcium release in the presence of caffeine suggests the appearance of a significant component of cytosolic Ca2+-induced calcium release in caffeine.     

Analysis of synaptic inputs to on-off amacrine cells of the carp retina

To elucidate the synaptic transmission between bipolar cells and amacrine cells, the effect of polarization of a bipolar cell on an amacrine cell was examined by simultaneous intracellular recordings from both cells in the isolated carp retina. When either an ON or OFF bipolar cell was depolarized by an extrinsic current step, an ON-OFF amacrine cell was transiently depolarized at the onset of the current but no sustained polarization during the current was detected. The current hyperpolarizing the OFF bipolar cell also produced the transient depolarization of the amacrine cell at the termination of the current. These responses had a latency of approximately 10 ms. The amplitude of the current-evoked responses changed gradually with current intensity within the range used in these experiments. They were affected by polarization of the amacrine cell membrane; the amplitude of the current-evoked responses as well as the light-evoked responses was increased when the amacrine cell membrane was hyperpolarized, while the amplitude was decreased when the cell was depolarized. These results confirm directly that ON-OFF amacrine cells receive excitatory inputs from both ON and OFF bipolar cells: the ON transient is due to inputs from ON bipolar cells, and the OFF transient to inputs from OFF bipolar cells. The steady polarization of bipolar cells is converted into transient signals during the synaptic process.     

TEA prevents inactivation while blocking open K+ channels in human T lymphocytes.

The whole-cell recording mode of the patch-clamp technique was used to study the effect of external tetraethylammonium ([TEA+]o) on the inactivating, voltage-dependent K+ channels of human T lymphocytes. TEA+ reduced the peak amplitude and slowed the time course of the K+ current decay during a depolarizing pulse, resulting in a crossover of the current records in the presence and absence of TEA+. In solutions with different [TEA+]o both the peak K+ current amplitude, lKpeak, and the time constant of the decay of the K+ current, tau d, were reduced in a dose-dependent manner, both with apparent binding constants, KD, of 12 mM. The integral of K+ current during a prolonged depolarizing pulse was unaltered in solutions with different [TEA+]o. The concentration dependence of [TEA+]o on lKpeak, tau d, and the unchanged current integral can be explained with a kinetic scheme in which open channels blocked by TEA+ cannot inactivate.     

Regulation of transmitter release from retinal bipolar cells.

Mb1 bipolar cells (ON-type cells) of the goldfish retina have exceptionally large (approximately 10 microns in diameter) presynaptic terminals, and thus, are suitable for investigating presynaptic mechanisms for transmitter release. Using enzymatically dissociated Mb1 bipolar cells under whole-cell voltage clamp, we measured the Ca2+ current (ICa), the intracellular free Ca2+ concentration ([Ca2+]i), and membrane capacitance changes associated with exocytosis and endocytosis. Release of transmitter (glutamate) was monitored electrophysiologically by a glutamate receptor-rich neuron as a probe. L-type Ca2+ channels were localized at the presynaptic terminals. The presynaptic [Ca2+]i was strongly regulated by cytoplasmic Ca2+ buffers, the Na(+)-Ca2+ exchanger and the Ca2+ pump in the plasma membrane. Once ICa was activated, a steep Ca2+ gradient was created around Ca2+ channels; [Ca2+]i increased to approximately 100 microM at the fusion sites of synaptic vesicles whereas up to approximately 1 microM at the cytoplasm. The short delay (approximately 1 ms) of exocytosis and the lack of prominent asynchronous release after the termination of ICa suggested a low-affinity Ca2+ fusion sensor for exocytosis. Depending on the rate of Ca2+ influx, glutamate was released in a rapid phasic mode as well as a tonic mode. Multiple pools of synaptic vesicles as well as vesicle cycling seemed to support continuous glutamate release. Activation of protein kinase C increased the size of synaptic vesicle pool, resulting in the potentiation of glutamate release. Goldfish Mb1 bipolar cells may still be an important model system for understanding the molecular mechanisms of transmitter release.     

Background sodium current stabilizes bursting in respiratory pacemaker neurons

Endogenous pacemaker properties have been proposed to generate rhythmic activity underlying many behaviors including respiration. For pacemakers to generate regenerative bursting, background currents maintain their membrane potential (Vm) within a range where bi-stable properties are expressed, thereby stabilizing rhythmogenesis. We previously found that the baseline Vm of respiratory pacemakers is stabilized against hyperpolarizing shifts in their Vm. In response to prolonged hyperpolarizing current injection synaptically isolated respiratory pacemakers steadily depolarize and resume bursting, suggesting a stabilizing background current is involved. What is the ionic basis of this background current in respiratory pacemakers? Here we demonstrate that in low-[Na(+)](o) ACSF, synaptically isolated respiratory pacemakers hyperpolarized and remained outside the bursting window, but could burst upon depolarizing current injection. These data suggest that pacemakers possess a background sodium current that is necessary to bring their Vm into a bursting range. Low-[Na(+)](o) ACSF also abolished the depolarizing shift evoked during prolonged hyperpolarizing current injection, and bursting did not resume. This depolarizing shift persisted in the presence of I(h)-current blockers, but was abolished in tetrodotoxin. Although, under control conditions, the Vm of synaptically isolated respiratory pacemaker neurons was not significantly affected when [K(+)](o) was changed from 3 to 8 mM, the Vm is altered when [K(+)](o) was raised in low-[Na(+)](o) ACSF. Thus, current-clamp studies suggest that respiratory pacemaker neurons possess a background sodium current that maintains their membrane potential within a range where they express bursting, thereby stabilizing rhythmogenesis.     

Regulation of intracellular Ca2+ by P2Y1 receptors may depend on the developmental stage of cultured rat striatal neurons

Mixed striatal cell cultures containing neurons and glial cells were grown either in neurobasal medium (NBM) or Dulbecco's modified Eagle's medium (DMEM). Whole-cell patch-clamp recordings indicated that, if at all, only a single, low amplitude spike was evoked shortly after starting the injection of a depolarizing current pulse into NBM neurons. In contrast, DMEM neurons fired series of high amplitude action potentials, without apparent spike frequency adaptation. The possible reason for the observed action potential failure in NBM neurons was a low density of Na+ channels per unit of membrane surface area. However, both in NBM and DMEM neurons, ATP did not induce inward current responses via P2X receptor-channels, although GABAA and N-methyl-D-aspartate (NMDA) receptor-channels could be activated by muscimol and NMDA, respectively. Ca2+ imaging experiments by means of the Fura-2 method were utilized to measure intracellular Ca2+ ([Ca2+]i) in neurons and glial cells. NBM, but not DMEM neurons responded to ATP with [Ca2+]i transients; glial cells grown in either culture medium were equally sensitive to ATP. ATP caused an increase of [Ca2+]i by a mechanism only partly dependent on external Ca2+; the residual ATP effect was blocked by cyclopiazonic acid (CPA) and was therefore due to the release of Ca2+ from its intracellular pools. The receptor involved was characterized by P2 receptor antagonists (PPADS, MRS 2179, AR-C69931MX) and was found to belong to the P2Y1 subtype. CPA caused an early [Ca2+]i response due to release from intracellular storage sites, followed by a late [Ca2+]i response due to the influx of this cation from the extracellular space, probably triggered by the opening of store-operated channels (SOCs) in the plasma membrane. It is concluded that in partial analogy with the effect of CPA, ATP releases [Ca2+]i via the Gq/phospholipase C/inositoltrisphosphate (IP3) pathway, thereby opening SOCs. It is hypothesized that this effect of ATP may have an important role for the proliferation and migration of striatal neuronal progenitors.     

Further characterization of the mechanisms mediating the rise in cytosolic free Na+ in thrombin-stimulated platelets. Evidence for inhibition of the Na+,K(+)-ATPase and for Na+ entry via a Ca2+ influx pathway.

Human platelets were loaded with the fluorescent Na(+)-sensitive dye sodium-binding benzofuran isophtalate (SBFI), and changes in the fluorescence excited at 345 and 385 nm were analyzed after manipulations that evoked predictable changes in the cytosolic Na+ concentration ([Na+]i). Raising [Na+]i by either gramicidin D or monensin specifically increased the fluorescence excited at 345 nm and decreased that excited at 385 nm. Hence, calculation of changes in the 345/385 nm excitation ratio yields an estimate of actual changes in [Na+]i. A transient activation of Na+/H+ exchange evoked by addition of acidified platelets to buffer, pH 7.4, evoked a transient rise in [Na+]i. The re-establishment of basal [Na+]i could be prevented by ouabain, indicating an involvement of the Na+,K(+)-ATPase. Upon stimulation by 0.5 unit/ml of thrombin, [Na+]i immediately increased by 16 +/- 4 mM and this rise continued for at least 60 min after addition of agonist, albeit at a lower rate. This latter sustained rise could not be curtailed by scavenging thrombin by means of hirudin. Addition of ouabain or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate induced a comparable slow rise in the 345/385 excitation ratio. This may indicate a protein kinase C-mediated inhibition by thrombin of the Na+,K(+)-ATPase. In the absence of extracellular Ca2+ (Ca2+o), the [Na+]i gain was augmented to 38 +/- 9 mM. This additional uptake of Na+ was prevented by (i) Mn2+ ions, (ii) La3+ ions, (iii) the blocker of receptor-mediated Ca2+ entry (1-[beta[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl]-1H-im ida zole hydrochloride), and (iv) by hirudin which reversed receptor occupancy by thrombin. These findings suggest that the additional thrombin-induced [Na+]i gain in the absence of Ca2+o is due to Na+ influx through a Ca2+ entry pathway. The increase in [Na+]i in the presence of Ca2+o results from Na+ influx via Na+/H+ exchange.