Effects of stimulating the acetylcholine receptor on the current-voltage relationships of the smooth muscle membrane studied by voltage clamp of potential recorded by micro-electrode.

1. A double sucrose-gap voltage-clamp technique is described for use on smooth muscle strips longer than about 2 mm. It involves intracellular recording by microelectrode of the membrane potential of a narrow region of the strip ("node") sandwiched between two streams of deionized sucrose solution. Current was passed into the node across one or both sucrose streams. 2. Preliminary experiments in which potential was recorded intracellularly at two points during polarization of a "short cable" preparation, formed by folding over a strip of smooth muscle, suggested that a node width of less than 0-15 mm was needed to achieve uniform potential during inward current flow. However, when node width between sucrose-gaps was reduced to 0-5 mm, spontaneous electrical activity was lost, and below 0-5 mm spike threshold was raised and the regenerative spike became graded. The currents flowing during the application of rectangular voltage-clamp command potentials were described. 3. Using taenia smooth muscle it was shown by recording with a second, independent micro-electrode that potential was not uniform for up to 200 ms or more following a step change in potential under voltage-clamp in nodes 0-4-0-5 mm wide where current was passed across both sucrose gaps. However, reasonably uniform nodal potentials were obtained using ramps with relatively slow rates of rise (25 mV/s). 4. Using such slow ramp commands under voltage clamp, the effects of carbachol on the current-voltage relationship of longitudinal muscle of ileum and taenia were studied in hypertonic solution. 5. In the presence of carbachol (10(-6) to 10(-5) g/ml.) additional inward current flowed across the membrane (in some experiments an equilibrium potential was observed at which this current reversed direction). The magnitude of this additional current was linearly related to potential at potentials negative to the resting potential. At potentials positive to the resting membrane potential, this additional current increased with depolarization over the range -40 to -10 mV; in ileum the effect of this additional inward current on the current-voltage relationship was to produce a region of net inward current where before, in the absence of carbachol, a net outward current existed. In taenia the additional inward current flowing in the presence of carbachol was too small to produce a region of net inward current; thus carbachol produced regenerative slow oscillations of potential (slow waves) in ileum but not in taenia. 6. These results support a previous suggestion that activation of the acetylcholine receptor of ileal smooth muscle produces an additional inward current in the membrane which increases with depolarization and is responsible for the regenerative slow waves seen when muscarinic stimulants are applied. A similar effect apparently operates in taenia but the additional inward current is too small to produce regenerative slow waves.     

Somatic sodium channels of frog olfactory receptor neurones are inactivated at rest

Membrane excitability of acutely isolated olfactory receptor neurones (ORNs) of the grass frog (R. pipiens) was studied with the use of the whole-cell tight-seal patch recording technique. ORNs of the frog had a mean resting membrane potential of –52 mV, a mean input resistance of 1–2 G, and a mean capacitance of 4.5 pF. In the majority of cells examined (over 70%), short duration (several milliseconds) action potentials were elicited at the end of a hyperpolarising pulse (offspike) or following hyperpolarization of the membrane potential by injection of current. Under voltage-clamp conditions, a fast inward current followed by an outward current could be evoked upon depolarisation of the membrane. The fast inward current decayed with a time constant of 1–2 ms, with an e-fold decrease per 52 mV increase in voltage, and was blocked by the selective voltage-dependent sodium channel blocker tetrodotoxin (0.5–1 M). Steady-state inactivation studies revealed that the mean voltage for half-inactivation (V _1/2) was –82 mV (range –72 to –98 mV), which indicates that the voltage-dependent Na⁺ channels in the cell body or soma of frog ORNs are not available for conducting currents at the resting membrane potential. This finding raises the possibility that voltage-dependent Na⁺ channels may not play a significant role in sensory transduction at the soma. Our results indicate that ORNs of the frog are very efficient in transducing signals towards the brain since currents generated at the cilia will be directed towards depolarising the axon. The data also could account for the low number of single-cell action potentials encountered in extracellular recording experiments in vivo.     

Space-clamp problems when voltage clamping neurons expressing voltage-gated conductances

The voltage-clamp technique is applicable only to spherical cells. In nonspherical cells, such as neurons, the membrane potential is not clamped distal to the voltage-clamp electrode. This means that the current recorded by the voltage-clamp electrode is the sum of the local current and of axial currents from locations experiencing different membrane potentials. Furthermore, voltage-gated currents recorded from a nonspherical cell are, by definition, severely distorted due to the lack of space clamp. Justifications for voltage clamping in nonspherical cells are, first, that the lack of space clamp is not severe in neurons with short dendrites. Second, passive cable theory may be invoked to justify application of voltage clamp to branching neurons, suggesting that the potential decay is sufficiently shallow to allow spatial clamping of the neuron. Here, using numerical simulations, we show that the distortions of voltage-gated K(+) and Ca(2+) currents are substantial even in neurons with short dendrites. The simulations also demonstrate that passive cable theory cannot be used to justify voltage clamping of neurons due to significant shunting to the reversal potential of the voltage-gated conductance during channel activation. Some of the predictions made by the simulations were verified using somatic and dendritic voltage-clamp experiments in rat somatosensory cortex. Our results demonstrate that voltage-gated K(+) and Ca(2+) currents recorded from branching neurons are almost always severely distorted.     

Functional roles of Kv1 channels in neocortical pyramidal neurons

Pyramidal neurons from layers II/III of somatosensory and motor cortex express multiple Kv1 alpha-subunits and a current sensitive to block by alpha-dendrotoxin (alpha-DTX). We examined functional roles of native Kv1 channels in these cells using current-clamp recordings in brain slices and current- and voltage-clamp recordings in dissociated cells. alpha-DTX caused a significant negative shift in voltage threshold for action potentials (APs) and reduced rheobase. Correspondingly, a ramp-voltage protocol revealed that the alpha-DTX-sensitive current activated at subthreshold voltages. AP width at threshold increased with successive APs during repetitive firing. The steady-state threshold width for a given firing rate was similar in control and alpha-DTX, despite an initially broader AP in alpha-DTX. AP voltage threshold increased similarly during a train of spikes under control conditions and in the presence of alpha-DTX. alpha-DTX had no effect on input resistance or resting membrane potential and modest effects on the amplitude or width of a single AP. Accordingly, experiments using AP waveforms (APWs) as voltage protocols revealed that alpha-DTX-sensitive current peaked late during the AP repolarization phase. Application of alpha-DTX increased the rate of firing to intracellular current injection and increased gain (multiplicative effects), but did not alter spike-frequency adaptation. Consistent with these findings, voltage-clamp experiments revealed that the proportion of outward current sensitive to alpha-DTX was highest during the interval between two APWs, reflecting slow deactivation kinetics at -50 mV. Finally, alpha-DTX did not alter the selectivity of pyramidal neurons for DC versus time-varying stimuli.     

Ionic currents and firing patterns of mammalian vagal motoneurons in vitro

The electrophysiological properties of guinea-pig dorsal vagal motoneurons were studied in an in vitro slice preparation. Antidromic, orthodromic and direct stimulation of the neurons demonstrated that the action potential is comprised of several distinct components: a fast initial spike followed by afterdepolarization and an early and a late afterhyperpolarizations. The fast initial spike and the early afterhyperpolarization were blocked by tetrodotoxin and tetraethylammonium ions, respectively. The afterdepolarization (present on the falling phase of the spike) and the late afterhyperpolarization were blocked by the addition of ions known to block calcium conductance (CdCl2, CoCl2 or MnCl2), indicating close association between these two potentials. Prolonged outward current injection through the recording electrode produced two different firing patterns, depending on the initial level of the membrane potential. From resting potential (usually -60 mV) the firing pattern was characterized by a short train of action potentials appearing shortly after the onset of the depolarization step. By contrast, when the depolarization was delivered from a hyperpolarized membrane potential level, a short train of repetitive firing appeared after an initial delay of 300-400 ms. The membrane current responsible for this initial reduction in excitability was studied by means of a single-electrode voltage-clamp technique. The magnitude, direction and kinetics of such current flow are consistent with the presence of early potassium current (IA), partly inactive at the resting potential. Synaptic activation of vagal motoneurons could be obtained by electrical stimulation of the tissue surrounding the vagal nucleus or by direct activation of the vagal nerve. Perivagal stimulation generated excitatory and inhibitory synaptic potentials which could be reversed by shifting the membrane potential. Vagal nerve stimulation, in addition to the antidromic activation of the cells, generated depolarizing responses which were unitary in nature and did not show much sensitivity to shifts in membrane potential. Perivagal and vagal nerve-evoked depolarizations could generate action potentials as well as partial dendritic spikes. We conclude that spike electroresponsiveness in vagal motoneurons is generated by voltage-dependent Na+ and Ca2+ conductances. In addition, the Ca2+-dependent current triggers a K+ conductance which is responsible for modulating the firing frequency obtained from the normal resting level.(ABSTRACT TRUNCATED AT 400 WORDS)     

Na+ and Ca2+ currents of acutely isolated adult rat nodose ganglion cells

The electrical properties of nodose ganglion cells acutely isolated from adult rats were studied using the whole-cell patch-clamp recording method. Current-clamp recordings revealed a mean resting membrane potential of -54.3 mV and an input resistance of 527 M omega. Depolarizing current steps evoked action potentials with the following properties (mean): amplitude 111 mV, threshold -36 mV, and rate of rise 117 V/s. Two types of action potentials were observed, short and long duration. These properties, with the exception of input resistance (527 M omega cf. 50 M omega), are similar to those reported previously using intracellular recording methods in intact nodose ganglia (11, 20, 28). Brief application of 10 microM 5-hydroxytryptamine resulted in a rapid depolarization and burst of action potentials in the majority of cells. With voltage-clamp recording, step depolarizations to potentials positive to -10 mV elicited a transient inward current that was followed by a sustained outward current. Inward Na+ current was isolated by ion substitution and pharmacological agents. Two types of Na+ current were observed. One current was completely abolished by 3-15 microM tetrodotoxin (TTX), had a rapid time course, activated over the potential range -60 to -10 mV, and attained half-maximal conductance at -30 mV. The other current persisted in the presence of 15 microM TTX, had a slower time course, activated over the potential range -30 to 0 mV, and attained half-maximal conductance at -15 mV. In addition, 500 microM Cd2+ and 5.0 mM Co2+ reduced the TTX-insensitive current to 53 and 42% of control, respectively. Inward Ca2+ current was isolated by ion substitution and pharmacological agents and was identified by a dependence on external Ca2+. Cd2+ (500 microM) and Co2+ (5 mM) reduced the maximal inward current to 5 and 20% of control, respectively. When Ba2+ was substituted for Ca2+ as the charge carrier, the maximal inward current increased to 175% of control. Some cells had two Ca2+ current components, an inactivating component that activated near -60 mV and a large sustained current that activated near -40 mV. The initial inactivating current appeared as a "hump" on the current-voltage (I-V) curve over the potential range of -60 to -30 mV. The results indicate that, following isolation of these adult mammalian neurons, the membrane surfaces are sufficiently clean to allow patch-clamp recording.(ABSTRACT TRUNCATED AT 400 WORDS)     

Excitable properties and voltage-sensitive ion conductances of horizontal cells isolated from catfish (Ictalurus punctatus) retina

External horizontal cells were enzymatically dissociated from intact catfish (Ictalurus punctatus) retina and pipetted onto a small chamber attached to the stage of an inverted phase-contrast microscope. Individual horizontal cells were recognized by their large size and restricted dendritic arborization. Low-resistance (3-12 M omega) patch-type electrodes were used to record intracellular potentials and to pass current across the cell membrane under either current or voltage-clamp conditions. The average resting potential of isolated horizontal cells was -67 V + 6.9 mV (mean +/- SD, n = 40). At the resting potential, the cell membrane appears to be mainly permeable to K. A depolarizing current step evoked an action potential in the cell. The maximum rate of rise of the action potential (dV/dt) in normal physiological solution was 6.5 +/- 1.8 V/s (means +/- SD, n = 24) and was reduced to 1.2 +/- 0.39 V/s (means +/- SD, n = 9) in 1-10 micron tetrodotoxin (TTX) and 3.2 +/- 1.4 V/s (means +/- SD, n = 6) in Ca-free solution. The maximum dV/dt was reduced in 10 mM extracellular K concentration [K]o to about half of that seen in standard saline, and values in 30 or 80 mM [K]o were similar to that measured in TTX. Following an action potential, the membrane potential reached a plateau potential of + 17.4 +/- 8.1 mV (means +/- SD, n = 17) and remained depolarized for variable periods of time lasting from less than a second to a few minutes. When the plateau potential was long lasting, the cell repolarized slowly and upon reaching zero rapidly repolarized to the original resting potential. The duration of the plateau potential decreased or was absent in saline containing one of the following calcium channel antagonists: La, Cd, Co, or Ni. The voltage-clamp technique was used to identify the membrane currents responsible for the membrane potential changes seen under current clamp. Experiments were carried out using either a single or two individual electrodes. Fast and steady-state inward currents were recorded from isolated horizontal cells in the voltage range between -20 and +20 mV. These currents were a result of increased membrane conductance to both Na and Ca ions. The Na channels are inactivated at depolarized potentials and are TTX sensitive. Ca channels are partially inactivated at depolarized potentials. The Ca conductance is decreased by Cd, Co, Ni, and La. Ba can substitute for Ca in the channel.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of intracellular calcium injection on steady state membrane currents in isolated single ventricular cells

The effect of intracellular calcium injection on steady state membrane currents was studied in isolated guineapig ventricular cells with a single-electrode voltage clamp method. When the membrane potential was clamped at the resting potential, an intracellular injection of Ca induced an inward current. The steady state current-voltage (I–V) curves before and after Ca injection crossed at around ?22 mV. Above this potential, Ca increased the current in the outward direction. A linear relationship was observed between the current mediated by the increase of intracellular Ca and membrane potentials from ?60 to +10 mV. This current component may correspond to the Ca-activated non-selective cation channels recorded by the patch clamp technique (Colquhoun et al., 1981).     

Calcium-dependent current generating the afterhyperpolarization of hippocampal neurons

A single-electrode voltage-clamp technique was employed on in vitro hippocampal slices to examine the membrane current responsible for the slow afterhyperpolarization (AHP) in CA1 pyramidal cells. This was achieved by using conventional procedures to evoke an AHP in current clamp, followed rapidly by a switch into voltage clamp (hybrid clamp). The AHP current showed a dependence on extracellular K+, which was close to that predicted for a K+ current by the Nernst equation. The AHP current could be blocked by Cd2+ or norepinephrine. Although the AHP current showed a requirement for voltage-dependent Ca2+ entry, the current did not show any clear intrinsic voltage dependence. Once activated, AHP current is not turned off by hyperpolarizing the membrane potential. The effects of norepinephrine, Cd2+, and tetraethylammonium (TEA) were used to identify an AHP current component to the outward current evoked by depolarizing voltage commands from holding potentials that approximate to the resting potential for these cells. The AHP current can contribute significantly to the outward current during the depolarizing command. Upon repolarization it is evident as a slow outward tail current. This slow tail current had the same time constant as AHP currents evoked by hybrid clamp. Fast components to the tail currents were also observed. These were sensitive to Cd2+ and TEA. They probably represent a voltage-sensitive gKCa, sometimes termed C-current. The strong sensitivity to voltage and TEA displayed by the conventionally described gKCa (IC) are properties inconsistent with the AHP. It seems likely that the AHP current (IAHP) represents a Ca2+-activated K+ current separate from IC and that these two currents coexist in the same cell.     

Patch-clamp study of postnatal development of CA1 neurons in rat hippocampal slices: membrane excitability and K+ currents.

1. The postnatal development of membrane properties and outward K+ currents in CA1 neurons in rat hippocampal slices was studied with the use of whole-cell patch-clamp techniques. 2. Neurons at all postnatal ages (2-30 days; P2-30) were capable of generating tetrodotoxin (TTX)-sensitive action potentials in response to intracellular injection of depolarizing current pulses. There was a gradual increase in the amplitude and a decrease in the duration of these action potentials with age. Stable values for spike duration were reached by P15, whereas spike amplitude increased until P20-25. In P2-5 neurons, the duration of action potentials was greatly prolonged by depolarization from the resting membrane potential, indicating a weak spike repolarizing mechanism at depolarized potentials. In contrast, the duration of spikes evoked in P20-30 neurons was not affected by similar changes in the membrane potential. 3. Application of tetraethylammonium (TEA, 10 mM) had no effect on the duration of spikes in P3-5 neurons, whereas application of 4-aminopyridine (4-AP, 2 mM) produced large increases in spike duration. In contrast, the duration of spikes in P26 neurons was greatly increased after TEA application, whereas 4-AP had smaller effects on spike duration in these neurons. 4. The input resistance and membrane time constant decreased with age from P2 to P15. The values for both parameters were considerably greater than those reported with conventional intracellular recording electrodes in the immature hippocampus. The resting membrane potential became more hyperpolarized with age. When the recording pipettes contained KCl (140 mM), the resting potential of P3-4 neurons was 34 mV depolarized compared with resting potentials observed with potassium gluconate-filled pipettes. Only a 13-mV change in resting potential was observed during similar comparisons in P27-28 neurons. 5. Outward currents activated by depolarization were examined with the use of voltage-clamp techniques in P2-30 neurons. In P2-5 cells, a small, slowly inactivating outward current was evoked with depolarizing commands from holding potentials near -50 mV. By preceding the depolarizing commands with a hyperpolarizing prepulse, an additional early transient outward current was evoked. The sustained and transient outward currents were separated by their kinetic properties and their sensitivity to cobalt (Co2+), TEA, and 4-AP.(ABSTRACT TRUNCATED AT 400 WORDS)     

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

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

Activation of a nonspecific cation conductance by intracellular Ca2+ elevation in bursting pacemaker neurons of Helix pomatia

The pacemaker current of a bursting neuron of Helix pomatia was investigated using voltage-clamp and pressure-injection techniques. In the steady state the net membrane current was zero near threshold of the action potential at -45 mV. Negative to this potential the membrane current was inward and steady. During burst activity a long-lasting inward current instantaneously appeared with voltage steps to membrane potentials below -20 mV. This inward current was already present when the clamp step fell into the rising phase of the first spike and became larger during the depolarizing phase of the spike. The repolarization phase and the interspike interval did not add much current. As the spike duration became longer in the course of the burst discharge the inward current grew in amplitude, but its increase was not proportional to that of the spike duration. This was observed with clamp steps to the potassium equilibrium potential (EK = -70 mV). The inward current decayed during a hyperpolarizing step with a half time of approximately 400 ms, which was invariant to voltage as measured between -40 and -100 mV. It decreased linearly from -100 to -40 mV with an extrapolated zero potential of about -20 mV. The inward current was not generated by spikes if the Ca2+ conductance was blocked by Ni2+. At membrane potentials positive to EK the development of an outward current, probably carried by K+, could be observed during the burst. It overlasted the inward current and decayed with time constants of 6-7 s. This current grew successively in amplitude in the course of the burst discharge and finally nullified the inward-current component at potentials around spike threshold, thus terminating the burst. An inward current with properties similar to the spike-induced inward current was produced by pressure injecting CaCl2 into the neurons. This current was unselectively carried by cations as shown by both ion-substitution experiments and measurements with ion-selective microelectrodes. Large cations such as choline, TEA, and Tris passed through the channels nearly as well as Na+. Changes in the H+ or Cl- concentration were not seen to affect the inward current. Spike as well as the injection-induced currents were largest in bursting pacemaker cells compared with other cells of similar size. Both currents were found to be small or absent in nonbursting but regularly firing pacemaker cells, albeit these cells reveal a larger Ca2+ current density than the bursting pacemaker cell.(ABSTRACT TRUNCATED AT 400 WORDS)     

Participation of a chloride conductance in the subthreshold behavior of the rat sympathetic neuron.

The presence of a novel voltage-dependent chloride current, active in the subthreshold range of membrane potential, was detected in the mature and intact rat sympathetic neuron in vitro by using the two-microelectrode voltage-clamp technique. Hyperpolarizing voltage steps applied to a neuron held at -40/-50 mV elicited inward currents, whose initial magnitude displayed a linear instantaneous current-voltage (I-V) relationship; afterward, the currents decayed exponentially with a single voltage-dependent time constant (63.5 s at -40 mV; 10.8 s at -130 mV). The cell input conductance decreased during the command step with the same time course as the current. On returning to the holding potential, the ensuing outward currents were accompanied by a slow increase in input conductance toward the initial values; the inward charge movement during the transient ON response (a mean of 76 nC in 8 neurons stepped from -50 to -90 mV) was completely balanced by outward charge displacement during the OFF response. The chloride movements accompanying voltage modifications were studied by estimating the chloride equilibrium potential (E(Cl)) at different holding potentials from the reversal of GABA evoked currents. [Cl(-)](i) was strongly affected by membrane potential, and at steady state it was systematically higher than expected from passive ion distribution. The transient current was blocked by substitution of isethionate for chloride and by Cl(-) channel blockers (9AC and DIDS). It proved insensitive to K(+) channel blockers, external Cd(2+), intracellular Ca(2+) chelators [bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)] and reduction of [Na(+)](e). It is concluded that membrane potential shifts elicit a chloride current that reflects readjustment of [Cl(-)](i). The cell input conductance was measured over the -40/-120-mV voltage range, in control medium, and under conditions in which either the chloride or the potassium current was blocked. A mix of chloride, potassium, and leakage conductances was detected at all potentials. The leakage component was voltage independent and constant at approximately 14 nS. Conversely, gCl decreased with hyperpolarization (80 nS at -40 mV, undetectable below -110 mV), whereas gK displayed a maximum at -80 mV (55.3 nS). Thus the ratio gCl/gK continuously varied with membrane polarization (2.72 at -50 mV; 0.33 at -110 mV). These data were forced in a model of the three current components here described, which accurately simulates the behavior observed in the "resting" neuron during membrane migrations in the subthreshold potential range, thereby confirming that active K and Cl conductances contribute to the genesis of membrane potential and possibly to the control of neuronal excitability.     

Proctolin activates a slow, voltage-dependent sodium current in motoneurons of the lobster cardiac ganglion

The two-electrode voltage-clamp technique was used to study the ionic basis of the slow depolarization caused by the neuropeptide proctolin. At negative membrane potentials, proctolin caused a dose-dependent slow inward current. This current reversed and became outward at membrane potentials positive to 0 to +20 mV. Current-voltage curves also showed the response to be voltage-dependent, with a reversal potential at positive membrane potentials. The response was blocked in sodium-free solutions. Solutions with reduced sodium concentration caused a shift of the reversal potential in a manner expected for a response that is largely sodium-dependent. We conclude that proctolin causes an inward current by increasing a voltage-dependent cation conductance that is predominantly permeable to sodium.     

Sodium currents in isolated rat CA1 pyramidal and dentate granule neurones in the post-status epilepticus model of epilepsy

Status epilepticus (SE) was induced in the rat by long-lasting electrical stimulation of the hippocampus. After a latent period of 1 week, spontaneous seizures occurred which increased in frequency and severity in the following weeks, finally culminating after 3 months in a chronic epileptic state. In these animals we determined the properties of voltage-dependent sodium currents in acutely isolated CA1 pyramidal neurones and dentate granule cells using the whole-cell voltage-clamp technique. The conductance of the fast transient sodium current was larger in SE rats (84+/-7 nS versus 56+/-6 nS) but related to a difference in cell size so that the neurones had a similar specific sodium conductance (control: 7.8+/-0.8 nS/pF, SE: 6.7+/-0.8 nS/pF). Current activation and inactivation were characterised by a Boltzmann function. After SE the voltage dependence of activation was shifted to more negative potentials (control: -45.1+/-1.4 mV, SE: -51.5+/-2.9 mV, P<0.05). In combination with a small shift in the voltage dependence of inactivation to more depolarised potentials (control: -68.8+/-2.3 mV, SE: -66.3+/-2.3 mV), it resulted in a window current that was much increased in the SE neurones (median: 64 pA in control, 217 pA in SE, P<0.05). The peak of this window current shifted to more hyperpolarised potentials (control: -44 mV, SE: -50 mV, P<0.05). No differences were found in the sodium currents analysed in dentate granule cells of control and SE animals. The changes observed in CA1 neurones after SE contribute to enhanced excitability in particular when membrane potential is near firing threshold. They can, at least partly, explain the lower threshold for epileptic activity in SE animals. The comparison of CA1 with DG neurones in the same rats demonstrates a differential response in the two cell types that participated in very similar seizure activity.     

Properties of subthreshold response and action potential recorded in layer V neurons from cat sensorimotor cortex in vitro

Properties of the action potential and subthreshold response were studied in large layer V neurons in in vitro slices of cat sensorimotor cortex using intracellular recording and stimulation, application of agents that block active conductances, and a single-microelectrode voltage clamp (SEVC). A variety of measured parameters, including action-potential duration, afterpotentials, input resistance, rheobase, and membrane time constant, were similar to the same parameters reported for large neurons from this region of cortex in vivo. Action-potential amplitudes and resting potentials were greater in vitro. Most measured parameters were distributed unimodally, suggesting that these parameters are similar in all large layer V neurons irrespective of their axonal termination. The voltage response to subthreshold constant-current pulses exhibited both time and voltage dependence in the great majority of cells. Current pulses in either the hyperpolarizing or subthreshold depolarizing direction cause the membrane potential to attain an early peak and then decay (sag) to a steady level. On termination of the pulse, the membrane response transiently overshoots resting potential. Plots of current-voltage relations demonstrate inward rectification during polarization on either side of resting potential. Subthreshold inward rectification in the depolarizing direction is abolished by tetrodotoxin (TTX). The ionic currents responsible for subthreshold rectification and sag were examined using the SEVC. Steady inward rectification in the depolarizing direction is caused by a persistent, subthreshold sodium current (INaP) (54). Sag observed in response to a depolarizing current pulse is due to activation of a slow outward current, which superimposes on and partially counters the persistent sodium current. Both sag in response to hyperpolarizing current pulses and rectification in the hyperpolarizing direction are caused by a slow inward "sag current" that is activated by hyperpolarizing voltage steps. The sag current is unaltered by TTX, tetraethylammonium, (TEA), Co2+, Ba2+, or 4-aminopyridine. Fast-rising, short-duration action potentials can be elicited by an intracellular current pulse or by orthodromic or antidromic stimulation. Spikes are blocked by TTX. The form of the afterpotential following a directly evoked spike varies among cells with similar resting potentials. Biphasic afterhyperpolarizations (AHPs) with fast and slow components were most frequently seen. About 30% of the cells displayed a depolarizing afterpotential (DAP), which was often followed by an AHP. Other cells displayed a purely monophasic AHP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Feedback effects of horizontal cell membrane potential on cone calcium currents studied with simultaneous recordings

Horizontal cell (HC) to cone feedback helps establish the center-surround arrangement of visual receptive fields. It has been shown that HC activity influences cone synaptic output by altering the amplitude and voltage dependence of the calcium current (ICa) in cones. In this study, we obtained voltage-clamp recordings simultaneously from cones and HCs to directly control the membrane potential of HCs and thereby measure the influence of HC membrane potential changes on ICa in adjacent cones. Directly hyperpolarizing voltage clamped HCs produced a negative activation shift and increased the amplitude of ICa in cones. Both of these effects were abolished by enhancing extracellular pH buffering capacity with HEPES. In contrast, addition of the gap junction blocker, carbenoxolone, did not significantly alter the shifts or amplitude changes in cone ICa produced by changes in HC membrane potential. These results support the hypothesis that changes in the HC membrane potential alter the voltage dependence and amplitude of cone ICa by altering extracellular pH levels at the synapse.     

Identification of different putative neuronal subtypes in cultures of the superior region of the hippocampus using electrophysiological parameters.

Cultured neurons offer many advantages over a slice preparation for whole-cell patch-clamp studies, such as better control over the environment and space clamp control. However, heterogeneous cultures of neurons present problems in distinguishing the cell type from which recordings are made. The present study uses correlations with data obtained in the hippocampal slice preparation to determine the feasibility of "identifying" different neuronal subtypes in cultures obtained from the superior region of postnatal two- to 13-day-old rat hippocampus. Whole-cell patch-clamp recording in the current-clamp mode after 24-96 h in culture was used to determine if the action potential duration would be a useful criterion in distinguishing cell types. Single action potentials were elicited by a 0.1-0.2 ms, 2-4 nA depolarizing pulse. The average membrane potential and input resistance were -46.8+/-1.2 mV (n = 58) and 576+/-56 Mohms (n = 57), respectively. A frequency distribution of the action potential duration measured at half-maximal amplitude showed four distinct groups of neurons (group 1, 1.36+/-0.03 ms, n = 17; group 2, 2.19+/-0.05 ms, n = 20; group 3, 3.17+/-0.10 ms, n = 16; group 4, 4.36+/-0.13, n = 5). Based on correlations with previous studies using intracellular recording in identified cells in slices, the data suggest that group 1 represents basket cells, group 2 represents vertical cells, group 3 represents a combination of stellate cells and pyramidal cells, and group 4 represents another unidentified class of cells. Further analysis of the fast afterhyperpolarization allows distinction between pyramidal cells and stellate cells in group 3. In contrast to the interneurons in a slice preparation, these cells offer good voltage control and environmental control. Future studies will record from these cells in current-clamp mode to quickly characterize the action potential before switching to voltage-clamp recording to characterize the currents present in the different types of interneurons.