Alterations in the action potential of Aplysia neurons evoked by a phorbolester are mediated by protein kinase C.

Alterations in the parameters of action potentials upon changes in protein kinase C (PKC) activity were studied on neurons of the visceral ganglion of Aplysia californica. The amplitude and maximum speed of the up-and downstroke of the action potentials (APs) were measured. Intracellularly injected PKC and intra- and extracellularly applied phorbol-12,13-diacetate (PDAc) had similar effects on the Aplysia neurons, the most prominent being an increase of the upstroke speed of the AP in every neuron. The non-PKC-activating 4 alpha-phorbol didecanoate had no effect, and the effects of the PKC blocker H-7 were opposite to those of PDAc. It was concluded that the changes of the AP evoked by PDAc are mediated through PKC activation.

Phorbol diacetate differentially regulates the N-methyl-D-aspartate (NMDA) and non-NMDA receptor-mediated components of the rat hippocampal excitatory postsynaptic currents.

The effects of phorbol 12,13-diacetate (PDAc) on evoked excitatory transmission were studied in neurons of the CA1 area of hippocampal slices of rats, using whole-cell voltage clamp of pyramidal neurons in situ and stimulation of the Schaffer collaterals. The application of PDAc (10 microM) increased the amplitude of the excitatory postsynaptic current (EPSC) and caused a lengthening of its decay, due to an increase in the contribution of the N-methyl-D-aspartate (NMDA) component to the total EPSC. The latter effect was depend upon the concentration of calcium in the extracellular medium. Experiments in which we separated the two components of the EPSCs by 6-cyano-7-nitroquinoxaline-2,3-dione and by 2-amino-5-phosphonopentanoic acid also demonstrated a more pronounced increase in the NMDA receptor-mediated current under PDAc. The effects of PDAc were markedly attenuated by the extracellular application of the protein kinase C inhibitor H-7 (300 microM), but not by intracellular perfusion with 20 mM of the same drug.

The action of a phorbol ester on voltage-dependent parameters of the sodium current in isolated hippocampal neurons.

The action of a phorbol ester (phorbol-12,13-diacetate) on the voltage-activated sodium current has been investigated by the voltage-clamp method in acutely isolated pyramidal neurons from rat hippocampus. The intracellular perfusion of isolated pyramidal neurons for 30-40 min induced a gradual 10-15 mV shift in both the current-voltage relationship and voltage-dependent steady-state inactivation to more negative potentials. The application of phorbol ester (1-10 microM) to isolated neurons for the same time increased the amplitude of sodium current by 15-20%, shifted the above-mentioned voltage-dependent parameters for an additional 10-15 mV in the same direction and changed the slope of the steady-state inactivation curve. In contrast, after prolonged incubation of slices in the phorbol ester-containing solution (1-10 microM) for 0.5-3 h, subsequent application of phorbol ester at the same concentration caused neither the addition shift of the voltage-dependent characteristics of sodium channels nor the change of the slope of the steady-state inactivation curve. However, in this case an increase in the amplitude of sodium current by 15-20% during 30-40 min intracellular perfusion was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase C activation in Aplysia neurons by phorbol diacetate: comparison of effects following extracellular or intracellular application.

The effects of protein kinase C activation on electrophysiological phenomena of neurons in Aplysia californica ganglions were studied. The enzyme was activated by phorbol-12,13-diacetate applied either extracellularly by perfusion, or by intracellular pressure injection. In both forms of application, an increase in the potential upstroke speed and amplitude as well as a reduction of the depolarization evoked by extracellular acetylcholine application was found. A protein kinase C blocker, H-7, had opposite effects on the action potential. All observed actions of the phorbol ester were consistently faster in intracellular application than extracellular.

Differential blockade of bicuculline and strychnine on GABA- and glycine-induced responses in dissociated rat hippocampal pyramidal cells.

The inhibitory effects of bicuculline (BIC) and strychnine (STR) on GABA- and glycine-induced responses were studied in the rat dissociated hippocampal CA1 pyramidal neurons in whole-cell mode by using the conventional patch-clamp technique. Both GABA and glycine elicited inward Cl- currents in a dose-dependent manner and had almost the same maximal responses. The half-maximum dose (Ka) and Hill coefficient were 6.4 microM and 1.1 for the GABA response, and 74 microM and 1.5 for the glycine response. BIC and STR antagonized both GABA and glycine responses in a competitive manner. The blocking potency of BIC and STR on the GABA response was comparable. The half inhibition dose (IC50) was 2.7 microM for BIC and 6.7 microM for STR. STR blocked the glycine response about 3,000 x more effectively than BIC. The IC50 was 28 nM for STR and 100 microM for BIC. The BIC and STR did not have voltage-dependent blocking effects on either GABA or glycine responses. Neither GABA nor glycine showed outward rectification in their current-voltage relationships. The functional role of glycine in the rat hippocampal CA1 region is discussed.

Voltage clamp analysis of the kinetics of piperidine-induced chloride current in isolated Aplysia neurons.

The effect of piperidine (Pip) on isolated Aplysia neurons was investigated using the voltage clamp and concentration clamp techniques in which neurons were perfused internally and externally with Na+,K+ free solution. Pip induced a Cl-current (ICl) in a dose-dependent manner for doses ranging from 10(-4) M to 10(-2) M. The dose-response curve gave an apparent dissociation constant of 8.4 x 10(-4) M and a Hill coefficient of 1.7. The current-voltage relationship was linear in the voltage range examined (-70 to +30 mV). The equilibrium potential for Pip induced current was close to the calculated equilibrium potential for chloride ions (ECl), (-10.7 mV). The activation phase of the ICl was characterized by a single exponential at all concentrations. The time constant of this phase decreased with increasing concentrations of Pip but did not depend on the membrane potential. The deactivation phase of the ICl proceeded on a single exponential curve at concentrations of Pip less than 5 x 10(-4) M, but on a double exponential at concentrations of 5 x 10(-4) M and higher. The deactivation time constant also decreased with increasing concentrations of Pip, but showed no potential dependence. Pip- and ACh-induced IClS were not blocked by 10(-4) M atropine. However, Pip-induced ICl was abolished with 10(-4) M d-tubocurarine (dTC), and the ACh-induced ICl was depressed by the same dose of dTC. These results suggest that Pip acts on at least two components of the nicotinic receptor-Cl channel complex in Aplysia neurons to elicit the ICl.

Piperidine discriminates between the transient and the persistent components of the ACh-induced chloride current in Aplysia neurons.

ACh-induced Cl- -current (ICl) is well known to desensitize with two components: an initial fast phase followed by a second, more slowly developing phase. In the present study, the influence of piperidine, a normal constituent in vertebrates and invertebrates, on ACh-induced ICl in isolated neurons of Aplysia was investigated by using the concentration clamp in combination with the voltage clamp technique. Pretreatment with piperidine in doses greater than 2 X 10(-4)M depressed the transient ACh-induced ICl but had little effect on the persistent ICl. Kinetic study of the desensitization phase of ACh-induced ICl showed that the slow time constant of the desensitization phase of ACh-induced ICl was not altered by pretreatment with piperidine. The present results indicate that piperidine can discriminate between the fast transient and slow persistent components of ACh-induced ICl in Aplysia neurons, and also suggest that two components of the desensitization phase of ACh-induced ICl function in an independent manner.

Valproate enhances inhibitory postsynaptic potentials in hippocampal neurons in vitro.

Effects of sodium valproate (0.5-10 mM) on hippocampal cells (CA3 pyramidal cells and granule cells) of the guinea pig in vitro were studied by intra- and extracellular recording. Inhibitory postsynaptic potentials were markedly and reversibly augmented. Their shunting action as well as duration increased by more than 150%. Effects of locally administered gamma-aminobutyric acid (GABA) did not change significantly. Membrane characteristics such as membrane potential, membrane input resistance and action potential did not alter. Valproate suppressed spontaneous spiking and repetitive discharges evoked by GABA antagonists. It is concluded that valproate enhances GABAergic synaptic transmission by a presynaptic mechanism. However, postsynaptic mechanisms might be involved in the limitation of repetitive firing.

Differences in baclofen-sensitivity between CA3 neurons and granule cells of the guinea pig hippocampus in vitro.

In the slice preparation of the guinea pig hippocampus, the effects of (+/-) baclofen added to the Krebs-Ringer solution on dentate granule cells and CA3 pyramidal cells were investigated by means of intracellular recording techniques. In a 10-25 microM concentration, baclofen reduces the inhibitory postsynaptic potentials of the granule cells evoked by electrical stimulation of the perforant path and hyperpolarizes the granule cell membrane slightly. The reduction of both, the excitatory and inhibitory postsynaptic potentials of CA3 pyramidal cells evoked by mossy fiber stimulation, however, is accompanied by a strong hyperpolarization and conductance increase. Further, repetitive discharges of granule cells elicited in the presence of the convulsant bicuculline (25 microM) are hardly affected by baclofen (50 microM), whereas those of CA3 neurons are blocked.

Bridge balance in intracellular recording; introduction of the phase-sensitive method.

Passing current through the microelectrode during intracellular recording produces an artifactual voltage-drop across the electrode's resistance. In modern biological amplifiers, the means of removing this artifact is through what is no more than an analog arithmetic unit which subtracts a voltage, having some fixed proportion to the current being injected, from the voltage appearing at the input to the amplifier. Various techniques have evolved for determining that fixed proportion, an operation which has come to be referred to as bridge balance, and these are reviewed. A new method is presented for determining bridge balance and another amplifier parameter which affects current injection, capacitance neutralization. Essentially, a sinusoidal current is passed through the electrode and preparation at a frequency high enough so that the cellular membrane presents negligable impedance but low enough so that the electrode appears as nearly a pure resistance. A set of analytical formulations is presented which describes the system and which is useful in choosing the best frequency of the test sinusoidal signal. This choice, part of the experimental design, is based on the predicted time constants of the recorded cell and the recording electrode. In using the phase-sensitive method, the capacitance neutralization of the recording amplifier is adjusted until there is no phase error in the injected current. Bridge balance can then be adjusted until no sinusoidal signal is visible at the output of the amplifier. Both bridge balance and correction of capacitance compensation can be automatically and continuously performed during intracellular recording by incorporating a phase-sensitive alternating current (AC) voltmeter (i.e. a lock-in amplifier) into the system. This permits accurate measurement of intracellular potential during current injection even though the resistance of the electrode might be changing.

GABA and lioresal actions on the identified Onchidium neuron.

Responses of identified single neurons in the isolated right cerebral ganglion of Onchidium to bath applied gamma-aminobutyric acid (GABA) and lioresal (LRS) were measured using conventional intracellular microelectrode techniques under current and voltage clamp conditions. In normal medium the neurons responded to both GABA and LRS. GABA induced two successive hyperpolarizations (phase I and II). Phase I was a short-lasting Cl- permeability increase while phase II was a long-lasting K+ permeability increase. LRS evoked only a long-lasting K+ permeability increase. In Ca2+-free high Mg2+ medium GABA produced only short-lasting hyperpolarization while LRS had no effect. GABA-induced phase I responses behaved as a simple Cl-electrode following changes of external Cl- concentrations. The Hill coefficient obtained from the relation between the relative change in the GABA increase phase in Ca2+-free high Mg2+ medium. These results indicate that GABA has a direct Cl- permeability increase action on the postsynaptic cell membrane of the identified Onchidium neuron while LRS has no such direct action.

[Effect of memantine on membrane properties and postsynaptic potentials in neurons of Aplysia californica]. / Die Wirkung von Memantin auf Membraneigenschaften und postsynaptische Potentiale von Nervenzellen der Aplysia californica.

The effect of 1,3-dimethyl-5-aminoadamantane (DMAA, D-145, memantine, Memantine) in the concentration range of 10(-6) to 10(-3) mol/l was investigated on membrane properties and synaptic transmission by the current or voltage clamp method on neurons in the visceral ganglion of Aplysia california. In the lower range (concentrations 10(-6) to 10(-5) mol/l) the substance increases the speed of voltage changes during the course of the action potential, increases spontaneous synaptic activity, and induces a slight membrane depolarization. With increasing concentration the effects reverse in that slope of action potential is slowed and synaptic transmission delayed or blocked. At the RC-R15 synapse, isolated from its interneuron, 10(-5) mol/l DMAA evokes spontaneously and regularly firing EPSPs; 10(-3) mol/l blocks both, spontaneous activity, and its responsiveness to electrical stimulation.

Single electrode voltage clamp by iteration.

A technique for providing conditions of voltage clamp which differs considerably from other voltage clamp schemes has been developed. The feedback network which determines the current which will clamp the cell to the desired voltage does not operate in real time. Instead, the system uses a form of discontinuous feedback. The event to be clamped, which must be one which can be made to repeat itself without change, is elicited and allowed to run to completion without the intervention of feedback. During each repetition of the event, a current waveform is injected whose shape is based on the foregoing trials (iterations). Successive repetitions of this process develop a current waveform which ever more closely clamps the voltage to the desired value. Implementation involves a means of converting the intracellular voltage signal to digital form (a transient recorder), a means of processing the digitalized voltage signal (a digital computer), and a means of delivering the clamping current back to the preparation. The system has two advantages over other voltage clamp techniques. First, that the feedback loop is open in real time confers great stability. This advantage is exploited in the use of iterative voltage clamp in single electrode preparations. Secondly, iterative voltage clamp is essentially unlimited in the speed with which it can respond to transients. This would make the technique of interest even in preparations such as squid giant axon, where two electrodes are used and very fast responsiveness is desired.

Heterosynaptic postactivation potentiation in hippocampal CA 3 neurons: long-term changes of the postsynaptic potentials.

CA 3 neurons were excited synaptically by stimulation in the dentate hilus and the stratum radiatum of CA 1 in guinea pig hippocampal slices. Following repetitive stimulation (10--20 c/s, 10 s) of either stimulation site, the amplitudes of orthodromic population spikes or the probability of unitary discharges increased. Changes of the intracellularly recorded potentials were either (a) increased EPSP amplitudes associated with decreased IPSP amplitudes, or (b) increased IPSP amplitudes. A cell showing enhanced IPSPs after repetitive activation could respond with increased EPSP amplitudes and decreased IPSP amplitudes upon further repetitive activation. The potentiation, which was always preceded by a 5--10 min depression, lasted up to 3 h. This potentiation was heterosynaptic, since the responses to the non-stimulated input also changed and since the inputs were found to excite the pyramidal cells through separate synapses in double shock experiments. The heterosynaptic mode of the potentiation as well as the changes of the IPSPs indicate that not only the excitatory pathway but also the inhibitory pathway must be considered in explaining postactivation potentiation in this hippocampal field.

Effect of temperature on postsynaptic potentials of cat spinal motoneurones.

The effect of spinal cord temperature on excitatory postsynaptic potentials (EPSP) and inhibitory postsynaptic potentials (IPSP) were measured by means of intracellular recordings from lumbar motoneurones of 43 cats. While body temperature and oil bath temperature were maintained between 37 and 38 degrees C, the temperature of the spinal segment under investigation was changed separately in the range between 30 and 42 degrees C. Cooling consistently produced an increase in amplitude and duration of both, mono- and poly-synaptic EPSPs and recurrent and direct IPSPs. Warming caused the opposite effect. The input resistance of the motoneurones was inversely related to the spinal cord temperature, while the latency of action potentials produced by intracellular injection of outward current was directly and exponentially related to spinal temperature. Although the data do not provide a quantitative differentiation of pre- versus postsynaptic temperature effects, they are consistent with the notion that temperature dependent changes on postsynaptic membrane properties contribute to the observed PSP changes. It is further suggested that similar postsynaptic temperature effects may be concerned in temperature sensitivity of proposed specific central neurones.