Role of ATP-dependent K(+) channels in the electrical excitability of early embryonic stem cell-derived cardiomyocytes.

Single, murine embryonic stem cell-derived early stage cardiomyocytes dissociated from embryoid bodies expressed two inward rectifier K(+) channels, I(K1) and the ATP dependent K(+) current. I(K1) exhibited low density in early stage cardiomyocytes, but increased significantly in late stage cells. In contrast, the ATP dependent K(+) current was expressed at similar densities in early and late stage cardiomyocytes. This current was found to be involved in the determination of the membrane potential, since glibenclamide depolarized early cardiomyocytes and exerted a positive chronotropic effect. Some cardiomyocytes displayed a bursting behavior of action potentials, characterized by alternating periods with and without action potentials. During the phases without action potentials, the membrane potential was hyperpolarized, indicating the involvement of K(+) channels in the generation of this bursting behavior. Extracellular recording techniques were applied to spontaneously contracting areas of whole embryoid bodies. In 20% of these bursting behavior similar to that seen in the single cells was observed. In regularly beating embryoid bodies, bursting could be induced by reduction of substrates from the extracellular medium as well as by superfusion with the positive chronotropic agents Bay K 8644 or isoproterenol. Perfusion with substrate-reduced medium induced bursting behavior after a short latency, isoproterenol and Bay K 8644 resulted in a positive chronotropic response followed by bursting behavior with longer latencies. The spontaneous bursting was blocked by glibenclamide. These experimental results suggest that intermittent activation of ATP dependent K(+) channels underlies the bursting behavior observed in single cardiomyocytes and in the whole embryoid body. Conditions of metabolic stress lead to the rhythmic suppression of action potential generation. Our data indicate that ATP dependent K(+) channels play a prominent role in the cellular excitability of early cardiomyocytes.

Action potential propagation failures in long-term recordings from embryonic stem cell-derived cardiomyocytes in tissue culture.

Three-dimensional cell aggregates (embryoid bodies, EBs) containing clusters of spontaneously beating cardiomyocytes were derived from permanent mouse embryonic stem (ES) cells. Extracellular recordings of the population action potentials of cardiomyocyte clusters were made using permanently mounted silver wire electrodes and microelectrode arrays integrated into the bottom of the culture dish. These techniques allowed long-term recordings (for up to several weeks) from individual EBs under cell culture conditions. The normal electrical activity consisted of regular spiking with a frequency of 0.5-5 Hz. However, most EBs (87%) spontaneously developed temporary or persistent complex activity patterns because of intermittent block of action potential propagation at narrow pathways connecting larger beating areas. Similar propagation blocks could also be reversibly induced in regularly spiking EBs by nimodipine (NDP). In addition to a slowing of pacemaker activity, NDP (20-200 nM) induced a stepwise decrease of the action potential frequency at the recording site. Perforated patch-clamp recordings from enzymatically isolated ES-cell-derived cardiomyocytes showed that similar activity patterns do not occur at the single-cell level. We suggest that this novel approach may provide a useful tool for in vitro studies of chronotropy and phenomena of propagation failure similar to AV block.

Rapid temperature changes induce adenosine-mediated depression of synaptic transmission in hippocampal slices from rats (non-hibernators) but not in slices from golden hamsters (hibernators).

Disturbances in neuronal communication induced by rapid temperature changes are a risk in the context of accidental hypothermia and would be fatal for hibernators during arousal from hibernation. Therefore, we investigated the effects of rapid temperature changes on synaptically induced CA1 population spikes in hippocampal slices from golden hamsters (hibernators) and rats (non-hibernators). Temperature was changed ramp-like by 0.3 degrees C/min, which corresponds to the rise of body temperature in golden hamsters during arousal from hibernation. During cooling from 35 to 10-15 degrees C, the population spike amplitude increased, reached maximal values at 25-30 degrees C and 20-25 degrees C in hamster and rat slices, respectively, and then decreased with further cooling. During rewarming, hamster slices displayed the same temperature dependence as during cooling. In contrast, in rat slices dynamic effects of the temperature change occurred. These were most obvious in a strong depression of the spike amplitude during rewarming as compared to cooling. Above 26-29 degrees C, the depression was superimposed by an excitatory effect. The depression was largely attenuated by theophylline (100-200 microM) and thus seems to be based on an increase of the concentration of endogenous adenosine, which in turn may result from an imbalance in energy metabolism during warming. The lack of warming-related depression in hamster slices can be explained by a lower sensitivity for adenosine as compared to rat slices. In addition, a better resistance of metabolic balance against rapid temperature changes may prevent large elevations of endogenous adenosine in the hamster hippocampus. For hibernators, the avoidance of temperature change-induced disturbances of neuronal communication may be a prerequisite for safe arousal from hibernation.

Histamine enhances synaptic transmission in hippocampal slices from hibernating and warm-acclimated Turkish hamsters.

The effects of histamine on synaptic transmission were studied at 37 degrees C and 22 degrees C with extracellular recordings of stimulus-induced population action potentials in area CA1 of hippocampal slices prepared from hibernating (HTH) and warm-acclimated Turkish hamsters (WTH) and rats. In rat slices, application of 50 microM histamine had no effects on population spikes and field excitatory postsynaptic potentials (EPSPs). In HTH as well as WTH slices, 50 microM histamine generally increased the population spike amplitude. The slope of the field EPSP was unchanged. At 37 degrees C, the sensitivity for histamine was significantly higher in HTH slices than in WTH slices. At 22 degrees C, the effects of histamine were less pronounced in HTH as well as WTH slices. Hibernation-related improvement of sensitivity for histamine is interpreted as supporting hippocampal function during arousal from hibernation.

Effects of T-type, L-type, N-type, P-type, and Q-type calcium channel blockers on stimulus-induced pre- and postsynaptic calcium fluxes in rat hippocampal slices.

The contribution of T-, L-, N-, P-, and Q-type Ca2+ channels to pre- and postsynaptic Ca2+ entry during stimulus-induced high neuronal activity in area CA1 of rat hippocampal slices was investigated by measuring the effect of specific blockers on stimulus-induced decreases in extracellular Ca2+ concentration ([Ca2+]o). [Ca2+]o was measured with ion-selective electrodes in stratum radiatum (SR) and stratum pyramidale (SP), while Ca2+ entry into neurons was induced with stimulus trains (20 Hz for 10 s) alternately delivered to SR and the alveus, respectively. The [Ca2+]o decreases recorded in SR in response to SR stimulation represented mainly presynaptic Ca2+ entry (Capre), while [Ca2+]o decreases recorded in SP in response to alvear stimulation were predominantly based on postsynaptic Ca2+ entry (Capost). Ethosuximide and trimethadione were ineffective in concentrations up to 1 mM. At 10 mM, they reduced Capost and, much less, also Capre. Nimodipine (25 microM) reduced Capost and, to a minor extent, Capre. omega-Agatoxin IVA (0.4-1 microM) and omega-contoxin MVIIC (1 microM) also reduced both Capre and Capost, but with a stronger action on Capre. omega-Conotoxin GVIA (3-8 microM) reduced Capost without effect on Capre. We conclude that during stimulus-induced, high-frequency neuronal activity Capost is carried by P/Q-, N-, and L- type channels and probably a further channel type different from these channels. Capre includes at least P/Q- and possibly L-type channels. N-type channels did not contribute to Capre in our experiments. Since ethosuximide and trimethadione were only effective in high concentrations, their action may be unspecific. Thus, T-type channels do not seem to play a major part in Ca2+ entry in this situation.

Synaptic transmission and paired-pulse behaviour of CA1 pyramidal cells in hippocampal slices from a hibernator at low temperature: importance of ionic environment.

To investigate the effects of ionic changes possibly associated with hibernation, hippocampal slices prepared from golden hamsters were studied in artificial cerebrospinal fluid (ACSF) of variable composition (K+ 3-5 mM, Ca2+ 2-4 mM, Mg2+ 2-4 mM, pH 7.0-7.7) at temperatures of 15-20 degrees C, just above the temperature below which synaptic transmission is blocked. Population action potentials (population spikes, PSs) of CA1 pyramidal cells were evoked by stimulation of the Schaffer collaterals/commissural fibers with paired pulses (interpulse interval 50 ms, interval between pairs 30 s). The responses evoked at given temperatures were investigated as a function of extracellular ion concentrations. In ACSF containing 3 mM K+, 2 mM Ca2+ and 2 mM Mg2+, PSs could be evoked at temperatures of > approximately 16 degrees C whereas at lower temperatures synaptic transmission was blocked. The threshold temperature was slightly higher for the first (PS1) than for the second PS (PS2) evoked by paired-pulse stimulation. The slices displayed paired-pulse facilitation (PPF) at all temperatures. Elevation of [K+]o from 3 to 5 mM depressed the amplitudes of both PS1 and PS2, with a stronger effect on PS2. PPF was reduced and, at near-threshold temperatures, turned into paired-pulse depression (PPD). Elevation of [Ca2+]o from 2 to 4 mM increased the amplitude of PS1. The amplitude of PS2, in contrast, was reduced at near-threshold temperatures. PPF turned into PPD. Elevation of [Mg2+]o from 2 to 4 mM reduced the amplitudes of both PS1 and PS2, with a stronger effect on PS1. Accordingly, PPF was increased. Acidification by 0.3 pH units strongly depressed the amplitudes of PS1 as well as PS2 and increased PPF. Alkalization by 0.4 pH units had only weak effects in the opposite direction. Changes in the ionic composition comparable to those investigated in the present study presumably occur in the brain interstitium of hamsters during entrance into hibernation. According to our results, such changes depress synaptic transmission at low temperatures in the hamster hippocampus in vitro. This modulation may be important for the regulation of neuronal activity during entrance into hibernation.

Modulation of synaptic transmission at low temperatures by hibernation-related changes in ionic microenvironment in hippocampal slices of golden hamsters.

In hamsters, the entrance into hibernation is associated with a respiratory acidosis and elevation of the blood plasma concentrations of potassium, calcium, and magnesium. To investigate the effects of presumed hibernation-related ionic changes in the brain interstitium on neuronal function, the transmission properties of hippocampal slices prepared from golden hamsters were studied at low temperatures in vitro. Slices were investigated at 15-20 degrees C in artificial cerebrospinal fluid (ACSF) of variable composition (K+, 3-5 mM; Ca2+, 2-4 mM; Mg2+, 2-4 mM; pH 7.0-7.7). Population action potentials (population spikes, PS) of CA1 pyramidal cells were continuously evoked with 100-microseconds stimulus pulses delivered to the Schaffer collaterals/commissural fibers in intervals of 30 s. The PS amplitude was measured as a function of extracellular ion concentrations at given temperatures or as a function of temperature at a given ACSF composition. Elevation of [K+]o, [Mg2+]o, or [H+]o all reduced the PS amplitude at low temperatures, whereas elevation of [Ca2+]o increased the PS amplitude. In conclusion, changes in the ionic microenvironment occurring during entrance into hibernation presumably result in depression of synaptic transmission at low temperatures in the hamster hippocampus. The modulatory effect of ionic changes may be an important factor supporting a general depression of the brain during entrance into hibernation.

Long-term potentiation at low temperature is stronger in hippocampal slices from hibernating Turkish hamsters compared to warm-acclimated hamsters and rats.

Tetanus-induced long-term potentiation (LTP) of population action potentials at 22 degrees C was investigated in area CA1 of hippocampal slices prepared from hibernating (HH) and warm-acclimated Turkish hamsters (WH) and rats. LTP elicited at this temperature was significantly stronger in HH slices compared to WH and rat slices. Hibernation-related improvement of the ability to develop long-lasting enhancement of synaptic transmission at low temperatures is interpreted as supporting hippocampal function during arousal from hibernation.

Effects of adenosine on synaptic transmission in hippocampal slices from hibernating and warm-acclimated Turkish hamsters and rats.

The effects of adenosine on synaptic transmission were studied with extracellular recordings of stimulus-induced population action potentials in area CA1 of hippocampal slices prepared from hibernating (HH) and warm-acclimated Turkish hamsters (WH) and rats. In HH as well as WH and rat slices, adenosine generally reduced the population spike amplitude and the slope of the field EPSP. The sensitivity for adenosine was significantly lower in HH slices than in WH and rat slices. The results are discussed with regard to the involvement of endogenous adenosine in the regulation of neuronal activity during entrance into and arousal from hibernation.

Pharmacological and electrographic properties of epileptiform activity induced by elevated K+ and lowered Ca2+ and Mg2+ concentration in rat hippocampal slices.

We studied some of the physiological and pharmacological properties of an in vitro model of epileptic seizures induced by elevation of [K+]0 (to 8 mM and 10 mM) in combination with lowering of [Mg2+]0 (to 1.4 mM and 1.6 mM) and [Ca2+]0 (to 0.7 mM and 1 mM) in rat hippocampal slices. These concentrations correspond to the ionic constitution of the extracellular microenvironment during seizures in vivo. The resulting activity was rather variable in appearance. In area CA3 recurrent discharges were observed which resulted in seizure-like events with either clonic-like or tonic-clonic-like ictaform events in area CA1. With ion-sensitive electrodes, we measured the field potential and the changes in extracellular ion concentrations which accompany this activity. The recurrent discharges in area CA3 were accompanied by small fluctuations in [K+]0 and [Ca2+]0. The grouped clonic-like discharges in area CA1 were associated with moderate increases in [K+]0 and small decreases in [Ca2+]0 in the order of 2 mM and 0.2 mM, respectively. Large, negative field-potential shifts and increases in [K+]0 to 13 mM, as well as decreases in [Ca2+]0 by up to 0.4 mM, accompanied the tonic phase of ictaform events. The ictaform events were not blocked by D-2-aminophosphonovalerate (2-APV) but were sensitive to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) alone and in combination with 2-APV and ketamine. In order to determine the pharmacological characteristics of the ictaform events we bath-applied most clinically employed anticonvulsants (carbamazepine, phenytoin, valproate, phenobarbital, ethosuximide, trimethadione) and some experimental anticonvulsants (losigamone, vinpocetine, and apovincaminic acid). Carbamazepine, phenytoin, valproate, and phenobarbital were effective at clinically relevant doses. The data suggest that the high-K+ model of epileptiform activity is a good model of focal convulsant activity.

Effects of changes in extracellular potassium, magnesium and calcium concentration on synaptic transmission in area CA1 and the dentate gyrus of rat hippocampal slices.

The dependence of stimulus-induced synaptic potentials on changes of extracellular ionic concentrations of potassium ([K+]o 3, 5, 8 mM), magnesium ([Mg2+]o 2, 4, 8 mM) and calcium [Ca2+]o (2 mM and continuous lowering by washing with Ca2(+)-free solutions) was investigated in area CA1 and dentate gyrus of rat hippocampal slices. Field potentials (fps), [K+]o and [Ca2+]o were measured with double-barreled ion selective/reference microelectrodes. Paired pulse stimulation (interval 50-ms) was applied either to the lateral perforant path or to the Schaffer collaterals. Elevation of [K+]o from 5 to 8 mM and of [Mg2+]o from 2 to 8 mM depressed the rise of excitatory postsynaptic potentials, as well as the amplitude of population spikes. With elevation of [K+]o, the effect was stronger in the dentate gyrus, while with elevation of [Mg2+]o, the reduction was more pronounced in area CA1. During washout of Ca2+, synaptic potentials became reduced and finally depressed. The [Ca2+]o at which synaptic transmission was blocked increased with higher [Mg2+]o and decreased with a change of [K+]o from 3 to 5 mM, whereas with an elevation of [K+]o from 5 to 8 mM, it rose in area CA1 but was reduced in dentate gyrus. All ionic changes also affected frequency habituation and potentiation in paired pulse experiments. In dentate gyrus, frequency habituation was reversed to frequency potentiation with moderate lowering of [Ca2+]o and with elevation of [Mg2+]o and [K+]o. In contrast, in area CA1 frequency potentiation was reduced upon elevation of [K+]o.