E. A. Lukyanetz

Pilocarpine-induced epileptiform activity of isolatedCA1 hippocampal neurons

Cellular mechanisms of pilocarpine-induced epileptiform activity in isolatedCA1 andCA3 hippocampal neurons were studied using a current-clamp technique. Isolated unipolar neurons dominate after enzymatic treatment of theCA1 tissue, whereas large multipolar neurons are mainly located in theCA3 area. Measurements of the membrane potential in these two groups of neurons showed that most of them are “silent” cells. Only a small group of neurons from theCA3 area displayed spontaneous electrical activity. Pilocarpine, a well known epileptogenic compound, induced rhythmic waveform changes in the membrane potential in some “silent” unipolar neurons from theCA1 area, whereas in another more numerous group of neurons it induced only steady depolarization of the membrane. Application of tetrodotoxin, the selective blocker of Na+ channels, blocked generation of action potentials induced inCA1 neurons by pilocarpine, but exerted no effect on waveform shifts of the membrane potential. It is suggested that the mechanism underlying epileptogenic action of pilocarpine on the brain activity in rats is based on the induction of waveform changes in the membrane potential in unipolar neurons of theCA1 hippocampal area.

The Involvement of Calcium Transport Systems of the Plasma Membrane in Calcium Exchange in Neurons of the Carassius gibelio Cerebellum

We studied the role of Na+/Ca2+ exchanger (NCX) and Ca2+-ATPase of the plasma membrane (РМСА), known to be the most important intracellular systems controlling calcium exchange in cerebellar neurons of a fish species tolerant to hypoxia, Carassius gibelio. In our experiments, we used the corresponding blockers of these transport systems, ions of lithium and lanthanum. The intracellular Ca2+ concentration ([Ca2+]і) was measured using a calcium-sensitive dye, Fura-2AM, and a microfluorescence technique. We found that neurons of the Carassius cerebellum possess an effective system of cleaning of the cytoplasm from excessive Ca2+, which is provided by both NCX and РМСА functioning in the plasma membrane. Under conditions of the blockade of functioning of РМСА using lanthanum, the basal Ca2+ level in the cells increased, on average, by 31.4% with respect to the control, independently of the duration of test depolarizations. After switching off of the NCX functioning by the replacement of sodium ions in the extracellular solution by lithium ions, the Ca2+ level in the cell increased by 36.6% with respect to the control (also independently of the duration of depolarization). The obtained data indicate that the functioning of РМСА and NCX in Carassius cerebellar neurons significantly influences the intracellular calcium exchange providing the maintenance of an adequate basal Ca2+ level in these neurons.

Role of Mitochondria in the Generation of Acetylcholine-Induced Calcium Transients in Rat Chromaffin Cells

A fluorescent probe, FURA-2M, was used to examine the role of mitochondria in the generation of calcium transients evoked by acetylcholine (ACh) in isolated rat chromaffin cells. Our experiments showed that application of 10 μM CCCP (carbonyl cyanide m-chlorophenylhydrazone, a mitochondrial protonophore) caused significant intracellular calcium transients (F1/F2 wave ratio 1.05). Application of CCCP did not affect the successive responses to repeated ACh applications in a cell subpopulation with the domination of nicotinic receptors (F1/F2 = 0.90 in control, and F1/F2 = 0.89 after CCCP application). In cells with the domination of muscarinic receptors, responses to repeated ACh applications decreased under control conditions. Application of CCCP caused recovery of the successive ACh responses by 27%, as compared with the control. The results allow us to suggest that the mitochondria themselves are not directly involved in the ACh-induced calcium transients, but calcium release from the mitochondria during CCCP treatment can cause the replenishment of other intracellular stores (endoplasmic reticulum) and in such a way recover the ACh responses to repeated stimulations in the cells with dominating metabotropic receptors.

Calcium Signaling in Carassius Cerebellar Neurons: Role of the Mitochondria

We studied the involvement of the mitochondria playing the role of a calcium store in the control of calcium exchange in cerebellar neurons of a fish species tolerant to hypoxia, crucian (Carassius gibelio). In our experiments we used an ionophore, CCCP, that blocked accumulation of calcium by the above organelles. The intracellular concentration of free Ca2+ ([Ca2+]і) was measured using a calcium-sensitive dye, Fura-2AM, and the microfluorescent technique. We found that cerebellar neurons of Carassius gibelio possess a well-expressed system clearing the cytoplasm from excessive Ca2+, and the mitochondria are actively involved in this process. Under conditions of suppression of the process of accumulation of calcium by the mitochondria under the action of CCCP, the amplitude of calcium transients increased by about 50%. In addition, the decay phase of depolarization-induced intracellular calcium transients was slowed down considerably. Therefore, our experiments are indicative of the significant role of the mitochondria in the control of calcium dynamics in cerebellar neurons of Carassius gibelio in the course of functional activity of these cells.

Accommodation Properties of Isolated Hippocampal Neurons under Conditions of an Experimental Model of Epilepsy

A pilocarpine/lithium model of status epilepticus is an effective tool allowing one to study the principles of development of temporal epilepsy. It is believed that, in addition to the corresponding modifications of the efficacy of synaptic transmission, changes in the endogenous properties of neuronal activity can promote repetitive epileptiform activity. We measured the accommodation parameters of spike generation by isolated neurons of the CA1 hippocampal area obtained from 14-day-old rats 2 or 24 h after they had been subjected to an epileptization procedure, as well as from control rats of the same age. The spike activity of the neurons was initiated by their depolarization with a long-lasting stimulus in a current-clamp mode under conditions of perforated patch clamp. We found that the initial phase of accommodation manifested as a rapid increase in interspike intervals immediately after application of the depolarizing stimulus became significantly shorter in rats 24 h after epileptization; at the same time, the characteristics of the late phase of accommodation underwent no changes. In addition, the mean number of generated action potentials dropped. Such changes were not found in neurons of rats 2 h after epileptization. It is hypothesized that the above effect is compensatory and not injuring; it can develop because of prolonged abnormal activation of neurons in the course of epileptic attacks.

Roles of the Mitochondria and Endoplasmic Reticulum in Calcium Elevation during Osmotic Shock in Adrenocortical Cells

Steroid hormones participate in various metabolic processes, and dysfunction of the adrenocortical system leads to numerous pathologies in humans. One of the factors that can influence the secretory properties of adrenocorticocytes is changes in the cell volume observed during osmotic shock. In our study, we tested the hypothesis that osmotic stress modifies intracellular Ca2+ signalling and in such a way can influence the secretion of steroids by adrenocorticocytes. The effects of hyperosmotic stress on the cytosolic Ca2+ concentration ([Ca]i) in cultured adrenocortical cells from the zona fasciculata of the rat adrenals were investigated using the indicator fura-2 technique. Our experiments have shown that exposure of the cells to a hyperosmotic solution caused a decrease in the cell volume, as well as a reversible rise in the [Ca]i. Calcium-free media partly eliminated [Ca]i responses. Pretreatment of the cells with thapsigargin or CCCP (blockers of internal calcium stores) significantly decreased the magnitude of responses induced by osmotic stress. These findings indicate that osmotic shock causes an increase in the [Ca]i in adrenocortical cells, mostly due to depletion of the intracellular stores, and may in such a way stimulate steroidogenesis.

Serotonin-induced changes in the activity of single Ca2+ channels inHelix pomatia neurons

The influence of the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) on single Ca2+ channel activity was studied on unidentified neurons of the snailHelix pomatia. Only one type of Ca2+ channels with the unitary conductance of 5 pS was identified using 100 mM Ca2+ in the patch pippette under patch-clamp in a cell-attached configuration. The amplitude histogram showed only one peak with the mean value of 0.5 pA at the testing potential of −30 mV. The distribution of channel open times monotonically declined with the mean time constant of 0.2 msec. The distribution of channel closed times could be fitted by a double-exponential curve with time constants of 1 and 12 msec. The study of the effect of 5-HT on Ca2+ single channel activity showed that 5-HT influenced the channel molecule indirectly, as the transmitter could exert its effect by being added to the bath solution, which did not come into contact with the tested membrane fragment under the micropipette tip. 5-HT prolonged the mean channel open time (up to 0.3 msec) and proportionally decreased both channel closed time constants to 0.4 and 6.0 msec, respectively. A conclusion is made that enhancement of Ca2+ macrocurrent by 5-HT is determined by three factors: (i) changes in kinetics of aiready existing channels, (ii) an increase in the number of active channels of the same type, and (iii) an increase in probability of a channel being open. At the same time, the unitary channel conductance was not affected by the transmitter.

Evoked Impulse Activity of Isolated Hippocampal Neurons in the Perforated Patch-Clamp Configuration

We describe the results obtained in studies of the impulse activity recorded from isolated neurons of the hippocampal CA1 area in the perforated patch-clamp configuration. Spiking was initiated by two modes of intracellular stimulation, namely by application of rectangular 2.5-sec-long current pulses or a ramp protocol (linear current ramp within the 10.0-sec-long interval). We proposed schemes of recording allowing experimenters to obtain reliable quantitative characteristics of the activity of isolated neurons taking into account the problem of reproducibility of the results. We also examined the rhythmicity of generation of action potentials by the studied neuron and typical phenomena of its disturbances; the techniques for selection of an optimum mode of ramp stimulation of the neuron making it possible to obtain detailed characteristics of generation of impulse activity are also described.

Involvement of the Endoplasmic Reticulum of Chromaffin Cells of the Rat Adrenal Gland in Calcium Signaling

We studied the involvement of the endoplasmic reticulum (ER) in calcium signaling in rat chromaffin cells. For this purpose, the following agents influencing the activity of the ER were used: (i) Caffeine that activates the release of Ca2+ from the endoplasmic store and (ii) thapsigargin that suppresses accumulation of calcium in the ER. The intracellular Ca2+ concentration was measured with the help of a calcium-sensitive dye, Fura-2AM, using the microfluorescent technique. Applications of caffeine led to a rise in the level of free Ca2+ in the cell cytosol and also to a decrease in the amplitude of calcium transients induced by depolarization of the plasma membrane under the action of a hyperpotassium solution. Under conditions of repeated caffeine applications, the amplitude of transients decreased to 9% of its initial value, which is explained by exhaustion of the calcium stores. The action of caffeine was restored when the calcium stores were re-filled under the action of depolarization of the plasma membrane. Thapsigargin completely removed the effect of caffeine and did not influence KCl-induced transients. Therefore, our experiments are indicative of a significant importance of the ER calcium stores for calcium signaling in chromaffin cells, which allows us to hypothesize that these stores play an important role in the control of secretion of catecholamines.

Comparative studies of calcium transients induced by acetylcholine in rat chromaffin cells

We used an intracellular fluorescent probe, FURA-2M, to examine the responses of isolated rat chromaffin cells to applications of 1 mM acetylcholine (ACh). Our data showed two different populations of the cell responses to such stimulation. Responses of the first type demonstrated fast rise and decay phases of the Ca2+ transients and no significant decrease in their amplitude during repetitive stimulation of the cell with ACh. Cell responses of the second type showed remarkably slower rise and decay phases of the Ca2+ transients and a noticeable drop of the cell responses during repetitive ACh stimulation that could be recovered after KCl depolarization. We find no significant differences in the amplitudes of the transients in these two populations of the cells. We conclude that there is heterogeneity of the chromaffin cells according to their ACh receptors: the first subpopulation predominantly expresses ionotropic (nicotinic) receptors (n cells), whereas the second cell population has mainly metabotropic (muscarinic) ones (m cells), which are associated with Ca2+ release from the intracellular stores.