Research Interests


Calcium Regulating Systems in Excitable Cells
Long Term Depression (LTD)
Glial Cells
Long-Lasting Calcium Concentration Gradients
New Method Designed to Monitor the Distribution of Calcium Efflux
Localisation of Ca2+ Extrusion Sites
Calcium Release via Exocytosis
Modulation of GABAergic Transmission in Primary Hippocampal Cultures
The mechanisms that regulate the intracellular cytoplasmic calcium ion concentration ([Ca2+]i) changes in different types of excitable and nonexcitable cells were the main subject of my investigations for the last several years. Acute isolated nerve, cardiac and exocrine secretory cells or primary cultures of these cells have been used in our experiments. Mainly, optical methods (digital imaging and confocal microscopy) based on the measurements of hydrogen and calcium ion concentrations by means of fluorescent probes (Quin-2, Fura-2, Calcium Green Dextrans) and conventional electrophysiological methods (microelectrodes, iontophoresis, patch clamp) have been employed in our studies.
Investigations that I conducted as a Ph.D. Student and a Research Associate in Prof. Kostyuk’s Department at the Bogomoletz Institute of Physiology (Kiev, Ukraine) allowed us to reveal and explain some interesting phenomena in Calcium Regulating Systems in Excitable Cells such as [Ca2+]i oscillations and dependence [Ca2+]i changes on G-proteins and cAMP. The new interdependences between the different second messengers systems (cyclic AMP, inositol-1,4,5-trisphosphate and calcium) have been also shown. Introduction of two new methods to study Ca2+ movement gave us the possibility to measure the calcium ion fluxes through plasma membrane and the value of the cytoplasmic mobilization of these ions in single isolated living cells.
My work as a Postdoctoral Fellow at Roche Institute of Molecular Biology (USA) involved a commercial confocal and a cooled CCD camera microscopy for the purpose to get the spatial and temporal resolution images of acute isolated hippocampal neurons and cells in the primary cerebellar culture. We monitored the dynamics of [Ca2+]i in these objects during the electrophysiological and ligand stimulation in order to understand how calcium ions are involved in triggering the use-dependent changes. My projects concerned to investigations of (i) Purkinje cerebellar neurons in primary culture (we were interested in studying of the biochemical processes underlying Long Term Depression (LTD) of the excitatory amino acid induced currents), (ii) the Glial Cells in the same culture (in order to understand the nature of [Ca2+]i oscillations and waves in these cells) and (iii) CA1 neurons in acute dissociated preparations. The latter experiments involved the studying of the Long-Lasting Calcium Concentration Gradients that occur after repeated local glutamate applications.
At the moment I work in Prof. Kostyuk’s Department as a Scientific Researcher.
For several years in co-operation with Prof. Petersen’s Department, the University of Liverpool (UK) we have developed a New Method Designed to Monitor the Distribution of Calcium Efflux from cells or small cellular aggregates. The idea behind this method was to use a fluorescent calcium indicator, bound to dextrans of large molecular weight to slow down calcium diffusion. Due to the decrease in diffusion rate, Ca2+ ions should be held close to the site of their release from the cells for a relatively long time, enough for the confocal microscope to detect such a local increase in Ca2+ concentration.
Employing the new technique we have investigated the Localisation of Ca2+ Extrusion Sites in single pancreatic and submadibular acinar cells. It was found that during agonist stimulation of pancreatic cells Ca2+ is primarily extruded by Ca2+ pumps from the secretory pole . We propose that this process is useful for maintaining a high Ca2+ concentration in the acinar lumen, which is necessary for promotion of endocytosis. On the contrary, the results obtained on submandibular cells indicate that calcium efflux from b-adrenergic-stimulated cells and a significant part of the cholinergic-stimulated efflux is due to exocytosis of the calcium contents of secretory granules. I.e. we have shown that in some types of cells, Calcium Release via Exocytosis can be one of the main mechanisms extruding calcium from cells to the extracellular milieu.
Now we are continuing the calcium efflux studies. We have also started a new neuroscience project devoted to Modulation of GABAergic Transmission in Primary Hippocampal Cultures. The topics of our studies are as follows:
The whole-cell patch-clamp technique was used to record monosynaptic inhibitory postsynaptic currents (IPSCs) from pairs of hippocampal neurons cultured for 2-3 weeks. The application of fresh physiological solution for 2-3 min reversibly reduced the amplitude of evoked GABAergic IPSCs to 72.5 % of control value. The amplitude and frequency of spontaneous IPSCs decreased too. The depression of evoked IPSCs was significantly smaller or absent if conditioned solution was applied (physiological solution, which had been previously in contact with neurons for 30 min). Currents evoked by exogenously applied GABA were unaffected by fresh solution. These results suggest that hippocampal neurons release some endogenous substance(s), by which they up regulate presynaptically their own inhibitory synaptic transmission.
Despite a large number of experimental results in respect of cholinergic modulation of GABAergic transmission in the hippocampus some questions remain unclear. As it was suggested earlier, opposite acetylcholine (Ach) effects on evoked and spontaneous (in the absence of tetrodotoxin (TTX)) GABAergic IPSCs in slices may result from opposite ACh effects on two types of GABAergic inhibitory interneurons in hippocampus. This possibility can not be easily addressed in slices where multiple interneurons are involved in the evoked responses. A possible contribution of the postsynaptic changes in the inhibitory ACh effect on GABA evoked responses was not examined, thus general question of relative contribution of pre- and postsynaptic changes in overall modulation of the GABAergic transmission was not studied. Since we investigated the effect of ACh on evoked GABAergic responses in cell cultures of dissociated hippocampal neurons this preparation allowed us to study GABAergis IPSCs evoked by stimulation of a single presynaptic neuron. We found that ACh inhibited evoked GABAergic IPSCs in the most of the connections. This was usually accompanied by an enhanced spontaneous input to the postsynaptic neurons. These results indicate that opposite ACh effects on spontaneous and evoked IPSCs are unlikely due to its opposite effects on two types of interneurons. Both, a decrease of the evoked responses and an increase of the spontaneous input, persisted in presence of APV and CNQX, although tended to be less pronounced under these conditions. ACh-induced changes of the evoked responses in our experiments were often accompanied by changes in paired pulse depression (PPD), which are thought to reflect some presynaptic modulation mechanisms. Time course of the PPD changes, however, did not always match that of the IPSC changes, suggesting a possible contribution of the postsynaptic mechanism. This suggestion can be supported by our observation that ACh in part of the neurons tested reversibly decreased responses evoked by exogenous GABA application. Thus, we conclude that postsynaptic mechanism may also contribute to the inhibitory ACh effect on GABAergic transmission.
We have been also studying glutamate modulation of GABAergic synaptic transmission. Extracellular glutamate application (0.5-100 uM) reduced evoked IPSCs in all synaptic connections tested. This reduction consists of the fast (less than 1sec) and the slow (t about 80sec) phases. In 84% of pairs studied there was not a complete recovery of evoked IPSC amplitudes to the control level after washing glutamate out (10 to 64 min). Glutamate application in the presence of ionotropic glutamate blockers DL-APV and CNQX eliminated the fast phase of the glutamate effect. Only slow reduction was observed in this case in the most part of the synaptic pairs. The reduction persisted in 73% of the experiments when glutamate was removed from the extracellular solution. We have concluded that both ionotropic and metabotropic glutamate receptors take part in GABAa-ergic synaptic transmission modulation - the former is involved in the fast and reversible whereas the later in the slow and prolonged IPSC inhibition.

Dr. Belan's Home Page | Publications | Bogomoletz Intst.of Physiology | Dept. of Gen. Physiol. of Nervous System