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XXIII Congress of I.P. Pavlov Physiology Society
New Trends in the Analysis of Function and Modulation of the Cells of Nervous System
P. Bregestovski
Aix Marseille University, INSERM, Institute of System Neurosciences, Marseille, France; Department of Physiology, Kazan; Medical State University, Kazan, Russia.
Several new areas for analysis and functional regulation of nervous system function have been developed in recent years. Among them connectomics, clarity, optogenetics, optosensorics and optopharmacology provide an excellent tools for detailed investigation of the nervous system organization, on clarifying of neuronal networks functioning and control of some biological organisms behaviour.
A crucial factor for the establishment of optogenetics become the discovery of algal light-gated ion channels (chan-nelrhodopsins) and pumps (halorhodopsins), which can be easily incorporated in different cell types. This provided unprecedented tolls for the control of neural activity with light. Consequently, a number of other light-sensitive modules were discovered and engineered to generate new photoswitches to control protein activity, protein localization, and gene expression. With the help of light one can investigate the function of cells controlling their activity, to measure the concentration of ions, ATP and other cellular components, to control the behaviour of organisms, as well as to seek for novel ways to treat certain diseases. Optogenetic approaches have been used in many models with medical orientation, including the study of stress, schizophrenia, memory disorders, drug addiction, psychiatry and motor functions, vision, pain, functional recovery after stroke and epilepsy.
Photopharmacology is a direction based on the creation of chemical compounds capable of controlling the functions of biological molecules possessing photosensitive switches. Photochromic compounds that activate or inhibit the activity of key cellular proteins, especially ion channels, represent a powerful tools for non-invasive control of neuronal network activity and, consequently, functional control of organisms and behavior. Photochromic switches can be divided into two main classes: (i) soluble photochromic ligands and (ii) compounds covalently bounding to target proteins. On this basis, potassium channel blockers, modulators of glutamate and GABA receptors, as well as cationic TRP channels were created. Photopharmacology offers great opportunities in the regulation of pain, restoration of the functions of the retina and other physiological functions.
Optosensorics uses genetically encoded biosensors for non-invasive imaging of concentrations of ions, the activity of enzymes, distribution of small molecules, proteins and organelles, and the protein interactions in living cells. These fluorescent molecules are used either at the transient expression in cultured cells or organisms or at stable expression producing transgenic animals possessing heritable and functional biosensors. Using the mouse Thy1 mini-promoter, we generated two lines of transgenic mice for the monitoring of intracellular chloride (Cl-) and for the simultaneous measurements of intracellular and pH. To reveal the ClopHensor expression pattern across the brain of transgenic mice we obtained transparent brain samples using CLARITY method and imaged them with confocal and light-sheet microscopy. This analysis provides the map of the brain areas available for non-invasive monitoring of intracellular Cl'/pH in normal and pathological conditions.
These and other directions will be presented in the talk.
Spatiotemporal Characteristics of Astrocyte Calcium Dynamics in Chronic Epilepsy
P. Denisov*, A. Lebedeva, A. Pimashkin, A. Semyanov
Institute of Neuroscience, Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia. * Presenting e-mail: denisov@neuro.nnov.ru
Epilepsy is diagnosed annually in 2.4 million people (WHO 2017). Epileptic status (ES) is the main symptom and is the most severe manifestation of generalized epilepsy. It is a series of seizures with tonic and clonic phases, leading to neurodegeneration and astrogliosis. The influence of these processes on the neuron-astrocytic interaction, in particular on the calcium events in the astrocytic network, remains poorly studied. The study of astrocytic activity can give new knowledge about the mechanisms of epilepsy and determine the direction of creating new methods of treatment.
As an experimental model in this project, a lithium-pilocarpine model of epileptogenesis was chosen. It reflects the various phenomenological features characteristic of temporal epilepsy in humans.
Sprague-Dawley rats (age 18-25 days) were used in the experiments. 2-4 weeks after ES, the changes in calcium dynamics in the neuron-astrocytic network stratum radiatum of the CA1 field on the hippocampal slices were stud-
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XXIII Congress of I.P. Pavlov Physiology Society
ied. The thickness of the sections was 350 |jm, the staining was performed with a calcium indicator Oregon Green 488 BAPTA-1 AM and an astrocytic marker sulforodamine 101. A confocal fluorescence microscope LSM Zeiss 510 DuoScan was used to visualize the calcium events. To analyze the space-time characteristics of calcium dynamics in the network of astrocytes, software was developed on the basis of Matlab, which allowed identifying the space-time characteristics of individual events. The following parameters were investigated: duration of events, maximum projection of events and frequency.
The distribution in accordance with the power law was observed both for the sizes and for the maximum projection of the calcium events. In particular, with ES, a statistically significant increase in the maximum projection was observed, indicating a decrease in the number of large events in comparison with the control. The change in the frequency of events is not statistically significant. This change is pathological in terms of the functioning of the calcium signaling of the astrocytic network.
The work was supported by the Russian Science Foundation (project No. 15-14-30000)
K+ Mediated Signaling within Tripartite Synapse
Alexey Semyanov
Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia.
Living cells had evolved with high intracellular concentration of K+ and with low concentration of Na+. Transmembrane gradients of these ions drive cellular excitability. Therefore, general belief is that K+ efflux is mostly needed for membrane repolarization during action potential. If fact, significant amount of K+ is also released via postsynaptic glutamate receptors during excitatory synaptic transmission in the CNS. This reduces postsynaptic depolarization making synaptic transmission less efficient and more energy costly. Why such K+ permeability is preserved in these receptors? Here, we report that NMDA receptor-dependent K+ efflux can provide a retrograde signal in the synapse. In hippocampal CA3-CA1 synapses, the bulk of astrocytic K+ current triggered by synaptic activity reflects K+ efflux through local postsynaptic NMDA receptors. The local extracellular K+ rise produced by activation of postsynaptic NMDA receptors boosts action potential evoked presynaptic Ca2+ transients and neurotransmitter release from Schaffer collaterals. Perisynaptic K+ accumulation during synaptic transmission also affects astrocytic transporter currents, making them slower. This suggests activity dependent enhancement of glutamate spillover also depends on postsynaptic cell. Our findings indicate that postsynaptic NMDA receptor-mediated K+ efflux contributes to use-dependent synaptic facilitation and increased glutamate dwell time, thus revealing a fundamental form of ionic signaling within tri-partite synapse.
Rapid Astrocyte Morphology Changes Support Epileptic Activity
Stefanie Anders1, Björn Breithausen1, Michel Herde1, Daniel Minge1, Tushar Deshpande1, Anne Boehlen1, Peter Bedner1, Christian Steinhäuser1, Christian Henneberger1,2,3
1 Institute of Cellular Neurosciences, University of Bonn Medical School, Bonn, Germany;
2 Institute of Neurology, University College London, London, United Kingdom;
3 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany.
Astrocytes actively contribute to neuronal network function. The close contact of individual astrocytes to thousands of neurons enables them to maintain and modulate neuronal function effectively by, for example, buffering potassium and glutamate clearance. A disruption of this spatial relationship could be of pathophysiological significance. Indeed, astrocyte dysfunction and long-term morphology changes have been implicated in numerous diseases including epilepsy. How rapid astrocyte morphology is altered by the onset of epileptiform activity and to what degree it contributes to aberrant network behavior is largely unknown. Combining established protocols of hippocampal epileptogenesis, electrophysiology and two-photon excitation fluorescence microscopy allowed us to monitor astrocyte morphology changes during the induction of epileptiform activity in acute hippocampal slices. Analysis revealed that small and medium-sized astrocyte processes shrink acutely within minutes after epileptiform discharges appeared in the CA1 region. Importantly, similar astrocyte morphology changes were also detected shortly after induction of status epilepticus in vivo by intracerebral kainate injection. In vitro, these astrocyte morphology changes outlasted the induction of epilep-tiform activity, persisted after pharmacological termination of epileptic activity by TTX and were sensitive to inhibition
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