Showing papers by "Pavel M. Balaban published in 2017"

Thermogenetic neurostimulation with single-cell resolution.

Abstract: Thermogenetics is a promising innovative neurostimulation technique, which enables robust activation of neurons using thermosensitive transient receptor potential (TRP) cation channels. Broader application of this approach in neuroscience is, however, hindered by a limited variety of suitable ion channels, and by low spatial and temporal resolution of neuronal activation when TRP channels are activated by ambient temperature variations or chemical agonists. Here, we demonstrate rapid, robust and reproducible repeated activation of snake TRPA1 channels heterologously expressed in non-neuronal cells, mouse neurons and zebrafish neurons in vivo by infrared (IR) laser radiation. A fibre-optic probe that integrates a nitrogen-vacancy (NV) diamond quantum sensor with optical and microwave waveguide delivery enables thermometry with single-cell resolution, allowing neurons to be activated by exceptionally mild heating, thus preventing the damaging effects of excessive heat. The neuronal responses to the activation by IR laser radiation are fully characterized using Ca2+ imaging and electrophysiology, providing, for the first time, a complete framework for a thermogenetic manipulation of individual neurons using IR light.

Insertion of the voltage-sensitive domain into circularly permuted red fluorescent protein as a design for genetically encoded voltage sensor.

Abstract: Visualization of electrical activity in living cells represents an important challenge in context of basic neurophysiological studies. Here we report a new voltage sensitive fluorescent indicator which response could be detected by fluorescence monitoring in a single red channel. To the best of our knowledge, this is the first fluorescent protein-based voltage sensor which uses insertion-into-circular permutant topology to provide an efficient interaction between sensitive and reporter domains. Its fluorescent core originates from red fluorescent protein (FP) FusionRed, which has optimal spectral characteristics to be used in whole body imaging techniques. Indicators using the same domain topology could become a new perspective for the FP-based voltage sensors that are traditionally based on Forster resonance energy transfer (FRET).

Cued memory reconsolidation in rats requires nitric oxide.

Abstract: It is well-known that the reactivation of consolidated fear memory under boundary conditions of novelty and protein synthesis blockade results in an impairment of memory, suggesting that the reactivated memory is destabilized and requires synthesis of new proteins for reconsolidation. We tested the hypothesis of nitric oxide (NO) involvement in memory destabilization during the reconsolidation process in rats using memory reactivation under different conditions. We report that administration of NO-synthase selective blockers 3-Br-7-NI or ARL in the conditions of reactivation of memory under a protein synthesis blockade prevented destabilization of fear memory to the conditioned stimulus. Obtained results support the role of NO signaling pathway in the destabilization of existing fear memory triggered by reactivation, and demonstrate that the disruption of this pathway during memory reconsolidation may prevent changes in long-term memory.

Role of Atypical Protein Kinases in Maintenance of Long-Term Memory and Synaptic Plasticity.

Abstract: Investigation of biochemical mechanisms underlying the long-term storage of information in nervous system is one of main problems of modern neurobiology. As a molecular basis of long-term memory, long-term changes in kinase activities, increase in the level and changes in the subunit composition of receptors in synaptic membranes, local activity of prion-like proteins, and epigenetic modifications of chromatin have been proposed. Perhaps a combination of all or of some of these factors underlies the storage of long-term memory in the brain. Many recent studies have shown an exclusively important role of atypical protein kinases (PKCζ, PKMζ, and PKCι/λ) in processes of learning, consolidation and maintenance of memory. The present review is devoted to consideration of mechanisms of transcriptional and translational control of atypical protein kinases and their roles in induction and maintenance of long-term synaptic plasticity and memory in vertebrates and invertebrates.

Encoding of High Frequencies Improves with Maturation of Action Potential Generation in Cultured Neocortical Neurons.

Abstract: The ability of neocortical neurons to detect and encode rapid changes at their inputs is crucial for basic neuronal computations, such as coincidence detection, precise synchronization of activity and spike-timing dependent plasticity. Indeed, populations of cortical neurons can respond to subtle changes of the input very fast, on a millisecond time scale. Theoretical studies and model simulations linked the encoding abilities of neuronal populations to the fast onset dynamics of action potentials. Experimental results support this idea, however mechanisms of fast onset of action potentials in cortical neurons remain elusive. Studies in neuronal cultures, that are allowing for accurate control over conditions of growth and microenvironment during the development of neurons and provide better access to the spike initiation zone, may help to shed light on mechanisms of action potential generation and encoding. Here we characterize properties of action potential encoding in neocortical neurons grown for 11-25 days in culture. We show that encoding of high frequencies improves upon culture maturation, which is accompanied by the development of passive electrophysiological properties and action potential generation. The onset of action potentials becomes faster with culture maturation. Statistical analysis using correlations and linear model approaches identified the onset dynamics of action potentials as a major predictor of age-dependent changes of encoding. Encoding of high frequencies strongly correlated also with the input resistance of neurons. Finally, we show that maturation of encoding properties of neurons in cultures is similar to the maturation of encoding in neurons studied in slices. These results show that maturation of action potential generators and encoding is, to a large extent, determined genetically and takes place even without normal micro-environment and activity of the whole brain in vivo. This establishes neuronal cultures as a valid experimental model for studying mechanisms of action potential generation and encoding, and their maturation.

Responses of Withdrawal Interneurons to Serotonin Applications in Naïve and Learned Snails Are Different.

Abstract: Long-term changes in membrane potential after associative training were described previously in identified premotor interneurons for withdrawal of the terrestrial snail Helix. Serotonin was shown to be a major transmitter involved in triggering the long-term changes in mollusks. In the present study we compared the changes in electrophysiological characteristics of identifiable premotor interneurons for withdrawal in response to bath applications of serotonin (5-HT) or serotonin precursor 5-hydroxytryptophan (5-HTP) in preparations from naive, neurotoxin-injected or associatively trained snails. It was found that 5-HT or 5-HTP applications caused a significant decrease of membrane potential in premotor interneurons of naive snails, associatively trained snails and snails with impaired serotonergic system by injection of a selective neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) 1 week before the experiments. Applications of 5-HT or 5-HTP did not cause significant changes in the action potential (AP) threshold potential of these neurons in naive snails. Conversely, applications of 5-HT or 5-HTP to the premotor interneurons of previously trained or 5,7-DHT-injected snails caused a significant increase in the firing threshold potential in spite of a depolarizing shift of the resting membrane potential. Results demonstrate that responsiveness of premotor interneurons to extracellularly applied 5-HT or 5-HTP changes for days after the associative training or serotonin depletion. Similarity of the effects in trained and 5,7-DHT-injected animals may be due to massive release of serotonin elicited by 5,7-DHT injection. Our results suggest that serotonin release due to aversive conditionining or elicited by the neurotoxin administration triggers similar changes in resting membrane potential and AP threshold in response to bath applications of 5-HT or its precursor 5-HTP.

Adaptive Changes in the Vestibular System of Land Snail to a 30-Day Spaceflight and Readaptation on Return to Earth.

Abstract: The vestibular system receives a permanent influence from gravity and reflexively controls equilibrium. If we assume gravity has remained constant during the species' evolution, will its sensory system adapt to abrupt loss of that force? We address this question in the land snail Helix lucorum exposed to 30 days of near weightlessness aboard the Bion-M1 satellite, and studied geotactic behavior of postflight snails, differential gene expressions in statocyst transcriptome, and electrophysiological responses of mechanoreceptors to applied tilts. Each approach revealed plastic changes in the snail's vestibular system assumed in response to spaceflight. Absence of light during the mission also affected statocyst physiology, as revealed by comparison to dark-conditioned control groups. Readaptation to normal tilt responses occurred at ~20 h following return to Earth. Despite the permanence of gravity, the snail responded in a compensatory manner to its loss and readapted once gravity was restored.

Nitric Oxide Upregulates Proteasomal Protein Degradation in Neurons

Abstract: Nitric oxide (NO) is involved in many neuronal functions such as neuromodulation and intracellular signaling. Recent studies have demonstrated that nitric oxide is involved in regulation of proteasomal protein degradation. However, its role in neuronal protein degradation still remains unclear. In our study, we investigated the influence of endogenous nitric oxide production in this process. We have shown that nitric oxide synthase blockade prevents decline of the UbG76V-GFP fluorescence (GFP-based proteasomal protein degradation reporter) in neuronal processes of the cultured hippocampal neurons. It suggests that nitric oxide may regulate ubiquitin-dependent proteasomal protein degradation in neurons. Also, we have confirmed that the NO synthesis blockade alone significantly impairs long-term potentiation, and demonstrated for the first time that simultaneous blockade of the NO and proteins synthesis leads to the long-term potentiation amplitude rescue to the control values. Obtained results suggest that nitric oxide is involved in the protein degradation in proteasomes in physiological conditions.

Physiological Aspects of the Use of the Hodgkin–Huxley Model of Action Potential Generation for Neurons in Invertebrates and Vertebrates

Abstract: Recent studies have shown that the dynamics of action potential generation in neurons in vertebrates, in contrast to invertebrates, is significantly different from the slow exponential dynamics predicted by the Hodgkin–Huxley equations and is characterized by a sudden kink-like origin in the form of a steep linear increase. In this context, new and important aspects of studies of the links between the dynamics of action potential generation and the frequency coding abilities of neurons and neuronal networks have been found. This review addresses contemporary models describing the kink-type dynamics of action potential generation, including an alternative model of cooperative activation of potential-dependent sodium channels and the effects of the dynamics of action potential generation on the processing abilities of neural networks. The relevance of this direction comes from the fact that despite the rapid development of neuron simulation in recent years, generally accepted models of nerve cells cannot provide a realistic description of the complete dynamics of action potential generation in mammalian neurons or correct assessments of the ability of these cells to encode high-frequency signals. Contemporary experimental and theoretical analyses of action potential generation and neuronal encoding, as summarized in the present work, are highly significant for improving our understanding of nerve cell physiology and assisting the creation of more accurate and correct models of neurons.

Extinction and Reconsolidation of Memory

Abstract: Retrieval of memory followed by reconsolidation can strengthen a memory, while retrieval followed by extinction results in a decrease of memory performance due to weakening of existing memory or formation of a competing memory. In our study we analyzed the behavior and responses of identified neurons involved in the network underlying aversive learning in terrestrial snail Helix, and made an attempt to describe the conditions in which the retrieval of memory leads either to extinction or reconsolidation. In the network underlying the withdrawal behavior, sensory neurons, premotor interneurons, motor neurons, and modulatory for this network serotonergic neurons are identified and recordings from representatives of these groups were made before and after aversive learning. In the network underlying feeding behavior, the premotor modulatory serotonergic interneurons and motor neurons involved in motor program of feeding are identified. Analysis of changes in neural activity after aversive learning showed that modulatory neurons of feeding behavior do not demonstrate any changes (sometimes a decrease of responses to food was observed), while responses to food in withdrawal behavior premotor interneurons changed qualitatively, from under threshold EPSPs to spike discharges. Using a specific for serotonergic neurons neurotoxin 5,7-DiHT it was shown previously that the serotonergic system is necessary for the aversive learning, but is not necessary for maintenance and retrieval of this memory. These results suggest that the serotonergic neurons that are necessary as part of a reinforcement for developing the associative changes in the network may be not necessary for the retrieval of memory. The hypothesis presented in this review concerns the activity of the "reinforcement" serotonergic neurons that is suggested to be the gate condition for the choice between extinction/reconsolidation triggered by memory retrieval: if these serotonergic neurons do not respond during the retrieval due to adaptation, habituation, changes in environment, etc., then we will observe the extinction; while if these neurons respond to the CS during memory retrieval, we will observe the reconsolidation phenomenon.

An analysis of the effect of the internal ribosome entry site of the encephalomyocarditis virus on the expression of the second gene in the bicistronic matrix in neurons of primary hippocampal cultures

Abstract: Molecular biological experiments sometimes require expression of two or more genes in a single cell with an accurate ratio between their expression levels. One of the methods to provide this control is the use of the internal ribosome entry site (IRES) from the encephalomyocarditis virus as a separating insert between two target genes in the expression vector. Previously, it was shown that the efficacy of translation of the gene after IRES varies considerably in a range from 6 to 100% depending on the cell type. In neurons, the exact ratio between the expression levels of genes that are located before and after the IRES in the expression vector is unknown. Here, we analyzed the ratio between the amounts of products of the first and second genes located before and after the IRES in a plasmid that was used to transfect neurons in a primary hippocampal culture. We created two plasmid vectors that contain genes of the yellow (Venus) and red (mCherry) fluorescent proteins in different orders, which are separated by the IRES. We found that the unmodified IRES sequence of the encephalomyocarditis virus decreases the expression in the second cistron by a factor of 2.7 in a primary culture of hippocampal neurons. These data will help us to use currently available libraries of mutant IRES sequences for accurate control of the relationships between the expression of different target genes in neurons.