Epigenetic Regulation as a Basis for Long-Term Changes in the Nervous System: In Search of Specificity Mechanisms.

Adaptive long-term changes in the functioning of nervous system (plasticity, memory) are not written in the genome, but are directly associated with the changes in expression of many genes comprising epigenetic regulation. Summarizing the known data regarding the role of epigenetics in regulation of plasticity and memory, we would like to highlight several key aspects. (i) Different chromatin remodeling complexes and DNA methyltransferases can be organized into high-order multiprotein repressor complexes that are cooperatively acting as the "molecular brake pads", selectively restricting transcriptional activity of specific genes at rest. (ii) Relevant physiological stimuli induce a cascade of biochemical events in the activated neurons resulting in translocation of different signaling molecules (protein kinases, NO-containing complexes) to the nucleus. (iii) Stimulus-specific nitrosylation and phosphorylation of different epigenetic factors is linked to a decrease in their enzymatic activity or changes in intracellular localization that results in temporary destabilization of the repressor complexes. (iv) Removing "molecular brakes" opens a "critical time window" for global and local epigenetic changes, triggering specific transcriptional programs and modulation of synaptic connections efficiency. It can be assumed that the reversible post-translational histone modifications serve as the basis of plastic changes in the neural network. On the other hand, DNA methylation and methylation-dependent 3D chromatin organization can serve a stable molecular basis for long-term maintenance of plastic changes and memory.

Ca2+-activated KCa3.1 potassium channels contribute to the slow afterhyperpolarization in L5 neocortical pyramidal neurons.

Layer 5 neocortical pyramidal neurons are known to display slow Ca2+-dependent afterhyperpolarization (sAHP) after bursts of spikes, which is similar to the sAHP in CA1 hippocampal cells. However, the mechanisms of sAHP in the neocortex remain poorly understood. Here, we identified the Ca2+-gated potassium KCa3.1 channels as contributors to sAHP in ER81-positive neocortical pyramidal neurons. Moreover, our experiments strongly suggest that the relationship between sAHP and KCa3.1 channels in a feedback mechanism underlies the adaptation of the spiking frequency of layer 5 pyramidal neurons. We demonstrated the relationship between KCa3.1 channels and sAHP using several parallel methods: electrophysiology, pharmacology, immunohistochemistry, and photoactivatable probes. Our experiments demonstrated that ER81 immunofluorescence in layer 5 co-localized with KCa3.1 immunofluorescence in the soma. Targeted Ca2+ uncaging confirmed two major features of KCa3.1 channels: preferential somatodendritic localization and Ca2+-driven gating. In addition, both the sAHP and the slow Ca2+-induced hyperpolarizing current were sensitive to TRAM-34, a selective blocker of KCa3.1 channels.

Bicistronic Construct for Optogenetic Prosthesis of Ganglion Cell Receptive Field of Degenerative Retina.

To perform optogenetic prosthetics of the retinal ganglion cell receptive field, a bicistronic genetic construct carrying the genes encoding the excitatory (channelrhodopsin-2) and inhibitory (Guillardia theta anion channelrhodopsin GtACR2) rhodopsins was created. A characteristic feature of this construct was the combination of these two genes with a mutant IRES insertion between them, which ensures the exact ratio of expression levels of the first and second genes in each transfected cell. Illumination of the central part of the neuron with light with a wavelength of 470 nm induced the action potential generation in the cell. Stimulation of the peripheral neuronal region with light induced the inhibition of action potential generation. Thus, using optogenetics methods, we simulated the ON-OFF interaction in the retinal ganglion cell receptive field. Theoretically, this construct can be used for optogenetic prosthetics of degenerative retina in the case of its delivery to the ganglion cells with lentiviral vectors.

Lynx1 Prevents Long-Term Potentiation Blockade and Reduction of Neuromodulator Expression Caused by Aß1-42 and JNK Activation.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels. Many neurodegenerative diseases are accompanied by cognitive impairment associated with the dysfunction of nAChRs. The human membrane-tethered prototoxin Lynx1 modulates nAChR function in the brain areas responsible for learning and memory. In this study, we have demonstrated for the first time that the ß-amyloid peptide Aß1-42 decreases Lynx1 mRNA expression in rat primary cortical neurons, and that this decrease is associated with the activation of c-Jun N-terminal kinase (JNK). In addition, we have demonstrated that the Lynx1 expression decrease, as well as the blockade of the long-term potentiation underlying learning and memory, caused by Aß1-42, may be prevented by incubation with a water-soluble Lynx1 analogue. Our findings suggest that the water-soluble Lynx1 analogue may be a promising agent for the improvement of cognitive deficits in neurodegenerative diseases.

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

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.

Chloride conducting light activated channel GtACR2 can produce both cessation of firing and generation of action potentials in cortical neurons in response to light.

Optogenetics is a powerful technique in neuroscience that provided a great success in studying the brain functions during the last decade. Progress of optogenetics crucially depends on development of new molecular tools. Light-activated cation-conducting channelrhodopsin2 was widely used for excitation of cells since the emergence of optogenetics. In 2015 a family of natural light activated chloride channels GtACR was identified which appeared to be a very promising tool for using in optogenetics experiments as a cell silencer. Here we examined properties of GtACR2 channel expressed in the rat layer 2/3 pyramidal neurons by means of in utero electroporation. We have found that despite strong inhibition the light stimulation of GtACR2-positive neurons can surprisingly lead to generation of action potentials, presumably initiated in the axonal terminals. Thus, when using the GtACR2 in optogenetics experiments, its ability to induce action potentials should be taken into account. Our results also open an interesting possibility of using the GtACR2 both as cell silencer and cell activator in the same experiment varying the pattern of light stimulation.

Comparative characteristics of two anion-channel rhodopsins and prospects of their use in optogenetics.

Anion-selective opsins slow ChloC and ACR2 were expressed in rat brain cortical neurons by electroporation in utero. It is shown that the light-activated channel ACR2 has pronounced advantages in terms of both the inactivation kinetics and the neuron inhibition intensity, which is associated with a more negative value of the light-activated current reversal potential compared to the slow ChloC channel. The identified properties of opsin ACR2 indicate that it can be used for strictly controlled suppression of neuronal activity in optogenetic experiments, including the expression in the retinal ganglionic cells for reconstituting the OFF-component of their receptive field, which is essential for optogenetic prosthetics of degenerative retina.

[Physiological Aspects of the Application of Hodgkin-Huxley Model of Action Potential Initiation for Invertebrate and Vertebrate Neurons].

Recent studies have revealed.that in contrast to invertebrate systems, the initiation of action potentials in vertebrate neurons significantly differ from the relatively slow exponential dynamics predicted by Hodgkin-Huxley equations, but rather is characterized by a sharp onset with a kink. These data provided new insights into the link between action potential initiation and abilities of neurons and neuronal networks to encode. high frequency signals. Here, we review recent models describing sharp onset dynamics of action potential initiation, including an alternative model of cooperative activation of sodium channels, as well as the influence of the dynamics of action potential initiation on computational abili- ties of neuronal networks. The importance this topic is due to the fact that, despite the rapid development of neuronal modeling during last decades, the well established models are unable to capture experimentally observed details of the onset dynamics of action potentials in mammalian neurons and the abilities of neurons to reliably encode code high frequency signals Recent advances of experimental and theoretical analysis of generation of action potentials and neuronal encoding, presented in this review, are ofgreat importance for better understanding of neuronal processing and development of a more precise and realistic neuronal model.

[Neurophilosophy].

In the paper the role of philosophical approaches in analyses of the MRT data, neuropathologies is dis cussed. Importance of teleological approaches is stressed.

[Extinction and Reconsolidation of Memory].

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.

Anion-selective channelrhodopsin expressed in neuronal cell culture and in vivo in murine brain: Light-induced inhibition of generation of action potentials.

Anionic channelrhodopsin slow ChloC was expressed in the culture of nerve cells and in vivo in mouse brain. We demonstrated ability of slow ChloC to suppress effectively the activity of the neuron in response to the illumination with the visible light. It has been shown for a first time that slow ChloC works equally efficiently in both neuronal culture and in the whole brain being expressed in vivo. Thus, slow ChloC could be considered as an effective optogenetic tool capable in response to light stimulation to inhibit the generation of action potentials in the neuron.

Nitric oxide is necessary for long-term facilitation of synaptic responses and for development of context memory in terrestrial snails.

Correlated electrophysiological and behavioral experiments in the snail Helix lucorum were conducted to investigate the contribution of nitric oxide (NO) to synaptic plasticity during withdrawal reflex and aversive context memory development. Time, stimulation frequency and number of tetani/electrical shocks were determined in vitro and in vivo. In isolated brain preparations, nerve tetanization accompanied by bath application of serotonin induced long-term facilitation (LTF) of the excitatory postsynaptic potential (EPSP) in withdrawal interneurons. Bathing with either the NO-synthase inhibitor N-omega-nitro-L-arginin (L-NNA) or the NO-scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide (PTIO) before the tetanization prevented tetanus-induced long-term increase of EPSP. Withdrawal interneurons are key elements in the network underlying aversive behavior, with LTF considered the basis for aversive learning. We hypothesized that L-NNA injections in free-behaving snails could influence aversive learning. Snails were trained for 1 or 5days to remember the context in which they were shocked. In one-day training experiments, the snails received 5 electrical shocks in one context. Different groups of snails were sham-injected or L-NNA-injected before or after training. After training, the sham-injected groups demonstrated a significant increase in behavioral responses compared to the L-NNA-injected groups. On the following day, only sham-injected snails demonstrated altered behavioral responses, but no associative context differences were observed. These results correlated with the electrophysiological results. In another series of experiments, the snails received electrical shocks for 5days. Testing on the second day after training demonstrated that the sham-injected group maintained selective aversive context memory, whereas the L-NNA-injected snails were not different between the two contexts. Together these results demonstrated that inhibition of NO synthesis prevents memory formation and influences synaptic plasticity in the withdrawal interneurons that underlie the behavioral changes. This suggests that NO influences the behavior via regulation of synaptic plasticity.

[Mismatch negativity and its hemodynamic equivalent (based on fMRI) in research of speech perception in healthy and in speech disorders].

The review is devoted to the use of electrophysiological index of auditory discrimination, known as "mismatch negativity" (MMN), and its hemodynamic equivalent obtained by functional magnetic resonamce imaging (fMRI) to study speech perception in normal and pathological conditions. Most attention is paid to works with using MMN as a neurophysiological index of the phonemic hearing impairment in patients with sensory aphasia. The MMN applicability for examination of speech compensation degree is substantiated. Also the perspectives of simultaneous EEG-fMRI registration in exploring speech pathologe are considered.

[The central pattern generators].

The central pattern generator (CPG) is defined as a set of neurons involved in joint production of patterned motor output. The roundtable discussion on the CPG was a part of the 5th All-Russian Conference on Animal Behavior (Moscow, Nov. 21, 2012). The discussion centred on three core themes: 1) the mechanisms of the organization and reconfiguration of pattern generating neuronal ensembles, 2) extrapolations that extend the CPG concept beyond the motor systems, and 3) evolutionary and developmental aspects of CPG.

[Advanced optical recording of neuronal activity with voltage-sensitive dyes].

Currently, the studies of electrical activity and plasticity of neuronal networks are impossible without employing of imaging techniques to visualize functional signals that allowing revealing electrical events in multiple neurons, as well as in their tiny dendrites and axons placed on their morphological picture. Imaging with voltage-sensitive dyes (VSD) is one of unique available methods that providing both high spatial resolution and ultrafast sampling (< 0.1 ms) in realtime with perfect S-to-N ratio. During the last decade a significant progress in VSD application has been achieved due to major method improvements and new probe synthesis especially in the field of research of initiation and propagation of action potentials. There was evidence of the method efficiency and usability while the method was added to the toolbox of modern neuroscience for research in hottest topics.

[Compartmentalization of neral non-synaptic plasticity at subcellular level].

One of the most efficient ways of CNS response to the variety of external and internal stimuli is a selective increase of excitability of neurons, acquired, for instance, during learning process. It is now well established, that persistent non-synaptic plasticity that emerging after learning, as well as synaptic one may serve an a substrate for long-term memory. Though, it remains unknown how the non-synaptic plasticity contributes to the alteration of the states of neuronal networks, which the long-term memory depends directly on. The explanation of how the non-synaptic plasticity is translated into the modified states of the neuronal networks and modified behavior remains one of the most important challenges of the contemporary research in the field of learning and memory. Also, little is known about the specific neuronal compartments ofthe axon and dendretic tree of that subjected to plastic changes in the context of morphological features of individual neuron and the efficiency of its input and output synapses subjected to indirect modifications due to non-synaptic plasticity and the influence of non-synaptic plasticity on the efficiency of neuronal synapses.

[Fast and aimed delivery of voltage-sensitive dyes to mammalian brain slices by biolistic techniques].

Fast voltage-sensitive dyes (VSD) are widely used in modern neuroscience for optical recording of electrical potentials at many levels, from single cell compartment to brain areas, containing populations of many neural cells. The more lipophilic a VSD, the better signal-to-noise ratio of the optical signal, but there are no effective ways to deliver a water-insoluble dye into the membrane of live cell. Here we report a new protocol based on rapid biolistic delivery of VSDs, which is optimal for further recordings of optical signals from live neurons of rat brain slices. This protocol allows us to stain locally (150 mkm) neural somata of brain structures with a Golgi-like pattern, and a VSD propagates even to distant neurites of stained cells very quickly. This technique also can be used for rapid local delivery of any lipophilic and water-insoluble substances into live cells, further optical recording of neural activity, and analysis of potential propagation in a nerve cell.

[Changes in intracellular calcium concentration during generation of high-amplitude EPSPs in grape snail neurons].

Change in intracellular calcium concentration is the main trigger of majority of physiological processes in the neuron including altering of gene expression and synaptic plasticity. We have found out that high amplitude EPSPs in command neurons of terrestrial snail similar to action potentials were accompanied by prominent calcium transients. It was shown that the amplitude of calcium transients accompanying high amplitude EPSPs in command neurons linearly depends on the strength of synaptic stimulation whereas dynamic of changes of EPSP amplitudes demonstrates pronounced saturation with increase of stimulus intensity. It means that in the certain range of membrane potentials calcium transients more accurately reflect stimulus strength then the level of membrane depolarization. We propose that calcium transients induced by high amplitude EPSPs are able to induce some biochemical changes in the neurons and therefore can mediate cell response at the subthreshold for action potential levels of membrane potential.

[Network, cellular and molecular mechanisms of plasticity in simple nervous systems].

In the present study we will try to single out several principles of the nervous system functioning essential for describing mechanisms of learning and memory basing on our own experimental investigation of cellular mechanisms of memory in the nervous system of gastropod molluscs and literature data: main changes in functioning due to learning occur in effectivity of synaptic inputs and in the intrinsic properties of postsynaptic neurons; due to learning some synaptic inputs of neurons selectively change its effectivity due to pre- and postsynaptic changes, but the induction of plasticity always starts in postsynapse, maintaining of long-term memory in postsynapse is also shown; reinforcement is not related to activity of the neural chain receptor-sensory neuron-interneuron-motoneuron-effector; reinforcement is mediated via activity of modulatory neurons, and in some cases can be exerted by a single neuron; activity of modulatory neurons is necessary for development of plastic modifications of behavior (including associative), but is not needed for recall of conditioned responses. At the same time, the modulatory neurons (in fact they constitute a neural reinforcement system) are necessary for recall of context associative memory; changes due to learning occur at least in two independent loci in the nervous system. A possibility for erasure of memory with participation of nitroxide is experimentally and theoretically based.

[Two-faced nitric oxide is necessary for both erasure and consolidation of memory].

One of ways of nitric oxide (NO) influence on neuronal activity is S-nitrosylation, the covalent attachment of NO group to the thiol side chain of cysteine, changes function of existing proteins, inhibiting their normal role in physiological functions including memory. Influence of NO via GC activates intracellular signaling cascades and triggers increased synthesis ofproteins, influencing the memory. In the present paper we want to express and test the hypothesis that the NO is necessary both for erasure and development of memory. In our experiments in terrestrial snail Helix we tested the idea that NO besides well shown participation in memory development is involved in erasure/lockout of memory during relearning and reconsolidation.