CADENCE is an open Python 3-written neuroinformatics tool with Qt6 graphic user interface for supervised calcium events detection. In neuronal ensembles recording during calcium imaging experiments, the output of instruments such as Celena X, Zeiss LSM 5 Live confocal microscope and Miniscope is a movie showing flashing cells somata. There are few pipelines to convert video to relative fluorescence ΔF/F, from simplest ImageJ plugins to sophisticated tools like MiniAn (Dong et al. in Elife 11, https://doi.org/10.7554/eLife.70661 , 2022). Minian, an open-source miniscope analysis pipeline. Elife, 11.). While in some areas of study relative fluorescence ΔF/F may be the desired result in itself, researchers of neuronal ensembles are typically interested in a more detailed analysis of calcium events as indirect proxy of neuronal electrical activity. For such analyses, researchers need a tool to infer calcium events from the continuous ΔF/F curve in order to create a raster representation of calcium events for later use in analysis software, such as Elephant (Denker, M., Yegenoglu, A., Grün, S. (2018). Collaborative HPC-enabled workflows on the HBP Collaboratory using the Elephant framework. Neuroinformatics, 19.). Here we present such an open tool with supervised calcium events detection.

Gene therapy offers a potential alternative to the surgical treatment of epilepsy, which affects millions of people and is pharmacoresistant in ~30% of cases. Aimed at reducing the excitability of principal neurons, the engineered expression of K + channels has been proposed as a treatment due to the outstanding ability of K + channels to hyperpolarize neurons. However, the effects of K + channel overexpression on cell physiology remain to be investigated. Here we report an adeno-associated virus (AAV) vector designed to reduce epileptiform activity specifically in excitatory pyramidal neurons by expressing the human Ca 2+ -gated K + channel KCNN4 (KCa3.1). Electrophysiological and pharmacological experiments in acute brain slices showed that KCNN4-transduced cells exhibited a Ca 2+ -dependent slow afterhyperpolarization that significantly decreased the ability of KCNN4-positive neurons to generate high-frequency spike trains without affecting their lower-frequency coding ability and action potential shapes. Antiepileptic activity tests showed potent suppression of pharmacologically induced seizures in vitro at both single cell and local field potential levels with decreased spiking during ictal discharges. Taken together, our findings strongly suggest that the AAV-based expression of the KCNN4 channel in excitatory neurons is a promising therapeutic intervention as gene therapy for epilepsy.

ABSTRACT Virtually all major processes in cells and tissues are regulated by calcium ions (Ca 2+ ). Understanding the influence of Ca 2+ on cell function requires technologies that allow for non-invasive manipulation of intracellular calcium levels including the formation of calcium patterns, ideally in a way that is expandable to intact organisms. The currently existing tools for optical and optogenetic Ca 2+ manipulation are limited with respect to response time, and tissue penetration depth. Here we present G enetically E ncoded C alcium Co ntroller ( GECCO ), a system for thermogenetic Ca 2+ manipulation based on snake TRP channels optically controlled by infrared illumination. GECCO is functional in animal and plant cells and allows studying how cells decode different profiles of Ca 2+ signals. GECCO enabled the shaping of insulin release from β-cells, the identification of drugs that potentiate Ca 2+ -induced insulin release, and the generation of synthetic Ca 2+ signatures in plants.

To date, intracellular calcium reporters are the most commonly used optical probes for detecting neuron activity. Synthetic low molecular weight and genetically encoded protein compounds provide an enormous variety of accessible methods for recording signals of different intensity at different levels of studying the nervous system, from detecting activity in synaptic boutons and dendritic spines to recording the activity of the central nervous system in free behavior in vivo. This paper presents a comparative description of methods for optical recording of changes in the intracellular calcium concentration and compares original experimental data obtained by each of the methods described. Advantages and drawbacks are described, and the potential areas of application of each of the commercially available and widely used types of calcium-sensitive reporters are outlined.

According to modern concepts, the dorsal hippocampus, specifically the CA1 field, plays a crucial role in the formation and reactivation of contextual fear conditioning (CFC) memory [1–5]. However, the extent to which the neurons of the dorsal hippocampus participate in CFC learning or memory reactivation remains poorly understood. The aim of this study was to examine the in vivo activity of neurons in the hippocampal CA1 field during CFC memory training and testing. The study conducted experimentations on male mice of the C57Bl/6 line (N=4). Miniature fluorescence microscopes, also known as miniscopes, were used to monitor neuronal activity in the CA1 field. The CA1 field in the hippocampus was injected with an AAV vector carrying the GCaMP6s calcium sensor, and implanted with a GRIN lens in the same area as the miniscope lens. The mice underwent CFC task training and the duration of freezing was then measured. After the training session, the mice exhibited a notable increase in freezing duration, suggesting the formation of context aversive memory. Throughout the training, a total of 591 active neurons were recorded (147.8±74.9 neurons per mouse), while 512 (128.0±40.6 neurons per mouse) neurons were recorded. The average frequency of calcium events per second during the complete duration of training session was 0.037±0.003, while for the testing, it was 0.042±0.015 events/second. Around 46% of the registered neurons remained active throughout the complete training procedure. The mean frequency of calcium events in these neurons surged considerably following the application of an electric shock (from 0.035±0.007 events/sec to 0.086±0.013 events/sec). Using k-means clustering, certain neurons showed increased activity after electric shock exposure, while others showed decreased activity. However, the type of activity change did not affect subsequent neuronal dynamics during memory retrieval. During memory retrieval, we observed that an average of 30–40% of neurons were reactivated. The number of active neurons notably decreased during episodes of freezing and almost all registered neurons were activated during episodes of movement. The average frequency of calcium events in the reactivating neurons did not change from the training to testing session. Thus, new data was obtained on the activation of neurons in the hippocampal CA1 area during memory formation and retrieval in CFC.

Epigenetic regulation plays an important role in cognitive brain functions. The increase in histone acetylation by histone deacetylase inhibitors, such as sodium butyrate, was shown previously to be highly correlated with memory enhancement. Currently, the data on sodium butyrate effects on neuronal activity in vivo are limited. This study investigates how sodium butyrate affects the activity of CA1 hippocampal neurons in freely behaving mice. We performed in vivo calcium imaging of CA1 neurons with miniscopes in mice during arena exploration trials. The first trial was followed by an intraperitoneal injection of sodium butyrate or saline. After 45 and 90 minutes, CA1 neurons were recorded again in the homecage. The second trial was made 24 hours later. The injection of sodium butyrate resulted in immediate inhibition of neuronal activity for the following 90 minutes. However, the population activity analysis revealed that neurons that were active in the first behavioral trial demonstrated significantly more correlated activity during the second trial in the sodium butyrate group, but not in the saline group. Thus, for the first time, we demonstrated the effects of sodium butyrate on neuronal activity in vivo .

The role of astrocytes in modulating synaptic plasticity is an important question that until recently was not addressed due to limitations of previously existing technology. In the present study, we took an advantage of optogenetics to specifically activate astrocytes in hippocampal slices in order to study effects on synaptic function. Using the AAV-based delivery strategy, we expressed the ionotropic channelrhodopsin-2 (ChR2) or the metabotropic Gq-coupled Opto-a1AR opsins specifically in hippocampal astrocytes to compare different modalities of astrocyte activation. In electrophysiological experiments, we observed a depression of basal field excitatory postsynaptic potentials (fEPSPs) in the CA1 hippocampal layer following light stimulation of astrocytic ChR2. The ChR2-mediated depression increased under simultaneous light and electrical theta-burst stimulation (TBS). Application of the type 2 purinergic receptor antagonist suramin prevented depression of basal synaptic transmission, and switched the ChR2-dependent depression into potentiation. The GABAB receptor antagonist, phaclofen, did not prevent the depression of basal fEPSPs, but switched the ChR2-dependent depression into potentiation comparable to the values for TBS in control slices. In contrast, light stimulation of Opto-a1AR expressed in astrocytes led to an increase in basal fEPSPs, as well as a potentiation of synaptic responses to TBS significantly. A specific blocker of the Gq protein downstream target, the phospholipase C, U73122, completely prevented the effects of Opto-a1AR stimulation on basal fEPSPs or Opto + TBS responses. To understand molecular basis for the observed effects, we performed an analysis of gene expression in these slices using quantitative PCR approach. We observed a significant upregulation of "immediate-early" gene expression in hippocampal slices after light activation of Opto-a1AR-expressing astrocytes alone (cRel, Arc, Fos, JunB, and Egr1) or paired with TBS (cRel, Fos, and Egr1). Activation of ChR2-expressing hippocampal astrocytes was insufficient to affect expression of these genes in our experimental conditions. Thus, we concluded that optostimulation of astrocytes with ChR2 and Opto-a1AR optogenetic tools enables bidirectional modulation of synaptic plasticity and gene expression in hippocampus.

Currently, the reporters of intracellular calcium are the most widely used optical probes for the detection of neuronal activity. Synthetic molecules and genetically encoded proteins provide a large variety of methods available for recording of neuron-related signals of different shapes and amplitudes at several levels of neuronal studies, starting from single-cellular level and ending up with the in vivo recordings in the freely behaving animals. Here, we compare different methods of optical recording of intracellular calcium transients based on to the original data obtained with those methods. We describe the advantages and limitations of each of the commercially available and widely employed calcium-sensitive probes.

Subcellular targeting of opsins in optogenetics provides new possibilities for investigating the function of nerve cells. One of the widely used motifs for central targeting of opsins is the motif of potential-dependent potassium channel Kv2.1. We have expressed construct CHR2-Venus-Kv2.1 in the layer 2/3 pyramidal neurons of the murine cerebral cortex by means of in utero electroporation. It was found that, although the majority of neurons expressing CHR2-Venus-Kv2.1 demonstrated mainly central localization of fluorescence in the soma, proximal dendrites and axon, there was also significant population of neurons with disruption of the “correct” targeting resulting in the fluorescent protein distributed uniformly throughout the entire cell surface. We have suggested that observed mislocalization was caused by overexpression of the construct. Indeed, a decrease in the plasmid concentration during the in utero electroporation procedure resulted in almost complete absence of neurons with altered targeting. Thus, the possibility of “incorrect” targeting of CHR2 by the potassium channel motif Kv2.1 should be taken into account when using this construct in optogenetic experiments.

Several recent studies showed that memory can be modulated by manipulating chromatin modifications using histone deacetylase (HDAC) inhibitors during memory formation, consolidation, and reconsolidation. We used a context fear conditioning paradigm with minimal non-painful current as a reinforcement, what elicited alertness to the context and freezing during tests in rats. Such paradigm resulted in a relatively weak memory in significant part of the rats. Here, we demonstrate that intraperitoneal administration of the HDAC inhibitor sodium butyrate immediately following memory reactivation, produced memory enhancement in rats with weak memory, however, not in rats with strong memory. Additionally, we investigated the ability of the HDAC inhibitor sodium butyrate to restore the contextual memory impaired due to the blockade of protein synthesis during memory reactivation. The results obtained evidence that the HDAC inhibitor sodium butyrate reinstated the impaired contextual memory. This enhancement effect is consistent with other studies demonstrating a role for HDAC inhibitors in the facilitation of contextual fear.

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.

Thermogenetics appeared recently as an evolutionary advance in methods of optical stimulation of nerve cells and uses focused light to activate light-sensitive cation channels in the neuron membrane. Light activation and opening of light-sensitive channels, which can be expressed genetically in any type of neuron, induces neuron membrane depolarization and a resultant action potential. In contrast to classical methods of optogenetics, which use the visible spectrum, thermogenetics uses channels sensitive to warming. This provides the opportunity for additional activation of these channels not only using IR light, but also other methods of warming nervous tissue, such as ultrasound or microwave radiation. The penetrability of living tissue to stimulation with IR radiation is an order of magnitude greater than that for visible spectrum radiation, which allows thermogenetics methods to be used in vivo without invasive surgical methods to “clear the way” for optogenetic stimulation. On the other hand, the thermal nature of the stimulation imposes additional limits on use of the method, as heating must be sufficiently gentle so as not to induce heat shock at the cellular level, and the threshold of activation must be sufficiently high for channel opening not to occur spontaneously at normal physiological temperatures.

Thermogenetics has emerged recently as an evolving extension of optical methods for neuronal stimulation that employs focused light to activate photosensitive cationic channels in neuronal membrane. Light-activation to open photosensitive channels that can be expressed genetically in all types of neurons is aimed to induce depolarization of neuronal membrane followed by a neuronal discharge. Unlike conventional optogenetics that employs visible spectrum, thermogenetics uses channels gated by heating. It provides the possibility to activate the channels not only with IR-radiation, but also with any other way to heat nervous tissue such as ultrasound or microwave radiation. The permeability of living tissue to IR-radiation is orders higher than that to visible light spectra, which allows use of thermogenetic stimulation in the experiments in vivo without invasive surgery to make access for optogenetic stimulation. On the other hand, the thermal nature of stimulation imposes additional limitations to application of thermogenetics as heating should be quite reasonable to avoid heat shock response at cellular level, while the threshold of activation of channels should be high enough to rule out their activation at normal physiological temperatures.

In the present work, using in situ hybridization, we studied the expression patterns of three molluscan homologs of vertebrate immediate-early genes C/EBP, c-Fos, and c-Jun in the central nervous system (CNS) of terrestrial gastropod snail Helix. The molluscan C/EBP gene was described in literature, while c-Fos and c-Jun were studied in terrestrial snails for the first time. Localization of the expression was traced in normal conditions, and in preparations physiologically activated using stimulation of suboesophageal ganglia nerves. No expression was detected constitutively. In stimulated preparations, all three genes had individual expression patterns in Helix CNS, and the level of expression was stimulus-dependent. The number of cells expressing the gene of interest was different from the number of cells projecting to the stimulated nerve, and thus activated retrogradely. This difference depended on the ganglia studied. At the subcellular level, the labeled RNA was observed as dots (probably small clusters of RNA molecules) and shapeless mass of RNA, often seen as a circle at the internal border of the cell nuclei. The data provide a basis for further study of behavioral role of these putative immediate-early genes in snail behavior and learning.

Widening and deepening our understanding of how the brain works requires constant improvements not only in methods of recording neuron activity, but also improvements in experimental approaches to activating individual cells and their compartments. Optogenetic stimulation methods using finely focused light to trigger the opening of the light-activated depolarizing cation channel rhodopsin-2 (ChR2) have become widely used in recent years. Current molecular biological methods provide for the genetic expression of ChR2 in different cell types, which, along with the ability to carry out electrophysiological experiments with reproducible patterns of activation and stable levels of ChR2 expression, have developed optogenetics into an effective method for gathering physiological data previously unavailable to conventional methods. We report here the use of local activation of axons using an optogenetic stimulation method. Experiments were performed in combination with recording the electrical activity of neurons using the patch-clamp method, as well as laser scanning confocal microscopy. Experiments used the transgenic mouse strain Thy1-ChR2-YFP, in which ChR2 is expressed in only a small proportion of pyramidal cells. Direct studies of the effects of functional activity in the proximal branches of pyramidal neuron axons in layer 5 of the visual cortex and hippocampal field CA1 on the shape and generation of action potentials were carried out. We also describe methodological advances and means of solving problems encountered in the optogenetic stimulation of the axons of pyramidal neurons in the central nervous system of mammals.

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.

One version of the optogenetic prosthetization of the degenerative retina is an approach based on creation of an ON/OFF receptive field for ganglion neurons by targeting the expression of an excitatory light-activated protein in the central part of the ganglion cell and an inhibitory protein at the periphery. Within the framework of this approach, we investigated the possibility of using various anchoring motifs to support the somatic or dendritic location of opsins. The motif of the Kv2.1 potassium channel does not always provide an exclusively central location for the light-activated protein, which may be linked with significant overexpression of the construct when powerful universal promoters are used. In addition, we have demonstrated the possibility of using a postsynaptic density protein, Homer1, as the anchoring motif for peripheral targeting of rhodopsins.

Protein kinase Mζ is considered important for memory formation and maintenance in different species, including invertebrates. PKMζ participates in multiple molecular pathways in neurons, regulating translation initiation rate, AMPA receptors turnover, synaptic scaffolding assembly, and other processes. Here, for the first time, we established the sequence of mRNA encoding PKMζ homolog in land snail Helix lucorum. We annotated important features of this mRNA: domains, putative capping sites, translation starts, and splicing sites. We discovered that this mRNA has at least two isoforms, and one of them lacks sequence encoding C1 domain. C1 deletion may be unique for snail because it has not been previously found in other species. We performed behavioral experiments with snails, measured expression levels of identified isoforms, and confirmed that their expression correlates with one type of learning.

For the purpose of optogenetic prosthetics of the receptive field of the retinal ganglion cell, we have created a bicistronic genetic construct that carries genes of excitatory (channelorhodopsin2) and inhibitory (anionic channelorhodopsin) rhodopsins. A distinctive feature of this construct is the combination of two genes into one construct with the mutant IRES inserted between them, which ensures precise ratio of the expression levels of the first and second gene in each transfected cell. It was found that the illumination of the central part of transfected neuron with light with a wavelength of 470 nm causes the generation of action potentials in the cell. At the same time, light stimulation of the periphery of the neuron causes cessation of the generation of action potentials. Thus, we were able to simulate the ON-OFF interaction of the receptive field of the retinal ganglion cell using optogenetic methods. Theoretically, this construction can be used for optogenetic prosthetics of degenerative retina in case of its delivery to ganglion cells using lentiviral vectors.