In vivo calcium imaging is widely used technique in neuroscience to evaluate the activity of neuronal networks. The miniscope, a single-photon miniature fluorescent microscope, has made it possible to conduct in vivo calcium imaging in freely moving animals. Various algorithms and software packages have been developed for the analysis of miniscope data. This study investigates the relationship between the sensitivity of neuron detection and the processing parameters utilized in the Minian analysis pipeline at different noise levels. To achieve this objective, we generated simulated data possessing certain attributes of an experimentally derived dataset. Simulated data was generated with various noise levels and processed through to the Minian analysis pipeline. Based on our findings, we provide recommendations for optimal values of Minian pipeline parameters depending on different noise levels. The results obtained in this study may serve as a preliminary guide for selecting appropriate parameter values during the processing of experimental data using the Minian analysis pipeline. The findings of this study are expected to be relevant to neuroscientists involved in the acquisition and processing of miniscope data.

The miniature single-photon fluorescent microscope (miniscope) enables the visualization of calcium activity in vivo in freely moving laboratory animals, providing the capability to track cellular activity during the investigation of memory formation, learning, sleep, and social interactions. However, the use of calcium sensors for in vivo imaging is limited by their relatively slow (millisecond-scale) kinetics, which complicates the recording of high-frequency spike activity. The integration of methods from single-photon miniature fluorescent microscopy with electrophysiological recording, which possesses microsecond resolution, represents a potential solution to this issue. Such a combination of techniques allows for the simultaneous recording of optical and electrophysiological activity in a single animal in vivo. In this study, a flexible polyimide microelectrode was developed and integrated with the gradient lens of the miniscope. The in vivo tests conducted in this research confirmed that the microelectrode combined with the gradient lens facilitates simultaneous single-photon calcium imaging and local field potential recording in the hippocampus of an adult mouse.

Recently, mounting evidence suggests that cognitive impairment may accompany traditional neurological diseases, such as neurodegenerative disorders, as well as result from previous infections (COVID-19, influenza). One approach to mitigating the neurological pathological state involves regulating abnormal neural activity. Nevertheless, addressing this issue directly may not always be feasible due to neuronal overexcitation or inadequate stimulation, leading to unfavorable outcomes. Meanwhile, astrocytes adapt their activation levels exclusively to the group of neurons requiring activation, boosting cognitive functions as an example [1]. Optogenetics was employed in this study to selectively stimulate metabotropic astrocyte receptors in acute hippocampal slices of mice with an Alzheimer’s model. The aim was to examine the effect on electrophysiological function of neurons, strength of synaptic contacts ex vivo, and cognitive performance in vivo. Several fundamentally different approaches exist for optogenetic stimulation of cells, including the use of molecular targets such as ionotropic receptors (e.g., ChR2) or metabotropic receptors (e.g., OptoGq). Our studies have shown enhanced activity of hippocampal pyramidal neurons and potentiated field excitatory potentials (fEPSP) following optogenetic activation of astrocytes expressing the metabotropic construct OptoGq. Conversely, the use of ChR2 resulted in an opposite effect [2]. For this reason, all subsequent investigations used a metabotropic construct. Astrocytes are known to respond to external stimuli via intracellular calcium [Ca2+] waves. The propagation of this wave results in the release of D-serine, cytokines, and lactate, subsequently modulating the activity of neurons. The role of astrocytes in regulating the function of NMDA receptors by releasing or removing glutamate from the extracellular environment is critical in modulating neural network excitation. Given the association of astrocytes with the pathogenesis and pathological mechanisms involved in neurodegenerative disorders, controlling their activity becomes a pressing and indispensable aspect of therapy. In the present investigation, optogenetic stimulation of hippocampal astrocytes transduced by AAV5_GfaABC1D_opto-a1AR-EYFP virus (which encodes a Gq-coupled metabotropic receptor) resulted in enhanced electrophysiological activity of hippocampal pyramidal neurons. This was evidenced by increased sEPSC of pyramidal neurons and the potentiation of field excitatory postsynaptic potentials (fEPSP) in the hippocampal region, following light activation of astrocytes [2]. A significant activation of early gene expression (cRel, Arc, Fos, JunB, and Egr1) was detected in hippocampal slices [3]. Additionally, optogenetic activation of the metabotropic receptor during behavioral tests in vivo restored cognitive functions in mice with an Alzheimer’s disease model. The activation of the Gq-coupled metabotropic receptor was found to be a molecular target that promotes positive changes in neuronal functioning at ex vivo and in vivo levels in both wild type mice and a mouse model of Alzheimer’s disease. Expression of OPTO-α1AR in astrocytes could potentially have a beneficial impact on other neuropathological conditions. In the future, alternative less-invasive methods, such as chemogenetics, could be employed to specifically activate astrocytes in distinct brain regions.

Single-fluorophore genetically encoded calcium indicators (GECIs), such as GCaMP, are widely used tools to investigate neuronal activity. Their primary advantage lies in their capability to provide real-time and highly sensitive responses to fluctuations in intracellular Ca2+ concentrations. This property is of particular importance when studying neuronal processes and ensembles wherein calcium signals play a crucial role in information transmission. This comprehensive review focuses on the GCaMP family, encompassing an analysis of their various types, distinctive features, and potential applications for visualizing neuronal activity. Special attention is given to the ongoing advancements in GCaMP technology, specifically, the endeavors to expand their spectral properties and enhance their capability to detect high-frequency spike activity.

Calcium (Ca2+) imaging is a commonly utilized neuroscience technique for in vivo recording of neuronal activity. It involves the optical measurement of calcium concentration using genetically encoded calcium indicators (GECI) [1]. However, the kinetics of changes in the fluorescence of GECI are relatively slow and limited by the biophysics of the calcium binding [2]. In response to single action potentials (APs) in pyramidal neurons, most of the widely used GECI have a fluorescence half-life of approximately 100 ms [3]. As a result, GECI cannot provide complete information about the dynamics of neural ensembles. To address this issue, new variants of GECI, such as jGCaMP7 [3], and jGCaMP8 [4], have been developed, or genetically encoded voltage indicators (GEVI), such as JEDI-2P [5], have been utilized. However, the speed of GECI or GEVI is still lower than that of electrophysiological registration methods. Thus, we have designed a microelectrode that can be utilized with a gradient lens for in vivo calcium imaging with a miniscope. The miniscope is a miniature microscope for single photon epifluorescence Ca2+ imaging, which enables recording of neuronal activity in freely moving laboratory animals, unlike the traditionally used two-photon imaging technique. The miniscope utilizes gradient-index (GRIN) lenses that are implanted directly into the brain of a laboratory animal, instead of a conventional lens. The gradient lens is a transparent cylinder with a diameter of 1.8 mm and a length of 3.8 mm. To facilitate electrophysiological recording, we developed a microelectrode that can be aligned with a GRIN lens. The microelectrode is a three-layer structure consisting of: 1) a polyimide film, 2) conductive copper tracks deposited through thermoforming, and 3) a polyimide film with cutouts for pads. On one side of the microelectrode, there are 12 gold-plated conductive contacts for registering local field potentials, while on the other side, a similar number of conductive tracks are present for connecting to a connector that transmits data to the processing board. The flexible microelectrode is wrapped around a gradient lens and fixed using thermoforming, after which it is implanted in the animal's brain. Using the developed microelectrode, our aim is to perform a comparative analysis of the calcium and electrophysiological activity of hippocampal neurons in freely moving wild-type mice and in a mouse model of Alzheimer's disease. This study will enable the identification of any abnormalities in Alzheimer's disease at the level of neural ensembles and may suggest new treatment approaches or mechanisms for the development of the progressive memory loss pathology associated with this disease. We would like to express our gratitude to Anastasia Viktorovna Bolshakova for her administrative assistance, and to the staff of the Laboratory of Molecular Neurodegeneration for their invaluable help and advices.

Astrocytes are most abundant glial cells in the central nervous system that reside between the microvascular network of the brain and neuronal synapses, thus mediating the absorption of nutrients from the systemic circulation. In addition, due to their unique anatomical location, astrocytes have a high enzymatic capacity for glycolysis, glycogenesis and lipid metabolism. This makes it possible to provide neurons with necessary nutrients as a source of energy, indicating a pivotal role of astrocytes in brain metabolism. Therefore, dysfunction of astroglia can lead to the development of neurodegenerative diseases in which metabolic disturbances speed up neuronal damage. Given the important role of astrocytes in the regulation of brain homeostasis and their close metabolic relationship with neurons, we address here the plasticity of astrocyte energy metabolism under physiological conditions and its effect on brain functions during the development of neurodegenerative diseases. A deeper insight into the mechanism underlying astrocyte metabolic plasticity will help identify novel potential diagnostic biomarkers and therapeutic targets to correct neurodegeneration and age-related brain dysfunctions.

A miniature fluorescence microscope (miniscope) is a promising tool for visualizing in vivo neuronal activity in behaving animals. The data obtained by a miniscope contain massive amounts of information about neuronal activity. However, the extraction and analysis of these data are complex tasks. There are various difficulties in the processing of data obtained by a miniscope, both in the extraction of neural activity data and in the subsequent analysis. A software page, constrained nonnegative matrix factorization for microendoscopic data (CNMF-E), was developed previously to assist in the processing of miniscope data. In this paper, we present a novel software package, NeuroInfoViewer (NIV), for high-level analysis and visualization of miniscope data following initial processing. We present an example of the analysis data flow, from raw miniscope imaging data to CNMF-E and to NIV. We suggest that NIV may serve as a useful tool for high-level analysis of miniscope data and we have deposited NIV in the public domain to facilitate its use by the neuroscience community.

The method of optogenetics has spread widely in neurobiology over the past 10 years and has found extensive application in various fields of this sciences. It allows to control and regulate cellular activity with high spatial and temporal resolution. In this study, optogenetic activation was applied to astrocytes expressing ChR2. Optogenetic stimulation parameters were determined, in which the frequency of spontaneous currents of hippocampal pyramidal neurons significantly changed. In the future, it is planned to use the obtained data on the modes of optogenetic stimulation of astrocytes to normalize the functions of the hippocampus in mice-models of Alzheimer’s disease.

Huntington’s disease is a hereditary, incurable, neurodegenerativedisease characterized by movement disorders—progressive choreichyperkinesia, as well as cognitive and mental disorders, includingmemory impairment, depression, panic attacks, obsessive compulsions,etc. According to the literature data, mild cognitive impairmentsbegin to manifest even before the appearance of first motor symptoms.Neurodegeneration of the cortex and striatum is believed to playa major role in the development of cognitive dysfunction. At thesame time, pathological changes in the hippocampus, which can alsocause cognitive impairments, have been studied to a much lesser extent.In the present study, using electrophysiological experiments, morphofunctionalanalysis, and behavioral tests, we performed a comprehensive assessmentof hippocampus-associated changes in YAC128 transgenic mice whichmodel Huntington’s disease. The revealed disturbances in the mechanismsof synaptic plasticity and changes in the morphology of synapsesin the hippocampus of YAC128 mice are progressive and occur beforemotor movement disorders. Thus, the obtained results support thehypothesis of the development of neurodegenerative changes in the hippocampus,which contribute to cognitive dysfunction in Huntington’s disease.

Astrocytes are most abundant glial cells in the central nervous system that reside between the microvascular network of the brain and neuronal synapses, thus mediating the absorption of nutrients from the systemic circulation. In addition, due to their unique anatomical location, astrocytes have a high enzymatic capacity for glycolysis, glycogenesis and lipid metabolism. This makes it possible to provide neurons with necessary nutrients as a source of energy, indicating a pivotal role of astrocytes in brain metabolism. Therefore, dysfunction of astroglia can lead to the development of neurodegenerative diseases in which metabolic disturbances speed up neuronal damage. Given the important role of astrocytes in the regulation of brain homeostasis and their close metabolic relationship with neurons, we address here the plasticity of astrocyte energy metabolism in physiological conditions and its effect on brain functions during the development of neurodegenerative diseases. A deeper insight into the mechanism underlying astrocyte metabolic plasticity will help identify novel potential diagnostic biomarkers and therapeutic targets to correct neurodegeneration and age-related brain dysfunctions.

Modern methods of fluorescence microscopy allow recording the activity of neurons in vivo, but their use has a number of serious limitations, specifically: the need of fixation an experimental animal, low scanning speed, complexity and high application cost. Most of these problems were solved after the appearance of single -photon miniature fluorescent microscopes (miniscopes), due to their small size, good resolution and the possibility of brain imaging in freely moving animals. This review examines variations and components of miniscopes, as well as discusses the ability of visualizing neuronal activity in vivo. Special attention is paid to the methods of processing data of neuronal activity in vivo: frames filtering from noise, compensation of shifts and distortions of the image. The methods of image processing of active neurons and interaction analysis of correlating neurons during an experimental series using the custom plugin are also described.

We report here a comparative (using chemical, electrical, and optical stimulation) study of the electrophysiological properties of cultured hippocampal neurons from transgenic animals expressing a mutant presenilin 1 protein. All three types of stimulation were found to elicit the following differences from the wild type: with overall more active electrophysiological behavior (especially at the beginning of stimulation), mutant cells differed from wild-type neurons by not maintaining consistent activity over time. However, only optogenetic stimulation increased the absolute number of action potentials and decreased their amplitude as compared with the corresponding values in wild-type neurons. The molecular-cellular mechanisms of this effect are suitable for further studies, both basic and applied (to identify regimes for controlling the electrical activity of neurons).

This article presents a basic scheme for a multichannel combined optoelectrode microimplant using feedback developed by the authors. An algorithm for stimulation and recording of neuron responses able to adjust the command signals to the light source and an optional system for administration of pharmacological compounds are described. The device can be used for autonomous adaptive optogenetic stimulation in chronic experiments on freely mobile animals, and has potential for use in treating patients. This report provides a detailed description of the preparation of a combined optoelectrode microimplant (optrode). The main advantage of this development is the ability to combine the stimulating and recording parts of the optogenetic system with adaptive control of light source parameters without the system being permanently connected to a personal computer. The system will later be tested on slices and in freely mobile animals in studies of the electrophysiological characteristics of hippocampal neurons in transgenic mice with different models of neurodegenerative diseases.

One of the promising options for treating pharmacoresistant forms of epilepsy is low-frequency stimulation of the brain. However, to avoid the side effects, it is required to minimize the stimulation of the brain. This can be achieved if stimulation occurs only at the time of increased probability of the ictal discharge and if it has a specific action on only one type of cells. A way to meet these conditions is to use a closed-loop system between the nervous tissue and a computer in the optogenetic experiment. We have developed a closed-loop system in the optogenetic experiment for effective and harmless suppression of epileptic activity and implemented it in slices of the rat hippocampus and the entorhinal cortex as well as in the primary hippocampal culture. First, we tested the closed-loop system in slices in 4-aminopyridine model of epilepsy. Epileptic activity was detected automatically via monitoring of spiking activity of a representative neuron in the entorhinal cortex. Since a neuron is firing only in response to synaptic input in the used epilepsy model, the spike threshold is a valid indicator of the beginning of epileptic discharge. The software realizing the closed-loop system starts the low-frequency stimulation in response to detection of an epileptic event. Next, we tested the developed system in an optogenetic experiment in culture after expression of channelrhodopsins. These results indicate the potential effectiveness of this approach for the suppression of ictal events

In this paper we gave a comparative study of the various physico-chemical properties of the hippocampal neurons in cell culture model of Alzheimer's Desease (Presenilin 1 mutations). It is shown that chemical, electrical and optical effects increase neuronal excitability differently for wild-type and KI (PS1-M146V neurons). In contrast to wild type KI neurons were more excitable, especially at the beginning of stimulation, but over time, this activity has been decreasing. At the same time, only in the case of optogenetic stimulation, exceeding the absolute values of their numbers has been registered, which made it possible to reduce their effect on the corresponding indices in wild type neurons. Molecular-cellular mechanisms of this phenomenon can serve as a material for further studies, both fundamental and practical (determination of the regimes of regulation of the electrical activity of neurons).