Encoding environmental information is a primary focus in neuroscience, as environmental context is crucial for the spatial orientation of animals. Place cells in the hippocampus play a key role in forming the spatial map of the surrounding space by mapping targets, environmental objects, various landmarks, and boundaries. However, it was previously unclear whether specific mapping properties exist for contrasting environmental elements that do not physically obstruct animal movement. In this study, we examined the calcium activity of CA1 hippocampal neurons in mice using miniscope imaging, alongside their behavior in an arena with contrasting colored elements on the floor. Our findings indicate that the place fields of CA1 hippocampal cells are concentrated in areas of environmental heterogeneity, rather than in the individual elements themselves or in areas preferred by the mice. This suggests that the contrasting surface signals of the environment are represented by CA1 hippocampal neurons within the overall spatial pattern.

A detailed analysis of animal behavior is a crucial step in understanding how the activity of brain structures and individual cells mediate animal behavior. Neurons can be selective to both the internal states of the animal and features of the environment. However, it is challenging to divide the continuum of animal behavior into discrete behavioral acts. We present Sphynx, a software tool for extracting a wide range of behavioral variables from video recordings of animal behavior and correlating these acts with environmental features. We demonstrate that detailed behavioral analysis using Sphynx reveals selectivity of neurons.

This study investigates the dynamics of non-spatial specializations in hippocampal place cells during exposure to novel environments. Hippocampal place cells, known for their role in spatial mapping, exhibit multi-modal responses to sensory cues. The research focuses on understanding how these cells adapt their specialization in response to novel stimuli, specifically examining non-spatial determinants such as odors and social interactions. Using a social-driven food odor recognition model in mice, the study records CA1 hippocampal neuron activity through miniscope imaging. The experimental design involves demonstrations of novel odors to mice, followed by observation sessions with food options. The analysis employs deep neural network tools for behavior tracking and the custom-developed INTENS software package for identifying neural specializations. Results indicate multiple specializations, particularly those related to odor, with differences observed between training and testing sessions. The findings suggest a temporal aspect to the formation of these specializations in novel conditions, necessitating further investigation for precise tracking.

We studied the population activity of hippocampal neurons in mice during novel arena exploration. To quantify the partial synchronization of neuronal activity, we calculated the effective dimensionality of a multidimensional time series of neuronal activity obtained using in vivo using calcium imaging. The effective dimensionality was calculated from the spectra of correlation matrices subjected to the consistent bias correction procedure. Applying this algorithm to mouse neural activity data, we found that the effective activity dimensionality of neuronal populations significantly increased during periods of animal stops. We attribute this finding to the presence of functional ensembles of neurons associated with movements, whose synchronised activity was disrupted during stops.

Of particular interest for researching the cognitive specializations of neurons is their correlation with environmental variables and animal behavior. Mutual information (MI) is a preferable method for measuring such correlations, as it allows for the assessment of non-linear relationships between variables, detects synchronization, and provides both significance and strength quantification. However, calculating MI for real data is significantly challenging. In this study, we used updated MI calculation techniques to analyze the connection between calcium fluorescence signaling and behavioral variables. Our approach encompasses novel strategies which we compiled into a software program known as INTENS (Information-Theoretic Evaluation of Neuronal Specializations), and it enabled to identify specialized neurons in mice hippocampal calcium activity data while they explored the arena with varying levels of novelty. Numerous methods exist for analyzing the relationship between neuron spikes and behavioral variables, including information-theoretical approaches [1]. Extracting information about the relationship between calcium fluorescent signals and behavior is of particular interest due to the signal’s ability to provide crucial information about subthreshold activations of the neuron. In this study, we use the GCMI Gaussian copula entropy method to calculate mutual information [2]. This method relies on the fact that mutual information between two random variables is independent of their marginal distributions and only depends on the type of copula used (a multidimensional distribution where each marginal distribution is uniform). The actual MI was compared to its corresponding values computed on the time-shifted signals for assessing the statistical significance of the computed information association between the calcium signal and the behavioral variable. Additionally, we devised a technique for gauging the strength of the coupling effect. This involved normalizing the mutual information between the fluorescence signal and the behavior with the entropy value of both variables, previously calculated as random variables. Importantly, the approach outlined earlier is effective for analyzing continuous variables such as calcium signal and animal speed, as well as pairs of continuous and discrete variables such as calcium signal and the presence or absence of grooming. The analysis of calcium signals recorded from the CA1 region of the hippocampus revealed neuronal specializations related to the animal’s external environment, such as place cells, and specializations related to its behavioral activities, including neurons activated during running, rearing, and freezing. Some neurons selectively activated in response to discrete parameters included the animal’s location within the arena (center, walls, and corners) and its speed (rest, slow, and fast). A total of 781 specializations were detected across 472 neurons throughout all four sessions of the experiment. Notably, a single neuron could have several specializations. However, more than half (55%) of the neurons were found to have only one specialization.

It is known that in the process of memory formation for new experiences, neurons in many regions of the brain are activated. In particular, neurons of the CA1 area of the hippocampus are activated during memory formation when animal first observe a new context [1]. However, it is not quite clear how the activity of CA1 neurons changes during memory formation and retrieval. Moreover, the question remains what changes in neuronal activity are observed in old animals during memory formation and retrieval. In our work, we investigated changes in the calcium activity of hippocampal CA1 neurons during the formation and retrieval of associative memory of the context model of context preexposure facilitation effect in young and aged mice. Calcium activity of individual neurons was recorded using a miniature microscope (miniscope), which allows optical detection of active neurons through the fluorescence of the calcium sensor. For this purpose, the mice underwent stereotactic surgery in which the calcium fluorescent sensor NCaMP7 was injected into the CA1 area of the hippocampus [2]. Then, a 0.5-mm-diameter GRIN lens was implanted into the studied area, and miniscope mounts were placed on the mouse head. The experiment was performed one week after the surgery. On the first day, the procedure of pretraining was performed: the mice were placed in a new context for 5 minutes free exploration, as a result of which the mice formed a spatial perception of the context. Three days later, the mice were briefly placed in the same context and immediately received footshock for 2s (1.5mA). Thus, the previously formed perception of the context was associated with the animal's state of fear. We tested associative memory three days after shock application: mice were placed in the same context for 5 minutes. A measure of formed associative memory was their level of freezing behaviour in the context. Young mice showed a low level of freezing on the first visit of the context. We also observed a low level of freezing in older mice on the first day of the experiment. At the same time, the retrieval of a previously formed memory significantly increased the level of the freezing in older mice, compared with the first day, indicating that this animal had formed an associative memory of the context. To analyze changes in calcium activity during memory formation and retrieval, we assessed changes in the level of neuronal activity in each individual animal freezing act. Calcium activity of individual CA1 area neurons was recorded during the first visit to the environment and during memory retrieval. In each mouse, about 20 neurons were recorded in the two groups under study in two sessions of the experiment. However, we did not find any significant changes in the number of active neurons in young and old animals at the moments of their fading into the environment. The results indicate that the processes of associative memory formation and retrieval do not manifest themselves in changes in the number of active cells at the moment of the animal freezing behaviour. Possibly, the studied processes of memory formation and retrieval are reflected in other forms of brain activity, such as cognitive maps of the context, which represent a network of cognitively specialized neurons (place fields).

A thorough analysis of animal behavior is essential for examining the relationship between specific neuron activations with the elements of the external environment, behavior, or internal state. Machine learning techniques have made some advancements in automatic segmentation of animal behavior based on data concerning the location of animal body parts [1–3]. At present, these methods cannot achieve the level of segmentation accuracy desired or make correlations between an animal’s behavioral acts and key environmental factors. To address this issue, the authors have created a software package that can extract a variety of behavioral variables from video recordings of animals in experimental settings, enabling mathematical analysis of a behavioral act continuum. The identification of specific aspects of an animal’s anatomy is crucial for extracting a vast array of behavioral variables. In order to accomplish this task, our team employed DeepLabCut, an accessible toolkit for tracking experimental animal behavior that operates on the principle of transfer learning through deep neural networks. We have devised a technique to ascertain the positions of animal body parts in diverse behavioral situations, resulting in a body parts collection meeting two criteria: offering superior responsiveness to small motor movements of the animal and delivering a high percentage of correct body part locations. In scenarios employing camera shooting from above, such a collection encompasses the nose, ears, tail base, body center, forelimbs, hind limbs, and both flanks of the animal’s body. Next, we created software tools to extract and annotate behavioral variables from data on animal kinematics in various cognitive tasks. Our automated system comprises two main scripting modules: CreatePreset and BehaviorAnalyzer. The CreatePreset module interacts with users to select the type of arena geometry, object location, and necessary temporal and spatial parameters for analysis. The script’s result saves as a mat-file for analyzing the behavior of all experiment videos, assuming a constant relative position of the arena and the video camera alongside the experiment’s design. The BehaviorAnalyzer module conducts initial processing on time series data consisting of coordinates of an animal’s body parts. This results in the formation of a kinematogram, which details the kinematics of the body parts. The module then isolates individual behavioral acts of the animal and annotates its behavior based on motivational and environmental factors. Using mutual information-based methods, we analyzed the specialization of hippocampal CA1 neurons in animals as they explored arenas with varying degrees of novelty. Through the analysis, we have identified neurons that exhibit selectivity in relation to specific continuous kinematic parameters governing the posture and trajectory of the animal. These parameters include the animal’s location in the arena space (X and Y coordinates), as well as the speed and angle of rotation of the animal’s head (i.e. absolute orientation in the arena). Neurons specialized in discrete acts of behavior were identified, including rests, locomotions, freezing, rears, and acts of interaction with objects. Furthermore, a selective activation of neurons was found with regard to an additional set of distinct parameters, which combine the animal’s location in the arena and its speed.

Hippocampal place cells are a well-known object in neuroscience, but their place field formation in the first moments of navigating in a novel environment remains an ill-defined process. To address these dynamics, we performed in vivo imaging of neuronal activity in the CA1 field of the mouse hippocampus using genetically encoded green calcium indicators, including the novel NCaMP7 and FGCaMP7, designed specifically for in vivo calcium imaging. Mice were injected with a viral vector encoding calcium sensor, head-mounted with an NVista HD miniscope, and allowed to explore a completely novel environment (circular track surrounded by visual cues) without any reinforcement stimuli, in order to avoid potential interference from reward-related behavior. First, we calculated the average time required for each CA1 cell to acquire its place field. We found that 25% of CA1 place fields were formed at the first arrival in the corresponding place, while the average tuning latency for all place fields in a novel environment equaled 247 s. After 24 h, when the environment was familiar to the animals, place fields formed faster, independent of retention of cognitive maps during this session. No cumulation of selectivity score was observed between these two sessions. Using dimensionality reduction, we demonstrated that the population activity of rapidly tuned CA1 place cells allowed the reconstruction of the geometry of the navigated circular maze; the distribution of reconstruction error between the mice was consistent with the distribution of the average place field selectivity score in them. Our data thus show that neuronal activity recorded with genetically encoded calcium sensors revealed fast behavior-dependent plasticity in the mouse hippocampus, resulting in the rapid formation of place fields and population activity that allowed the reconstruction of the geometry of the navigated maze.

The emergence of neuronal specializations is the key question in modern neuroscience. In our previous study we estimated the basic parameters of the emergence of spatial representations (place fields) of a novel one-dimensional environment in mice during a free navigating task. However, in one-dimensional environments place fields may show direction specificity which observation was obscured by arbitrariness of animals trajectory. Here we expanded our approach onto a two-dimensional environment with a complex topology and compared the properties of the emergence of place fields such as selectivity score, tuning latency and the amount of “instant” place fields with our previous results. As expected, all basic parameters in 2D environment indicated rapid dynamics of place field tuning, similar to 1D case, including gradual decreasing of the tuning latency of place fields within familiarization to a novel environment and notable (11%) amount of “instant” place fields.

A detailed analysis of animal behavior is a necessary step in understanding how the activity of brain structures and individual cells mediates animal behavior. The goal of the present study was to create a software tool that allows to extract information about the behavior of an animal in the fear conditioning paradigm.

Hippocampal place cells are a well-known object of plenty of studies about learning and memory. Their firing patterns, or place fields, constitute cognitive maps that support animal's navigation in space. However, most of the place cell studies used rewarded models, where animals were pre-trained to run back and forth for food or water reward. Thereby a question is still unclear about the speed of formation of such cognitive maps in a novel context, i.e., which time is required for place cells to tune on a special piece of the environment. To address this question and to exclude the influence of explicitly reinforced behavior, we developed a reward-free model where animals can explore a novel environment (custom made circular O-shaped track with visual cues on surrounding curtains, see Fig. 1А). Then we recorded the neural activity from the hippocampal field CA1 of mice with a head-mounted Inscopix nVista HD miniature microscope (see Fig. 1B), while the animals were placed in the track, which was a completely novel environment for them. We aligned these data with trajectories of mice and determined place cells as neurons that have a preferential activation zone (putative place field) and that activated in most of the moments the animal visited the field (i.e., more than in 50% of such cases). As expected, place fields were distributed uniformly across the track, without significant gaps and clusters which might be caused by the proximity of visual cues (Fig. 1C-1D). Then, we estimated the time which is required for each individual cell to begin firing with respect to its place field. The majority of cells got tuned within the first three minutes of context exploration, the distribution of times of first in-field activation can be seen on Fig. 1E. However, different animals demonstrate different exploration dynamics, so we re-calculated this distribution in terms of full laps along the circular track. More than 1/3 of cells got tuned within the first lap, while more than 60% of cells do that within the first three laps. Taken together, these data suggest that a cognitive map is mainly formed while an animal makes it first laps in a novel environment, which is consistent with previous findings in rats. Also, the obtained data should serve as a base for searching for possible behavior or intrinsic triggers for cognitive map formation in a novel environment.

Neuronal encoding of environmental information has long been in the focus of neuroscience. Neurophysiological studies showed that neuronal activity changes specifically when animals become familiar with space or objects. However, despite a considerable amount of experimental data, the specific neural basis of place and object encoding remains an open question. The goal of the present study was to examine how the hippocampus encodes information about the place and various types and values of objects at the level of individual neurons, and also to determine stability and variability of cognitive maps over time and the presentation of novel objects.

Genetically encoded calcium indicators (GECIs) have become a widespread tool for the visualization of neuronal activity. As compared to popular GCaMP GECIs, the FGCaMP indicator benefits from calmodulin and M13-peptide from the fungi Aspergillus niger and Aspergillus fumigatus, which prevent its interaction with the intracellular environment. However, FGCaMP exhibits a two-phase fluorescence behavior with the variation of calcium ion concentration, has moderate sensitivity in neurons (as compared to the GCaMP6s indicator), and has not been fully characterized in vitro and in vivo. To address these limitations, we developed an enhanced version of FGCaMP, called FGCaMP7. FGCaMP7 preserves the ratiometric phenotype of FGCaMP, with a 3.1-fold larger ratiometric dynamic range in vitro. FGCaMP7 demonstrates 2.7- and 8.7-fold greater photostability compared to mEGFP and mTagBFP2 fluorescent proteins in vitro, respectively. The ratiometric response of FGCaMP7 is 1.6- and 1.4-fold higher, compared to the intensiometric response of GCaMP6s, in non-stimulated and stimulated neuronal cultures, respectively. We reveal the inertness of FGCaMP7 to the intracellular environment of HeLa cells using its truncated version with a deleted M13-like peptide; in contrast to the similarly truncated variant of GCaMP6s. We characterize the crystal structure of the parental FGCaMP indicator. Finally, we test the in vivo performance of FGCaMP7 in mouse brain using a two-photon microscope and an NVista miniscope; and in zebrafish using two-color ratiometric confocal imaging.

Green fluorescent genetically encoded calcium indicators (GECIs) are the most popular tool for visualization of calcium dynamics in vivo. However, most of them are based on the EGFP protein and have similar molecular brightnesses. The NTnC indicator, which is composed of the mNeonGreen fluorescent protein with the insertion of troponin C, has higher brightness as compared to EGFP-based GECIs, but shows a limited inverted response with an ΔF/F of 1. By insertion of a calmodulin/M13-peptide pair into the mNeonGreen protein, we developed a green GECI called NCaMP7. In vitro, NCaMP7 showed positive response with an ΔF/F of 27 and high affinity (Kd of 125 nM) to calcium ions. NCaMP7 demonstrated a 1.7-fold higher brightness and similar calcium-association/dissociation dynamics compared to the standard GCaMP6s GECI in vitro. According to fluorescence recovery after photobleaching (FRAP) experiments, the NCaMP7 design partially prevented interactions of NCaMP7 with the intracellular environment. The NCaMP7 crystal structure was obtained at 1.75 Å resolution to uncover the molecular basis of its calcium ions sensitivity. The NCaMP7 indicator retained a high and fast response when expressed in cultured HeLa and neuronal cells. Finally, we successfully utilized the NCaMP7 indicator for in vivo visualization of grating-evoked and place-dependent neuronal activity in the visual cortex and the hippocampus of mice using a two-photon microscope and an NVista miniscope, respectively.

Neuronal encoding of environmental information has long been in the focus of neuroscience. Neurophysiological studies show that when animals become familiar with space or objects, neuronal activity changes due to these factors. However, despite a considerable amount of experimental data, the specific neural bases of space and objects encoding, and the nature of their disturbance in aging or pathology remains an open question. In this work, we test the hypothesis that neurons in different regions of the brain, hippocampus and retrosplenial cortex (RSC) encode spatial and object information respectively, and that natural aging impairs these two forms of memory differently. To test this hypothesis, we developed an approach combining optical recording of neuronal activity and assessment of animal behavior during novel object recognition (NOR) and object place recognition (OPR) tasks, as well as cognitive enhancement in young and old animals. First, we examined what forms of memory are affected in old mice, and whether it is possible to restore the age‐impaired memory pharmacologically. We trained young (2–3 months) and old (18–21 months) mice in NOR and OPR tasks and then tested short‐term 120 min memory and long‐term 24 h memory. We showed that both young and old mice demonstrate short‐term NOR and OPR memories, and that P2 cognitive enhancer that belongs to a new class of sAPP‐mimetics did not potentiate this memory. On the other hand, both old and young mice failed to form NOR and OPR long‐term memories. Interestingly, administration of P2 produced long‐term memory manifestation only in the old mice.Next, we recorded the object‐type and object‐place related calcium activity in the RSC and hippocampus CA1 of young mice using genetically encoded YtnC sensor and fiber‐optic photometry (Fig. 1, A). We found an increase in the number of calcium events in the RSC when animals examined new position of the familiar object. We also discovered decrease in the RSC activity during exploration of a novel object.Using multiphoton microscopy and NVista minimicroscopy we registered GCaMP6 calcium activity of identified RSC (Fig. 1, B) and CA1 (Fig. 1, C) neurons in young mice during NOR and OPR tasks performed in the Mobile HomeCage. Different classes of neurons were identified – those demonstrating an increase and decrease of activity during place and objects exploration, as well as cells that were active during specific behavioral acts.Thus, we showed that both object‐type and object‐place memory was specifically expressed in the activity of RSC and CA1 neurons. Both object‐type and object‐place long‐term memories were missing in the old mice but were brought to the surface by administration sAPP‐mimetic cognitive enhancer. In the future, we will test how memory‐related brain activity changes in NOR and OPR tasks in old animals, and whether these changes in neuronal activity could be compensated pharmacologically.Support or Funding InformationSupported by RSF 20‐15‐00283 and RFBR 17‐00‐00215, 19‐315‐80020, 17‐29‐07083, 18‐32‐20212Neuronal calcium activity in novel object recognition and object place recognition tasks. A: fiber‐optic photometry of YtnC calcium signal from retrosplenial cortex (RSC) and hippocampus CA1; red line – baseline. B: two‐photon microscopy of identified GCaMP6S neurons in the retrosplenial cortex; asterisks show calcium events – spikes. Ñ: NVista minimicroscopy of GCaMP6S neurons in hippocampus CA1; arrows show calcium spikes.Figure 1

Background: The recently developed genetically encoded calcium indicator (GECI), called NTnC, has a novel design with reduced size due to utilization of the troponin C (TnC) as a Ca2(+)-binding moiety inserted into the mNeonGreen fluorescent protein. NTnC binds two times less Ca2+ ions while maintaining a higher fluorescence brightness at the basal level of Ca2+ in neurons as compared with the calmodulin-based GECIs, such as GCaMPs. In spite of NTnC's high brightness, pH-stability, and high sensitivity to single action potentials, it has a limited fluorescence contrast (F-(Ca2+)/F+Ca2+) and slow Ca2+ dissociation kinetics. Results: Herein, we developed a new NTnC-like GECI with enhanced fluorescence contrast and kinetics by replacing the mNeonGreen fluorescent subunit of the NTnC indicator with EYFP. Similar to NTnC, the developed indicator, named iYTnC2, has an inverted fluorescence response to Ca2+ (i.e. becoming dimmer with an increase of Ca2+ concentration). In the presence of Mg2+ ions, iYTnC2 demonstrated a 2.8-fold improved fluorescence contrast in vitro as compared with NTnC. The iYTnC2 indicator has lower brightness and pH-stability, but similar photostability as compared with NTnC in vitro. Stopped-flow fluorimetry studies revealed that iYTnC2 has 5-fold faster Ca2+ dissociation kinetics than NTnC. When compared with GCaMP6f GECI, iYTnC2 has up to 5.6-fold faster Ca2+ association kinetics and 1.7-fold slower dissociation kinetics. During calcium transients in cultured mammalian cells, iYTnC2 demonstrated a 2.7-fold higher fluorescence contrast as compared with that for the NTnC. iYTnC2 demonstrated a 4-fold larger response to Ca2+ transients in neuronal cultures than responses of NTnC. iYTnC2 response in neurons was additionally characterized using whole-cell patch clamp. Finally, we demonstrated that iYTnC2 can visualize neuronal activity in vivo in the hippocampus of freely moving mice using a nVista miniscope. Conclusions: We demonstrate that expanding the family of NTnC-like calcium indicators is a promising strategy for the development of the next generation of GECIs with smaller molecule size and lower Ca2+ ions buffering capacity as compared with commonly used GECIs.

The NTnC genetically encoded calcium indicator has an advantageous design because of its smaller size, GFP-like N- and C-terminal ends and two-fold reduced number of calcium binding sites compared with widely used indicators from the GCaMP family. However, NTnC has an inverted and modest calcium response and a low temporal resolution. By replacing the mNeonGreen fluorescent part in NTnC with EYFP, we engineered an NTnC-like indicator, referred to as YTnC, that had a positive and substantially improved calcium response and faster kinetics. YTnC had a 3-fold higher calcium response and 13.6-fold lower brightness than NTnC in vitro. According to stopped-flow experiments performed in vitro, YTnC had 4-fold faster calcium-dissociation kinetics than NTnC. In HeLa cells, YTnC exhibited a 3.3-fold lower brightness and 4.9-fold increased response to calcium transients than NTnC. The spontaneous activity of neuronal cultures induced a 3.6-fold larger ΔF/F response of YTnC than previously shown for NTnC. On patched neurons, YTnC had a 2.6-fold lower ΔF/F than GCaMP6s. YTnC successfully visualized calcium transients in neurons in the cortex of anesthetized mice and the hippocampus of awake mice using single- and two-photon microscopy. Moreover, YTnC outperformed GCaMP6s in the mitochondria and endoplasmic reticulum of cultured HeLa and neuronal cells.

Cognitive maps are known as spatial patterns of activation of place cells, i.e. neurons that selectively fire only in certain spatial areas, which provide the navigation of animals in space. However, the dynamics of the formation of such maps when an animal is for the first time placed in a new environment remains mostly unknown. The technique of calcium in vivo imaging using head-mounted miniscopes allows the recording of a large number of neurons with cellular resolution from various brain structures of behaving animals without restrictions. In particular, this approach makes it possible to record the neural activity of the hippocampus in a model of free exploratory behavior in a new context. In this study, we obtained the neural activity of mice in a custom made circular track. Based on these data, the time series of place cell activity were reconstructed and the place fields constituting cognitive maps were identified. The properties of these cognitive maps were analyzed - specifically, it was found that place fields in the circular track are evenly distributed, without reference to the proximity of the prominent landmarks of the context. It was shown that the emergence of stable neural responses is relatively rapid and that most of the place fields emerge during the first three visits to the place field. These results provide important information for identifying cause-effect relationships between individual behavioral acts and the emergence of spatial specialization of neuronal ensembles.