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.

Animal behavior is driven by the simultaneous activation of neurons within networks that are distributed across various brain areas. The complexity of neuronal networks, involving various brain regions and different types of neurons, necessitates the ability to record the activity of specific neuronal subpopulations simultaneously. For this purpose the multi-fiber photometry systems have been proposed in several previous studies. Here we developed a new optical probe that combines two optic fibers with different length. We implanted the probes bilaterally addressing two different brain area in each hemisphere. We further applied this technique for calcium activity registration simultaneously in four brain areas during associative learning. We showed that the basolateral amygdala and primary somatosensory cortex become more active towards a previously neutral stimulus, implying their roles in associative memory formation during learning. Altogether, we showed the new optic-fiber probe for four-site calcium activity registration in brain of behaving mouse.

In the present study, we analyzed the differential involvement of hippocampal interneurons and pyramidal neurons in the retrieval of associative aversive context memory. For this purpose, we used a model of associative learning in which the formation of a neutral context memory and the subsequent association of this memory with the footshock US during a brief reminder of the context were significantly separated in time. The activation of hippocampal neurons during associative context memory retrieval in this task was addressed by immunohistochemical detection of the immediate early gene c-fos protein. Retrieval of associative context memory was accompanied by an increase in the number of c-Fos-positive cells in the CA1 region, but not in the CA3 region and the dentate gyrus of the hippocampus. Next, a protein marker, the product of the homeobox-containing gene Emx1, was used to specifically identify excitatory neurons, and the marker glutamate decarboxylase, GAD, the product of the GAD1 and GAD2 genes, was used to specifically identify inhibitory neurons. The results of double staining for cell markers and c-Fos protein showed that during retrieval of associative aversive context memory in the CA1 region of the hippocampus, both Emx1-positive excitatory neurons and, less, GAD-positive inhibitory interneurons were activated. At the same time, regardless of the type of behavioral procedure (retrieval of associative context memory, non-associative context memory, or exploration of context, where animals previously received the footshock but did not remember it), the proportion of activated excitatory and inhibitory neurons remained constant, only the number of activated cells of each type changed. Altogether, our results indicate the specific role of hippocampal CA1 neurons in associative context memory and demonstrate that both excitatory and inhibitory neurons are involved in the encoding of such memory.

A weak memory can be converted into a long-term form if it is preceded by another weak training episode or exploration of a new environment. Current hypotheses explain the mechanisms behind this phenomenon, suggesting that the formation of new memories involves a population of neurons that are pre-activated by previous cognitive experiences. We visualized neuronal activity from two distinct cognitive episodes separated in time using transgenic Arc-Cre-Tomato mice. Mice were trained using a weak cued fear conditioning protocol that typically does not lead to long-term memory formation. However, when re-trained with the same cue, significant memory improvement was observed. In contrast, different cues did not produce this effect. Our analysis revealed a higher percentage of overlapping neuronal populations in the prelimbic cortex, primary auditory cortex, and amygdala in mice trained with the same cue. These findings suggest that cumulative learning and memory potentiation depend on the involvement of overlapping neuronal populations and only occur when these events engage the same groups of brain neurons.

This study investigates the neuronal mechanisms underlying configural associative learning to complex conditioned stimuli (CS) and elemental learning to discrete CSs in mice. Using a fear conditioning model, mice were trained with a complex CS consisting of light and sound stimuli or discrete light and sound CSs. Fos imaging was employed to analyze brain activity in primary sensory cortical areas and associative cortical areas, as well as in amygdala during training. Results indicate that associative areas of the neocortex, such as the prelimbic cortex, but not primary sensory areas or the amygdala, may be the site for specific configural processes during complex CS associative learning.

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.

We report here an analysis of the differential involvement of interneurons and pyramidal neurons of different hippocampal areas in the retrieval of associative aversive contextual memory. This was addressed using a model of associative learning in mice, where formation of a memory of a neutral situation and the subsequent association between short-term presentation of the context and an unconditioned stimulus in the form of footshock (FS) were significantly separated in time. Activation of neurons in different areas of the hippocampus during retrieval of the resulting associative contextual memory was studied by immunohistochemical detection of the protein product of the immediate early gene c-fos. Retrieval of associative memory of a context was accompanied by an increase in the number of c-Fos-positive cells in field CA1 but not in field CA3 or the dentate fascia of the hippocampus. Next, a protein marker – the product of the homeobox-containing gene Emx1 – was used for the specific detection of excitatory neurons and glutamate decarboxylase (GAD) – the product of the genes GAD1 and GAD2 – was used as a marker for the specific detection of inhibitory neurons. Results obtained by double staining for cellular markers and c-Fos protein showed that extraction of the associative aversive memory of the context led to activation of both excitatory neurons positive for the Emx1 marker and, to a lesser extent, inhibitory interneurons positive for the GAD marker in hippocampal field CA1. Independently of the type of behavioral impact (retrieval of associative memory of the context, non-associative memory of the context, or exploration of the context combined with FS but not remembered by the animals), the proportions of activated excitatory and inhibitory neurons remained constant, only the numbers of activated cells of each type changing. These results indicate a specific involvement of neurons in hippocampal field CA1 in associative contextual memory and also demonstrate that both excitatory and inhibitory neurons are involved in encoding this type of memory.

Posttraumatic stress disorder (PTSD) is a debilitating psychosomatic condition characterized by impairment of brain fear circuits and persistence of exceptionally strong associative memories resistant to extinction. In this study, we investigated the neural and behavioral consequences of inhibiting protein synthesis, a process known to suppress the formation of conventional aversive memories, in an established PTSD animal model based on contextual fear conditioning in mice. Control animals were subjected to the conventional fear conditioning task. Utilizing c-Fos neural activity mapping, we found that the retrieval of PTSD and normal aversive memories produced activation of an overlapping set of brain structures. However, several specific areas, such as the infralimbic cortex and the paraventricular thalamic nucleus, showed an increase in the PTSD group compared to the normal aversive memory group. Administration of protein synthesis inhibitor before PTSD induction disrupted the formation of traumatic memories, resulting in behavior that matched the behavior of mice with usual aversive memory. Concomitant with this behavioral shift was a normalization of brain c-Fos activation pattern matching the one observed in usual fear memory. Our findings demonstrate that inhibiting protein synthesis during traumatic experiences significantly impairs the development of PTSD in a mouse model. These data provide insights into the neural underpinnings of protein synthesis-dependent traumatic memory formation and open prospects for the development of new therapeutic strategies for PTSD prevention.

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.

The retention of information represents a defining trait of cognitive systems. The primary function of an organism’s memory is to retain engrams of unique experiences for extended periods, without interrupting memory trace during new learning processes. One of the cardinal unsolved issues in neuroscience lies in unveiling the mechanisms responsible for long-term memory. Despite this, a trustworthy experimental animal model for single-trial long-term memory remains undeveloped. To address this challenge, we aimed to create an animal model to study lifelong memory formation with a single trial. We used a mouse model of post-traumatic stress disorder (PTSD) for this purpose [1]. In this model, mice exposed to a powerful electrical foot shock develop highly persistent traumatic memories, which results in long-term behavioral changes [1]. Our hypothesis was that this model could uncover the mechanisms related to lifelong memory. The primary rationale for adopting the PTSD model as a model of lifelong memory pertains to the heightened durability of traumatic memories in comparison to conventional aversive memories [2]. Protein synthesis inhibitors, which are needed for long-term memory consolidation, can be used as amnesic agents to evaluate memory stability [3]. In a PTSD model using predator scent, the administration of a protein synthesis blocker prior to a traumatic experience disrupts the development of PTSD in mice [4]. However, it remains unclear how traumatic memory impairment affects PTSD development in the footshock model and whether the impairment persists long-term. Based on the evidence that the formation of PTSD necessitates the consolidation of associative memory, which is reliant on protein synthesis and coincides with changes in the stress response system, Siegmund and Watzhek [1] present a two-part proposal concerning the onset of PTSD. The two-part hypothesis regarding PTSD posits that PTSD formation involves sensory conditioning and sensitization processes that mutually reinforce one another. In line with this theorem, we considered the effects of studying context and exposure timing on the development of post-traumatic stress disorder, as it could potentially interfere with sensory conditioning formation [5]. The aim of this study is to create an experimental approach for developing enduring long-term memory through a single trial event on adult mice. We methodically analyzed behavioral expressions and the endurance of normal and traumatic fear memory, as well as their sensitivity to protein synthesis inhibition. Additionally, we investigated the effects of separating the timing of the associative and aversive elements of traumatic memory on PTSD development in a mouse model. The experiment involved male C57Bl/6 mice, aged between 3–4 months and 15–18 months (for an investigation into aged mice), which were placed in an electrified chamber. After 170 seconds, the mice experienced either one footshock (1.5 mA, 2 s) to elicit fear memory or three footshocks (1.5 mA, 10 s) for PTSD induction. After receiving the footshock, the mouse was held in the chamber for 60 seconds before being returned to its home cage. A memory test was conducted seven days later by placing the mice back in the same context. To assess the existence of standard PTSD symptoms, like sensitization and generalization, the creatures were exposed to an unfamiliar context and an unexpected auditory stimulus, respectively. In the experiment, animals were placed in unfamiliar or familiar safe contexts that differed from their previous ones. The duration of freezing was measured to assess fear and evaluate memory retention, sensitization, and generalization. Anxiety levels were evaluated using the elevated plus maze test. In the process of constructing a highly stable long-term memory model, we evaluated the behavioral performance of PTSD-induced (PTSD), fear-conditioned (FC), and active control (AC) groups of animals at 7 days, 1 month, and 3 months after exposure. As a result of PTSD induction, mice displayed increased fear levels for up to 3 months in the training context, along with heightened fear sensitization and generalization at 7 days following exposure, relative to the FC and AC groups. The PTSD group exhibited heightened freezing behavior within a month and a decreased number of entries to the closed arms during the initial three months following exposure when contrasted with the FC and AC groups. We additionally noted that the FC group displayed raised fear levels in contrast to the control animals up to 3 months post-exposure, albeit lower in magnitude than that of the PTSD group. The FC group, similar to the PTSD group, showed increased sensitization and generalization compared to control animals at 1 week post-exposure, but to a lesser extent than we observed in the PTSD group. The findings suggest that traumatic memory remains present for a minimum of three months, whereas fear memory in the fear conditioning paradigm diminishes in intensity during this time. Hence, PTSD induction effectively functions as an animal model of profoundly stable long-term memory, in opposition to the fear conditioning paradigm. As the high stable long-term memory model is designed for testing in senior animals, the impact of aging on traumatic memory formation and storage becomes an important inquiry at six months and one year following exposure. We analyzed the formation of both aversive and traumatic memory in mice aged 15–18 months, one week after footshock exposure. In the study involving older mice, the PTSD group exhibited increased levels of fear memory and sensitization when compared with both the FC and AC groups, in addition to displaying higher levels of generalization and anxiety when compared with the AC group. Furthermore, mice that underwent rear-conditioning showed heightened levels of fear during learning, fear sensitization, and generalization, although not anxiety. These findings demonstrate the formation of traumatic and aversive memory in aged mice, indicating that conducting memory tests six months and a year after exposure is an appropriate approach. To evaluate the durability of traumatic and aversive memory, we administered a protein synthesis inhibitor called cycloheximide (Chm) to interfere with memory formation. Mice were given a cycloheximide solution (95 mg/kg) via intraperitoneal injection 30 minutes before footshock (Chm-PTSD and Chm-FC groups) while control animals received a saline injection (Sal-PTSD, Sal-FC). After training, the Chm-FC group displayed a decrease in freezing rate when compared to the saline-injected group at the 7- and 30-day marks. The Chm-PTSD group exhibited fear levels comparable to those of the Sal-FC group in the training context, and did not display sensitization or generalization of fear. These findings suggest that inhibiting protein synthesis during memory formation resulted in complete amnesia for standard aversive memory and merely weakened traumatic memory. As a result, only the associative component of traumatic memory remained intact, while nonspecific symptoms of PTSD were absent. The strong resilience of memory in the PTSD model to severe disruptions, such as protein synthesis blockade, also confirms its stability, which facilitates exploration of lifelong memory mechanisms in the PTSD model. To examine the timing effect of the associative and aversive component of PTSD development, we evaluated traumatic memory formation when contextual memory formation was absent, and contextual exploration occurred three days before shock cessation. A week after exposure to immediate and intense footshock, mice in the experimental group exhibited lower levels of fear, reduced fear sensitization, and less anxiety compared to those in the PTSD-induced group. If an intense foot shock was preceded by a contextual study three days earlier, the animals exhibited lower levels of fear in an unfamiliar, safe context, and reduced anxiety compared to the mice induced with PTSD. In both cases, the fear level after the shock cessation was identical to that of the mice who experienced an immediate, moderate shock. Based on our findings, we concluded that aversive memory forms in the absence or prior formation of contextual memory in relation to traumatic exposure. However, PTSD induction does not occur under such circumstances. This indicates that contextual memory needs to be formed simultaneously with the traumatic event for the development of traumatic memory in the mouse model of PTSD. In summary, our findings indicate that aversive memory tends to fade over time, while traumatic memory remains stable for a minimum of 3 months following its induction. Therefore, PTSD proves to be a fitting candidate laboratory model for high, stable, and long-term memory. Aged mice aged between 15–18 months display the formation of traumatic memory and experience PTSD symptoms in a manner similar to adult animals aged between 3–4 months in the PTSD model. For the induction of PTSD in mice, contextual memory formation about the traumatic environment and the traumatic event occurrence may coincide, which is crucial.

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).

One of neurobiology’s primary objectives is to discover the mechanisms by which past experiences influence current behavior and learning. While it is acknowledged that previous cognitive experiences can facilitate the creation of new memories in both humans and animals, investigations into the behavioral and neuronal aspects of the phenomenon of new memory being dependent on past cognitive experiences remain to be explored. The aim is to evaluate the theory that the facilitating effects of prior experiences on the creation of new memories are solely evident if these occurrences involve shared groups of neurons in the brain. To this end, a novel method was devised to lightly instruct mice to instinctively freeze when presented with a cue by exposing them to a conditioned signal for a brief (5 seconds) period, followed immediately by a gentle electrodermal stimulation. We discovered that weak memory can be strengthened through repeated weak learning in cumulative learning, despite the lack of formation of long-term memory caused by weak learning itself. Strengthening of memory only occurs when the animal is trained on the same conditioned signal during both weak training times. On the other hand, if the conditioned signals used during the first and second weak training are insignificantly different, no memory formation will occur for either. However, memory reinforcement in cumulative learning depended on context, fully manifesting only when multiple training sessions occurred in the same environment. We examined if long-term memory formation of the conditioned signal during cumulative learning relied on the animals’ past experiences, which were subject to alteration through repeated training. We discovered that successful long-term memory formation for the conditioned signal occurred when two weak learnings were separated by more than 30 minutes (up to 30 days), but not when the interval between learnings was 30 seconds or 5 minutes. Thus, weak memory reinforcement in cumulative learning occurs only when the second training is separated from the first by a time interval sufficient to form a latent memory of the first. The data obtained suggest that weak training of the conditioned reflex freeze results in long-term plastic rearrangements in the brains of mice. These changes can be strengthened through repeated weak training to the same conditioned signal, resulting in the behavioral manifestation of memory. We then investigated the activity of different brain regions during cumulative learning. We demonstrated that repeated weak training elicits selective activation of mouse brain regions that are crucial for long-term memory formation, including associative cortical regions, amygdala, and hippocampus. Conversely, such activation was absent after a single weak training, where the brain activity was equivalent to the mice that received the conditioned stimulus without reinforcement and remained untrained. This study suggests that a weak single training can form a memory trace in the brain, which can be strengthened through repetition. However, identifying this trace at the level of entire brain structures appears to be unattainable. Therefore, we directly evaluated the main hypothesis of this study by examining the overlap of neuronal populations in transgenic Cre-lineage mice. We labeled neurons involved in the first weak learning with fluorescent protein expression and cells active in the second learning with immunohistochemical staining for the native Arc protein. It was demonstrated that during cumulative learning, over 30% of neurons in the prelimbic cortex, auditory cortex, and amygdala were reactivated, while only 10% of cells were reactivated when two weak learnings were performed on different conditioned signals that did not lead to the formation of memories. Consequently, the present study examines the phenomenon of newly formed memory dependence on individual experience history at both a behavioral and neuronal level. The study demonstrated that the enhancing effect of previous experiences on memory creation relies on the repeated activation of identical neurons.

Natural learning involves multiple sensory modalities receiving complex stimuli. Elemental learning theories suggest separate encoding and association of each component in a compound signal. Configural theories predict the formation of a representation of the entire complex signal, which is associated with the second event. We developed a mouse model of fear conditioning to a compound tone-light cue or its separate components to test these alternative theories. First, we investigated the memory dynamics of compound cues and their individual components and discovered that they mature at varying times following conditioning. We demonstrated that the memory of the components matures at different intervals after training: memory of the auditory stimulus and the auditory component of CCS is demonstrated behaviorally right after training, whereas memory of the light stimulus and the light component of CCS matures within three days. The memory of CCS, its components, and discrete conditioned stimuli persists for an extended period, up to one month. A similar dissociation was observed in extinction experiments, revealing that the extinction of memory for one CCS component did not affect the memory of the other component when the extinction procedure began a day after training. In addition, when the extinction procedure began seven days after training, while the memory was fully mature, the extinction of one component of contextual conditional stimuli led to the extinction of the other component. Next, c-Fos imaging was conducted to examine cellular activity across multiple brain regions, including the frontal, prelimbic, cingulate, retrosplenial, parietal, primary and secondary visual, primary and secondary auditory cortices, as well as the hippocampus and amygdala. This examination was performed following conditioning using either the entire compound cue or its individual components. We discovered different cortical activation patterns between compound-cue and single-cue conditioning. Conditioning to the compound cue activated prelimbic and frontal associative cortices, whereas single cues did not. Third, we demonstrated that retrieval of memory only through the entire compound cue, and not through single cues, activated the parietal cortex, primary visual cortex, mediolateral secondary visual cortices, and hippocampal CA1. Fourth, through in vivo two-photon imaging, we examined retrieval-induced c-Fos expression in the parietal cortex of fos-EGFP transgenic mice and identified at least three distinct neuronal populations with differential response specificity to the compound signal and its components. Taken together, our data suggest that intricate signals have the potential to establish both integral and elemental neuronal representations. These representations can be used separately in behavior and have different long-term memory dynamics.

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.

Post-traumatic stress disorder (PTSD) is defined by the International Classification as impairments to emotional state and reactions to a stressful situation in humans. the symptoms of PTSD include anxiety and intrusive memories of the traumatizing event, nightmares, irritability, increased awareness of danger or preoccupation with potential danger, impaired attention, and emotional blunting. The cause of PTSD is an acute psychological trauma induced by a powerful stress-inducing event such as participation in military combat or being in a military combat zone, terrorist acts, natural or technogenic catastrophes, domestic or sexual violence, and the sudden death of a close person, or even health problems. The mechanisms of PTSD have in recent years attracted ever more research attention. Despite great advances in studies of PTSD at the behavioral and psychological, as well as the physiological level, in humans and in a large number of models of PTSD in laboratory animals, the phenomenon of post-traumatic stress disorder remains poorly understood at the theoretical level. This article reviews current concepts of the mechanisms by which PTSD develops, theoretical approaches to understanding this disorder, and experimental data supporting these models.

Genetically encoded monomeric blue-to-red fluorescent timers (mFTs) change their fluorescent color over time. mCherry-derived mFTs were used for the tracking of the protein age, visualization of the protein trafficking, and labeling of engram cells. However, the brightness of the blue and red forms of mFTs are 2–3- and 5–7-fold dimmer compared to the brightness of the enhanced green fluorescent protein (EGFP). To address this limitation, we developed a blue-to-red fluorescent timer, named mRubyFT, derived from the bright mRuby2 red fluorescent protein. The blue form of mRubyFT reached its maximum at 5.7 h and completely transformed into the red form that had a maturation half-time of 15 h. Blue and red forms of purified mRubyFT were 4.1-fold brighter and 1.3-fold dimmer than the respective forms of the mCherry-derived Fast-FT timer in vitro. When expressed in mammalian cells, both forms of mRubyFT were 1.3-fold brighter than the respective forms of Fast-FT. The violet light-induced blue-to-red photoconversion was 4.2-fold less efficient in the case of mRubyFT timer compared to the same photoconversion of the Fast-FT timer. The timer behavior of mRubyFT was confirmed in mammalian cells. The monomeric properties of mRubyFT allowed the labeling and confocal imaging of cytoskeleton proteins in live mammalian cells. The X-ray structure of the red form of mRubyFT at 1.5 Å resolution was obtained and analyzed. The role of the residues from the chromophore surrounding was studied using site-directed mutagenesis.

A resting state network is a correlated activity of many neural structures in absence of external stimulation or functional tasks, and it is a fundamental endogenous feature of the human and animal brain. However, the nature and function of such spontaneous activity remain poorly understood. One of the hypothesis suggest that they reflect background replay and consolidation of individually acquired neural networks of prior experience. However, classical non-invasive methods used for resting state network detection cannot tag specific cellular elements of the neural network at the time when individual experience is acquired so that their activity can be subsequently investigated in the resting state. To overcome this limitation, we started a project on cellular imaging of mouse resting state networks and relating their activity to animal's past experiences. We used a large-scale c-Fos imaging of resting state neuronal activity in the mouse brain combined with graph analysis methods to get deeper insights into structure and functional significance of resting state brain networks. We characterized resting-state activity of 104 mouse brain structures and found that there was no direct relationship between anatomical attributes of examined areas and the level of their activity. We also analyzed individual variability of brain areas activity and showed that resting-state networks identified by c-Fos expression were stable and reproducible in all the animals. Next, c-Fos activity of 42 selected brain areas (sensory and motor cortices, hippocampus, parahippocampal cortex, amygdala, basal nuclei, associative and sensory thalamic nuclei, hypothalamic nuclei and midbrain) to characterize the major components and analyze functional connectivity of the resting state network. we identified several major groups of functionally connected areas in the resting state network of awake mouse brain: a cluster of medial prefrontal cortex and other medial associative neocortical areas, a cluster of visual areas, a tightly connected cluster of sensorimotor areas and basal nuclei, and a fully isolated cluster of auditory areas. Importantly, activity of structures known for their relationship to fear and threat learning (such as hippocampus, amygdala, and prelimbic cortex) was not correlated and did not comprise any functional group. This high variability in the activity of fear-related brain structures will be used at the next stage of the project to examine changes in the resting state network activity in relation to prior threat experience.

We demonstrate a versatile framework for cellular brain imaging in awake mice based on suitably tailored segments of graded-index (GRIN) fiber. Closed-form solutions to ray-path equations for graded-index waveguides are shown to offer important insights into image-transmission properties of GRIN fibers, suggesting useful recipes for optimized GRIN-fiber-based deep-brain imaging. We show that the lengths of GRIN imaging components intended for deep-brain studies in freely moving rodents need to be chosen as a tradeoff among the spatial resolution, the targeted imaging depth and the degree of fiber-probe invasiveness. In the experimental setting that we present in this paper, the head of an awake mouse with a GRIN-fiber implant is fixed under a microscope objective, but the mouse is free to move around an in-house-built flat-floored air-lifted platform, exploring a predesigned environment, configured as an arena for one of standard cognitive tests. We show that cellular-resolution deep-brain imaging can be integrated in this setting with robust cell-specific optical neural recording to enable in vivo studies with minimal physical restraints on animal models. The enhancement of the information capacity of the fluorescence signal, achieved via a suitable filtering of the GRIN-fiber readout, is shown to open routes toward practical imaging modalities whereby the deep-brain neuronal dynamics and axonal connections underpinning the integrative functions of essential brain structures can be studied in awake rodent models.

Associative learning is a fundamental mechanism for acquiring new experience by the brain. Though molecular, structural and synaptic aspects of this process have been thoroughly studied, less is known about how it happens at the level of cortical circuitries. According to the theory of predictive coding, associative learning leads to the formation of cortical representations or engrams that affect the supragranular (L2/3) and infragranular (L5/6) cortical layers in different ways. We utilized the cued (auditory) fear conditioning paradigm and layer-specific c-Fos neuroimaging of associative (prelimbic and cingulate) and auditory cortical areas after memory retrieval in mice to elucidate this issue. Retrieval of associative memory about auditory conditioned stimulus (CS) resulted in the preferential activation of cingulate and infralimbic cortices in conditioned mice compared to unpaired, CS-only controls and home cage mice. This activation was specific in all layers of cingulate cortex as well as in infragranular layers of prelimbic cortex. In auditory cortex in all mice, presented with auditory stimuli during retrieval (conditioned, unpaired and sound control groups) there was strong a c-fos activation. Such activation was found in all layers. Finally, we used correlation analysis and showed the c-fos expression in prelimbic and primary auditory but not cingulate cortex was highly-correlated across layers. The present findings indicate that associative memory cortical engram is unequal across layers and preferentially includes infragranular neurons.