Sex differences in spontaneous behavior and cognition in mice using an automated behavior monitoring system.

Isolation of sex differences as a key characteristic underlying neurobehavioral differentiation is an essential component of studies in neuroscience. The current study sought to address this concern by observing behavioral differences using an automated home cage system for neurobehavioral assessment, a method rapidly increasing in use due to advances in technology and advantages such as reduced handling stress and cross-lab variability. Sex differences in C57BL/6 mice arose for motor activity and circadian-linked behavior, with females being more active compared to males, and males having a stronger anticipatory increase in activity leading up to the onset of the light phase compared to females. These activity differences were observed not only across the lifespan, but also in different genetic background mouse strains across different testing sites showing the generalizability and robustness of these observed effects. Activity differences were also observed in performance on a spatial learning and reversal task with females making more responses and receiving a corresponding elevation in reward pellets. Notably, there were no sex differences in learning nor achieved accuracy, suggesting these observed effects were predominantly in activity. The outcomes of this study align with previous reports showcasing differences in activity between males and females. The comparison across strains and testing sites showed robust and reproducible differences in behavior between female and male mice that are relevant to consider when designing behavioral studies. Furthermore, the observed sex differences in performance on the learning and reversal procedure raise concern for interpretation of behavior differences between sexes due to the attribution of these differences to motor activity rather than cognition.

Cholinergic-Sensitive Theta Oscillations in Memory Encoding in Mice.

Cholinergic regulation of hippocampal theta oscillations has long been proposed to be a potential mechanism underlying hippocampus-dependent memory encoding processes. However, cholinergic transmission has been traditionally associated with type II theta under urethane anesthesia. The mechanisms and behavioral significance of cholinergic regulation of type I theta in freely exploring animals is much less clear. In this study, we examined the potential behavioral significance of cholinergic regulation of theta oscillations in the object location task in male mice that involves training and testing trials and provides an ideal behavioral task to study the underlying memory encoding and retrieval processes, respectively. Cholinergic regulation of hippocampal theta oscillations and the behavioral outcomes was examined by either intrahippocampal infusion of cholinergic receptor antagonists or knocking out cholinergic receptors in excitatory neurons or interneurons. We found that both muscarinic acetylcholine receptors (mAChRs) and α7 nicotinic AChRs (α7 nAChRs) regulated memory encoding by engaging excitatory neurons and interneurons, respectively. There is a transient upregulated theta oscillation at the beginning of individual object exploration events that only occurred in the training trials, but not in the testing trials. This transient upregulated theta is also the only theta component that significantly differed between training and testing trials and was sensitive to mAChR and α7 nAChR antagonists. Thus, our study has revealed a transient cholinergic-sensitive theta component that is specifically associated with memory encoding, but not memory retrieval, in the object location task, providing direct experimental evidence supporting a role for cholinergic-regulated theta oscillations in hippocampus-dependent memory encoding processes.

FiPhA: an open-source platform for fiber photometry analysis.

Significance: Fiber photometry (FP) is a widely used technique in modern behavioral neuroscience, employing genetically encoded fluorescent sensors to monitor neural activity and neurotransmitter release in awake-behaving animals. However, analyzing photometry data can be both laborious and time-consuming. Aim: We propose the fiber photometry analysis (FiPhA) app, which is a general-purpose FP analysis application. The goal is to develop a pipeline suitable for a wide range of photometry approaches, including spectrally resolved, camera-based, and lock-in demodulation. Approach: FiPhA was developed using the R Shiny framework and offers interactive visualization, quality control, and batch processing functionalities in a user-friendly interface. Results: This application simplifies and streamlines the analysis process, thereby reducing labor and time requirements. It offers interactive visualizations, event-triggered average processing, powerful tools for filtering behavioral events, and quality control features. Conclusions: FiPhA is a valuable tool for behavioral neuroscientists working with discrete, event-based FP data. It addresses the challenges associated with analyzing and investigating such data, offering a robust and user-friendly solution without the complexity of having to hand-design custom analysis pipelines. This application thus helps standardize an approach to FP analysis.

Genetic deletion of α7 nAChRs reduces hippocampal granule and pyramidal cell number in both sexes but impairs pattern separation in males only.

Introduction: Neurogenesis within the dentate gyrus is thought to play an important role in cognitive processes such as reversal learning and pattern separation. The α7 nicotinic acetylcholine receptor (α7 nAChR) is expressed early in newly formed granule cells of the dentate gyrus, though its role in neurogenesis and related cognitive function is not fully understood. Methods: To better characterize relevant function of α7 nAChRs, we performed unbiased stereology to quantify hippocampal granule cells, pyramidal cells, and total volume and used a touchscreen operant spatial discrimination/reversal task to test pattern separation in a global α7 nAChR knockout mouse line. Results: The knockout resulted in an ≈22% reduction in granule cells and a ≈ 20% reduction in pyramidal cells in both sexes, with no change in total hippocampal volume. However, the knockout impaired performance in the touchscreen task for males only. The sex-dependent difference in behavioral, but not stereological, results suggest a divergence in the structure-function relationship in males versus females. Detailed analyses revealed males were more biased by the initial reversal contingency relative to females indicating a potential source of the sex-specific interaction with the loss of α7 nAChRs. Discussion: These findings argue that the α7 nAChR plays a critical role in hippocampal development, not just granule cell neurogenesis, and plays a sex-dependent role in cognitive function.

FiPhA: An Open-Source Platform for Fiber Photometry Analysis.

Significance: Fiber photometry is a widely used technique in modern behavioral neuroscience, employing genetically encoded fluorescent sensors to monitor neural activity and neurotransmitter release in awake-behaving animals, However, analyzing photometry data can be both laborious and time-consuming. Aim: We propose the FiPhA (Fiber Photometry Analysis) app, which is a general-purpose fiber photometry analysis application. The goal is to develop a pipeline suitable for a wide range of photometry approaches, including spectrally resolved, camera-based, and lock-in demodulation. Approach: FiPhA was developed using the R Shiny framework and offers interactive visualization, quality control, and batch processing functionalities in a user-friendly interface. Results: This application simplifies and streamlines the analysis process, thereby reducing labor and time requirements. It offers interactive visualizations, event-triggered average processing, powerful tools for filtering behavioral events and quality control features. Conclusions: FiPhA is a valuable tool for behavioral neuroscientists working with discrete, event-based fiber photometry data. It addresses the challenges associated with analyzing and investigating such data, offering a robust and user-friendly solution without the complexity of having to hand-design custom analysis pipelines. This application thus helps standardize an approach to fiber photometry analysis.

Assessing the importance of sex in a hippocampus-dependent behavioral test battery in C57BL/6NTac mice.

Inclusion of male and female subjects in behavioral neuroscience research requires a concerted effort to characterize sex differences in standardized behavioral assays. Sex differences in hippocampus-dependent assays have been widely reported but are still poorly characterized. In the present study, we conducted a parametric analysis of spontaneous alternation, object recognition, and fear conditioning in a commonly used control strain, C57BL/6NTac. Our findings show largely similar performance between males and females across the majority of behavioral end points. However, we identified an important difference in nonassociative fear sensitization, whereby females showed an enhanced fear response to the 75-dB tone that is used as the conditional stimulus. In addition, we observed an impairment in object location performance in females that was ameliorated by more extensive habituation to handling. Together, these findings argue that sex differences in nonassociative fear responses to both novel auditory cues and novel objects need to be considered when designing and interpreting cognitive assays in C57BL/6 mice. Furthermore, this elevated fear sensitization could serve as a novel approach to model the increased incidence of anxiety disorders in women.

A brain-specific pgc1α fusion transcript affects gene expression and behavioural outcomes in mice.

PGC1α is a transcriptional coactivator in peripheral tissues, but its function in the brain remains poorly understood. Various brain-specific Pgc1α isoforms have been reported in mice and humans, including two fusion transcripts (FTs) with non-coding repetitive sequences, but their function is unknown. The FTs initiate at a simple sequence repeat locus ∼570 Kb upstream from the reference promoter; one also includes a portion of a short interspersed nuclear element (SINE). Using publicly available genomics data, here we show that the SINE FT is the predominant form of Pgc1α in neurons. Furthermore, mutation of the SINE in mice leads to altered behavioural phenotypes and significant up-regulation of genes in the female, but not male, cerebellum. Surprisingly, these genes are largely involved in neurotransmission, having poor association with the classical mitochondrial or antioxidant programs. These data expand our knowledge on the role of Pgc1α in neuronal physiology and suggest that different isoforms may have distinct functions. They also highlight the need for further studies before modulating levels of Pgc1α in the brain for therapeutic purposes.

3D-Printed Capacitive Sensor Objects for Object Recognition Assays.

Object recognition tasks are widely used assays for studying learning and memory in rodents. Object recognition typically involves familiarizing mice with a set of objects and then presenting a novel object or displacing an object to a novel location or context. Learning and memory are inferred by a relative increase in time investigating the novel/displaced object. These tasks are in widespread use, but there are many inconsistencies in the way they are conducted across labs. Two major contributors to this are the lack of consistency in the method of measuring object investigation and the lack of standardization of the objects that are used. Current video-based automated algorithms can often be unreliable whereas manual scoring of object investigation is time consuming, tedious, and more subjective. To resolve these issues, we sought to design and implement 3D-printed objects that can be standardized across labs and use capacitive sensing to measure object investigation. Using a 3D printer, conductive filament, and low-cost off-the-shelf components, we demonstrate that employing 3D-printed capacitive touch objects is a reliable and precise way to perform object recognition tasks. Ultimately, this approach will lead to increased standardization and consistency across labs, which will greatly improve basic and translational research into learning and memory mechanisms.

Loss of α7 nicotinic acetylcholine receptors in GABAergic neurons causes sex-dependent decreases in radial glia-like cell quantity and impairments in cognitive and social behavior.

The dentate gyrus (DG) is a unique brain structure in that neurons can be generated postnatally and integrated within existing circuitry throughout life. The maturation process of these newly generated neurons (granule cells) is modulated by nicotinic acetylcholine receptors (nAChRs) through a variety of mechanisms such as neural stem pool proliferation, cell survival, signal modulation, and dendritic integration. Disrupted nAChR signaling has been implicated in neuropsychiatric and neurodegenerative disorders, potentially via alterations in DG neurogenesis. GABAergic interneurons are known to express nAChRs, predominantly the α7 subtype, and have been shown to shape development, integration, and circuit reorganization of DG granule cells. Therefore, we examined histological and behavioral effects of knocking out α7 nAChRs in GABAergic neurons. Deletion of α7 nAChRs resulted in a reduction of radial glia-like cells within the subgranular zone of the DG and a concomitant trend towards decreased immature neurons, specifically in male mice, as well as sex-dependent changes in several behaviors, including social recognition and spatial learning. Overall, these findings suggest α7 nAChRs expressed in GABAergic neurons play an important role in regulating the adult neural stem cell pool and behavior in a sex-dependent manner. This provides important insight into the mechanisms by which cholinergic dysfunction contributes to the cognitive and behavioral changes associated with neurodevelopmental and neurodegenerative disorders.

Alcohol Intake Increases in Adolescent C57BL/6J Mice during Intermittent Cycles of Phase-Delayed, Long-Light Conditions.

Adolescents naturally go to bed and awaken late, but are forced to awaken early for school and work. This leads to "social jetlag", a state of circadian desynchrony (CD), in which internal biological rhythms are out of sync with behavioral rhythms. CD is associated with increased alcohol intake in adults, but has been less well-studied in adolescents. The goal of this study was to model adolescent alcohol intake during similar CD conditions in male C57BL/6J mice. Free access alcohol intake, water intake and wheel-running activity were measured during a normal 12HR photoperiod or during alternating photoperiod (Experiment 1: 12 h light for 4 days followed by 18 h light for 3 days, with dark (activity onset) delayed 9 h during the 18HR photoperiod; Experiment 2: 12 h light for 4 days followed by 6 h light for 3 days, with dark onset delayed 3 h during the 6HR photoperiod). In Experiment 1, CD produced a small but significant increase in the total alcohol intake per day as well as in intake in bouts, with the greatest increase over controls in the hours following the 6HR dark period. Additionally, the pattern of alcohol intake in bouts shifted to increase alcohol intake during the shorter dark period. In Experiment 2, the opposite effect occurred-the longer dark cycle led to lower alcohol drinking in the second half of the dark period. However, in Experiment 2, CD produced no significant changes in either total alcohol intake or alcohol intake in bouts. CONCLUSION: shifts in the light cycle that disrupt the regular pattern of day and night, and increase the length of the night phase, are sufficient to increase both drinking in bouts and restricted drinking in adolescent mice, modeling increased alcohol intake in adolescents during CD.