Biology subjects

Badura, A.

Publications and source records attributed to Badura, A..

6 recordsLinked to original sources

bioRxiv2023

Detecting ataxia using automated analysis of motor coordination and balance of mice on the balance beam

BackgroundThe balance beam assay is a well-known paradigm to assess motor coordination in mouse models of neurodegenerative diseases. Classically, these experiments are quantified using manual annotation, which is time-consuming and prone to inter-experimenter variability. MethodsWe present an open-source analysis pipeline that allows for the automated quantification of motor function in mice crossing the balance beam. ResultsUsing an established Pcp2-Ppp3r1 ataxia mouse model, we validated the analysis pipeline by comparing the motor performance of Pcp2-Ppp3r1 animals to their wildtype littermates. Pcp2-Ppp3r1 animals showed a significant increase in the number of missteps and increased time to traverse the beam. Moreover, we compared the results of the automated classification of missteps and stops to that of 3 independent observers, which showed no significant differences between the classifier and the cumulative observer score for missteps. ConclusionWe show that our pipeline can reliably report crossing time and missteps, offering a high-throughput, automated option for the analysis of balance beam data. Method summaryOur method consists of an easy-to-follow and low-cost manual for building a balance beam setup and an analysis pipeline of mouse movement while crossing the beam. We present a MATLAB script combining FFMPEG and ImageJ (using the MIJ package) to pre-process the videos and extract the time for the mouse to cross the beam. Mice movements on the beam were then tracked using motr and stops and missteps were extracted using a trained JAABA classifier. The trained classifier, the code and all accompanying files were deposited in an open source repository

animal behavior and cognition↗

bioRxiv2021

Tsc1 haploinsufficiency leads to Pax2 dysregulation in the developing murine cerebellum

Tuberous sclerosis complex 1 (TSC1) is a tumour suppressor gene that inhibits the mechanistic target of rapamycin (mTOR) pathway. Mutations in TSC1 lead to a rare complex disorder of the same name, in which up to 50% of patients present with autism spectrum disorder (ASD). ASD is a highly prevalent, early-onset neurodevelopmental disorder, characterized by social deficits and repetitive behaviours, although the type and severity of symptoms show wide variability across individuals. Amongst different brain areas proposed to play a role in the development of ASD, the cerebellum is commonly reported to be altered, and cerebellar-specific deletion of Tsc1 in mice is sufficient to induce an ASD-like phenotype. Given that the mTOR pathway is crucial for proper cell replication and migration, this suggests that dysregulation of this pathway, particularly during critical phases of cerebellar development, could contribute to the establishment of ASD. Here, we used a mouse model of TSC to investigate gene and protein expression during embryonic and early postnatal periods of cerebellar development. We found that, at E18 and P7, mRNA levels of the cerebellar inhibitory interneuron marker Pax2 were dysregulated. This was accompanied by changes in the expression of mTOR pathway-related genes and downstream phosphorylation of S6. Differential gene correlation analysis revealed dynamic changes in correlated gene pairs across development, with an overall loss of correlation between mTOR- and cerebellar-related genes in Tsc1 mutants compared to controls. We corroborated the genetic findings by characterizing the mTOR pathway and cerebellar development on protein and cellular levels with Western blot and immunohistochemistry. We found that Pax2-expressing cells were hypertrophic at E18 while, at P7, their number was increased and maturation into parvalbumin-expressing cells delayed. Our findings indicate that E18 and P7 are crucial time points in cerebellar development in mice that are particularly susceptible to mTOR pathway dysregulation. Manuscript contribution to the fieldASD is one of the most prevalent neurodevelopmental disorders, however little is known about the shared mechanisms underlying its aetiology. At the anatomical level, the cerebellum has been identified as one of the key structures involved in the development of ASD, whereas at the molecular level, mutations in the mTOR signalling pathway, essential for cell growth and proliferation, carry a high genetic risk for this disorder. We used a haploinsufficient tuberous sclerosis complex 1 (Tsc1) mouse model to investigate the effects of mTOR overactivation in the developing cerebellum. Tsc1 inhibits the mTOR pathway, and mice with cerebellar-specific deletion of Tsc1 have been shown to harbour an ASD-like phenotype. We found that Pax2 expression in the cerebellum is dysregulated at prenatal and early postnatal time points, leading to a delayed maturation of inhibitory interneurons. Our findings indicate that mTOR overactivity in the cerebellum selectively affects the development of cerebellar interneurons. This finding is in line with other studies, which found decreased numbers of inhibitory interneurons in other models of ASD. Therefore, deficits in the maturation of the inhibitory signalling could be one of the mechanisms integrating high-risk mutations that underlie ASD aetiology.

neuroscience↗

bioRxiv2021

Behavioral and molecular underpinnings of performance variability in eyeblink conditioning in male and female mice

The functional and molecular sources of behavioral variability in mice are not fully understood. As a consequence, the predominant use of male mice has become a standard in animal research, under the assumption that males are less variable than females. Similarly, to homogenize genetic background, neuroscience studies have almost exclusively used the C57BL/6 (B6) strain. Here, we examined individual differences in performance in the context of associative learning. We performed delayed eyeblink conditioning while recording locomotor activity in mice from both sexes in two strains (B6 and B6CBAF1). Further, we used a C-FOS immunostaining approach to explore brain areas involved in eyeblink conditioning across subjects and correlate them with behavioral performance. We found that B6 male and female mice show comparable variability in this task and that females reach higher learning scores. We found a strong positive correlation across sexes between learning scores and voluntary locomotion. C-FOS immunostainings revealed positive correlations between C-FOS positive cell density and learning in the cerebellar cortex, as well as multiple previously unreported extra-cerebellar areas. We found consistent and comparable correlations in eyeblink performance and C-fos expression in B6 and B6CBAF1 females and males. Taken together, we show that differences in motor behavior and activity across brain areas correlate with learning scores during eyeblink conditioning across strains and sexes.

neuroscience↗

bioRxiv2021

A novel automated approach for improving standardization of the marble burying test enables quantification of burying bouts and activity characteristics

The marble burying test is a commonly used paradigm to screen phenotypes in mouse models of neurodevelopmental and psychiatric disorders. The current methodological approach relies solely on reporting the number of buried marbles at the end of the test. By measuring the proxy of the behavior (buried marbles), rather than the behavior itself (burying bouts), many important characteristics regarding the temporal aspect of this assay are lost. Here we introduce a novel, automated method to quantify mouse behavior throughout the duration of the marble burying test with the focus on the burying bouts. Using open-source software packages, we trained a supervised machine learning algorithm (the "classifier") to distinguish burying behavior in freely moving mice. In order to confirm the classifiers accuracy and uncover the behavioral meaning of the marble burying test, we performed marble burying test in three mouse models: Ube3am-/p+ (Angelman Syndrome model), Shank2-/- (autism model), and Sapap3-/- (obsessive-compulsive disorder model) mice. The classifier scored burying behavior accurately and consistent with the literature in the Ube3am-/p+ mice, which showed decreased levels of burying compared to controls. Shank2-/- mice showed a similar pattern of decreased burying behavior, which was not found in Sapap3-/- mice. Tracking mouse behavior throughout the test enabled us to quantify activity characteristics, revealing hypoactivity in Ube3am-/p+ and hyperactivity in the Shank2-/- mice, indicating that mouse activity is unrelated to burying behavior. Together, we demonstrate that our classifier is an accurate method for the analysis of the marble burying test, providing more information than the currently used methods. Significance StatementThe marble burying test is widely used in phenotyping neurodevelopmental and neuropsychiatric disorder mouse models. Currently, its analysis consists of manually scoring the number of buried marbles upon the completion of the assay. This approach is not standardized across laboratories, and leaves out important variables such as movement characteristics and information about the burying bouts. This leads to divergent interpretations of the marble burying test, ranging from anxiety to cognitive impairment. We introduce a method that reliably tracks mouse behavior throughout the experiment, classifies the duration and number of the burying bouts, and is generalizable across laboratories. Using machine learning for measuring the actual burying behavior standardizes this method, and provides rich information about the burying characteristics.

neuroscience↗

bioRxiv2021

Purkinje Cell Activity During Suppression of Voluntary Eye Movements in Rhesus Macaques

Volitional suppression of responses to distracting external stimuli enables us to achieve our goals. This volitional inhibition of a specific behavior is supposed to be mainly mediated by the cerebral cortex. However, recent evidence supports the involvement of the cerebellum in this process. It is currently not known whether different parts of the cerebellar cortex play differential or synergistic roles in planning and execution of this behavior. Here, we measured Purkinje cell (PC) responses in the medial and lateral cerebellum in two rhesus macaques during a pro- and antisaccade task. During an antisaccade trial, non-human primates were instructed to make a saccadic eye movement away from a target, rather than towards it, as in prosaccade trials. Our data shows that the cerebellum plays an important role not only during execution of the saccades, but also during the volitional inhibition of eye movements towards the target. Simple Spike (SS) modulation during the instruction and execution period of pro- and antisaccades was prominent in PCs of both medial and lateral cerebellum. However, only the SS activity in the lateral cerebellar cortex contained information about trial identity and showed a stronger reciprocal interaction with complex spikes. Moreover, SS activity of different PC groups modulated bidirectionally in both regions, but the PCs that showed facilitating and suppressive activity were predominantly associated with instruction and execution, respectively. These findings show that different cerebellar regions and PC groups contribute to goal-directed behavior and volitional inhibition, but with different propensities, highlighting the rich repertoire of cerebellar control in executive functions. Significance StatementThe antisaccade task is commonly used in research and clinical evaluation as a test of volitional and flexible control of behavior. It requires volitional suppression of prosaccades, a function that has been attributed to the neocortex. However, recent findings indicate that cerebellum also contributes to this behavior. We recorded from neurons in the medial and lateral cerebellum to evaluate their responses in this task. We found that both regions significantly modulated their activity during this task, but only cells in the lateral cerebellum encoded the stimulus identity in each trial. These results indicate that the cerebellum actively contributes to the control of flexible behavior and that lateral and medial cerebellum play different roles during volitional eye movements.

neuroscience↗

bioRxiv2021

Activated PI3Kδ syndrome, an immunodeficiency disorder, leads to sensorimotor deficits recapitulated in a murine model.

The phosphoinositide-3-kinase (PI3K) family plays a major role in cell signalling and is predominant in leukocytes. Gain-of-function (GOF) mutations in the PIK3CD gene lead to the development of activated PI3K{delta} syndrome (APDS), a rare primary immunodeficiency disorder. A subset of APDS patients also displays neurodevelopmental delay symptoms, suggesting a potential role of PIK3CD in cognitive and behavioural function. However, the extent and nature of the neurodevelopmental deficits has not been previously quantified. Here, we assessed the cognitive functions of two APDS patients, and investigated the causal role of the PIK3CD GOF mutation in neurological deficits using a murine model of this disease. We used E1020K knock-in mice, harbouring the most common APDS mutation in patients. We found that APDS patients present with visuomotor deficits, exacerbated by autism spectrum disorder comorbidity, whereas p110{delta}E1020K mice exhibited impairments in motor behaviour, learning and repetitive behaviour patterning. Our data indicate that PIK3CD GOF mutations increase the risk for neurodevelopmental deficits, supporting previous findings on the interplay between the nervous and the immune system. Further, our results validate the knock-in mouse model, and offer an objective assessment tool for patients that could be incorporated in diagnosis and in the evaluation of treatments.

neuroscience↗