Workflow for the unsupervised clustering of sleep stages identifies light and deep sleep in electrophysiological recordings in mice.

BACKGROUND: Sleep physiology plays a critical role in brain development and aging. Accurate sleep staging, which categorizes different sleep states, is fundamental for sleep physiology studies. Traditional methods for sleep staging rely on manual, rule-based scoring techniques, which limit their accuracy and adaptability. NEW METHOD: We describe, test and challenge a workflow for unsupervised clustering of sleep states (WUCSS) in rodents, which uses accelerometer and electrophysiological data to classify different sleep states. WUCSS utilizes unsupervised clustering to identify sleep states using six features, extracted from 4-second epochs. RESULTS: We gathered high-quality EEG recordings combined with accelerometer data in diverse transgenic mouse lines (male ApoE3 versus ApoE4 knockin; male CNTNAP2 KO versus wildtype littermates). WUCSS showed high recall, precision, and F1-score against manual scoring on awake, NREM, and REM sleep states. Within NREM, WUCSS consistently identified two additional clusters that qualify as deep and light sleep states. COMPARISON WITH EXISTING METHODS: The ability of WUCSS to discriminate between deep and light sleep enhanced the precision and comprehensiveness of the current mouse sleep physiology studies. This differentiation led to the discovery of an additional sleep phenotype, notably in CNTNAP2 KO mice, showcasing the method's superiority over traditional scoring methods. CONCLUSIONS: WUCSS, with its unsupervised approach and classification of deep and light sleep states, provides an unbiased opportunity for researchers to enhance their understanding of sleep physiology. Its high accuracy, adaptability, and ability to save time and resources make it a valuable tool for improving sleep staging in both clinical and preclinical research.

Glutamatergic and GABAergic Receptor Modulation Present Unique Electrophysiological Fingerprints in a Concentration-Dependent and Region-Specific Manner.

Brain function depends on complex circuit interactions between excitatory and inhibitory neurons embedded in local and long-range networks. Systemic GABAA-receptor (GABAAR) or NMDA-receptor (NMDAR) modulation alters the excitatory-inhibitory balance (EIB), measurable with electroencephalography (EEG). However, EEG signatures are complex in localization and spectral composition. We developed and applied analytical tools to investigate the effects of two EIB modulators, MK801 (NMDAR antagonist) and diazepam (GABAAR modulator), on periodic and aperiodic EEG features in freely-moving male Sprague Dawley rats. We investigated how, across three brain regions, EEG features are correlated with EIB modulation. We found that the periodic component was composed of seven frequency bands that presented region-dependent and compound-dependent changes. The aperiodic component was also different between compounds and brain regions. Importantly, the parametrization into periodic and aperiodic components unveiled correlations between quantitative EEG and plasma concentrations of pharmacological compounds. MK-801 exposures were positively correlated with the slope of the aperiodic component. Concerning the periodic component, MK-801 exposures correlated negatively with the peak frequency of low-γ oscillations but positively with those of high-γ and high-frequency oscillations (HFOs). As for the power, θ and low-γ oscillations correlated negatively with MK-801, whereas mid-γ correlated positively. Diazepam correlated negatively with the knee of the aperiodic component, positively to ß and negatively to low-γ oscillatory power, and positively to the modal frequency of θ, low-γ, mid-γ, and high-γ. In conclusion, correlations between exposures and pharmacodynamic effects can be better-understood thanks to the parametrization of EEG into periodic and aperiodic components. Such parametrization could be key in functional biomarker discovery.

Neurexin1α knockout rats display oscillatory abnormalities and sensory processing deficits back-translating key endophenotypes of psychiatric disorders.

Neurexins are presynaptic transmembrane proteins crucial for synapse development and organization. Deletion and missense mutations in all three Neurexin genes have been identified in psychiatric disorders, with mutations in the NRXN1 gene most strongly linked to schizophrenia (SZ) and autism spectrum disorder (ASD). While the consequences of NRXN1 deletion have been extensively studied on the synaptic and behavioral levels, circuit endophenotypes that translate to the human condition have not been characterized yet. Therefore, we investigated the electrophysiology of cortico-striatal-thalamic circuits in Nrxn1α-/- rats and wildtype littermates focusing on a set of translational readouts, including spontaneous oscillatory activity, auditory-evoked oscillations and potentials, as well as mismatch negativity-like (MMN) responses and responses to social stimuli. On the behavioral level Nrxn1α-/- rats showed locomotor hyperactivity. In vivo freely moving electrophysiology revealed pronounced increases of spontaneous oscillatory power within the gamma band in all studied brain areas and elevation of gamma coherence in cortico-striatal and thalamocortical circuits of Nrxn1α-/- rats. In contrast, auditory-evoked oscillations driven by chirp-modulated tones showed reduced power in cortical areas confined to slower oscillations. Finally, Nrxn1α-/- rats exhibited altered auditory evoked-potentials and profound deficits in MMN-like responses, explained by reduced prediction error. Despite deficits for auditory stimuli, responses to social stimuli appeared intact. A central hypothesis for psychiatric and neurodevelopmental disorders is that a disbalance of excitation-to-inhibition is underlying oscillatory and sensory deficits. In a first attempt to explore the impact of inhibitory circuit modulation, we assessed the effects of enhancing tonic inhibition via δ-containing GABAA receptors (using Gaboxadol) on endophenotypes possibly associated with network hyperexcitability. Pharmacological experiments applying Gaboxadol showed genotype-specific differences, but failed to normalize oscillatory or sensory processing abnormalities. In conclusion, our study revealed endophenotypes in Nrxn1α-/- rats that could be used as translational biomarkers for drug development in psychiatric disorders.

3D printed rodent skin-skull-brain model: A novel animal-free approach for neurosurgical training.

In neuroscience, stereotactic brain surgery is a standard yet challenging technique for which laboratory and veterinary personnel must be sufficiently and properly trained. There is currently no animal-free training option for neurosurgeries; stereotactic techniques are learned and practiced on dead animals. Here we have used three-dimensional (3D) printing technologies to create rat and mouse skin-skull-brain models, specifically conceived for rodent stereotaxic surgery training. We used 3D models obtained from microCT pictures and printed them using materials that would provide the most accurate haptic feedback for each model-PC-ABS material for the rat and Durable resin for the mouse. We filled the skulls with Polyurethane expanding foam to mimic the brain. In order to simulate rodent skin, we added a rectangular 1mm thick clear silicone sheet on the skull. Ten qualified rodent neurosurgeons then performed a variety of stereotaxic surgeries on these rat and mouse 3D printed models. Participants evaluated models fidelity compared to cadaveric skulls and their appropriateness for educational use. The 3D printed rat and mouse skin-skull-brain models received an overwhelmingly positive response. They were perceived as very realistic, and considered an excellent alternative to cadaveric skulls for training purposes. They can be made rapidly and at low cost. Our real-size 3D printed replicas could enable cost- and time-efficient, animal-free neurosurgery training. They can be absolute replacements for stereotaxic surgery techniques practice including but not limited to craniotomies, screw placement, brain injections, implantations and cement applications. This project is a significant step forward in implementing the replacement, reduction, and refinement (3Rs) principles to animal experimentation. These 3D printed models could lead the way to the complete replacement of live animals for stereotaxic surgery training in laboratories and veterinary studies.