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IntelliSleepScorer, a Software Package with a Graphic User Interface for Mice Automated Sleep Stage Scoring

Sleep stage scoring in rodents is the process of identifying the three stages: nonrapid eye movement sleep (NREM), rapid eye movement sleep (REM), and ...

Broad Institute of MIT and Harvard

T-type voltage-gated Ca channels have been implicated in many human disorders, and there has been increasing interest in developing highly selective and potent T-type Ca channel modulators for potential clinical use. However, the unique biophysical properties of T-type Ca channels are not conducive for developing high-throughput screening (HTS) assays to identify modulators, particularly potentiators. To illustrate, T-type Ca channels are largely inactivated and unable to open to allow Ca influx at -25 mV, the typical resting membrane potential of the cell lines commonly used in cellular screening assays. To address this issue, we developed cell lines that express K2.3 channels to hyperpolarize the membrane potential to -70 mV, thus allowing T-type channels to return to their resting state where they can be subsequently activated by membrane depolarization in the presence of extracellular KCl. Furthermore, to simplify the HTS assay and to reduce reagent cost, we stably expressed a membrane-tethered genetic calcium sensor, GCaMP6s-CAAX, that displays superior signal to the background compared to the untethered GCaMP6s or the synthetic Ca sensor Fluo-4AM. Here, we describe a novel GCaMP6s-CAAX-based calcium assay utilizing a high-throughput fluorometric imaging plate reader (Molecular Devices, Sunnyvale, CA) format that can identify both activators and inhibitors of T-type Ca channels. Lastly, we demonstrate the utility of this novel fluorescence-based assay to evaluate the activities of two distinct G-protein-coupled receptors, thus expanding the use of GCaMP6s-CAAX to a wide range of applications relevant for developing cellular assays in drug discovery.

Novel Fluorescence-Based High-Throughput FLIPR Assay Utilizing Membrane-Tethered Genetic Calcium Sensors to Identify T-Type Calcium Channel Modulators.

Motivated by the potential of objective neurophysiological markers to index thalamocortical function in patients with severe psychiatric illnesses, we comprehensively characterized key non-rapid eye movement (NREM) sleep parameters across multiple domains, their interdependencies, and their relationship to waking event-related potentials and symptom severity. In 72 schizophrenia (SCZ) patients and 58 controls, we confirmed a marked reduction in sleep spindle density in SCZ and extended these findings to show that fast and slow spindle properties were largely uncorrelated. We also describe a novel measure of slow oscillation and spindle interaction that was attenuated in SCZ. The main sleep findings were replicated in a demographically distinct sample, and a joint model, based on multiple NREM components, statistically predicted disease status in the replication cohort. Although also altered in patients, auditory event-related potentials elicited during wake were unrelated to NREM metrics. Consistent with a growing literature implicating thalamocortical dysfunction in SCZ, our characterization identifies independent NREM and wake EEG biomarkers that may index distinct aspects of SCZ pathophysiology and point to multiple neural mechanisms underlying disease heterogeneity. This study lays the groundwork for evaluating these neurophysiological markers, individually or in combination, to guide efforts at treatment and prevention as well as identifying individuals most likely to benefit from specific interventions.

Non-rapid eye movement sleep and wake neurophysiology in schizophrenia.

Machine learning has been applied in recent years to categorize sleep stages (NREM, REM, and wake) using electroencephalogram (EEG) recordings; however, a well-validated sleep scoring automatic pipeline in rodent research is still not publicly available. Here, we present IntelliSleepScorer, a software package with a graphic user interface to score sleep stages automatically in mice. IntelliSleepScorer uses the light gradient boosting machine (LightGBM) to score sleep stages for each epoch of recordings. We developed LightGBM models using a large cohort of data, which consisted of 5776 h of sleep EEG and electromyogram (EMG) signals across 519 unique recordings from 124 mice. The LightGBM model achieved an overall accuracy of 95.2% and a Cohen's kappa of 0.91, which outperforms the baseline models such as the logistic regression model (accuracy = 93.3%, kappa = 0.88) and the random forest model (accuracy = 94.3%, kappa = 0.89). The overall performance of the LightGBM model as well as the performance across different sleep stages are on par with that of the human experts. Most importantly, we validated the generalizability of the LightGBM models: (1) The LightGBM model performed well on two publicly available, independent datasets (kappa >  = 0.80), which have different sampling frequency and epoch lengths; (2) The LightGBM model performed well on data recorded at a lower sampling frequency (kappa = 0.90); (3) The performance of the LightGBM model is not affected by the light/dark cycle; and (4) A modified LightGBM model performed well on data containing only one EEG and one EMG electrode (kappa >  = 0.89). Taken together, the LightGBM models offer state-of-the-art performance for automatic sleep stage scoring in mice. Last, we implemented the IntelliSleepScorer software package based on the validated model to provide an out-of-box solution to sleep researchers (available for download at https://sites.broadinstitute.org/pan-lab/resources ).

IntelliSleepScorer, a software package with a graphic user interface for automated sleep stage scoring in mice based on a light gradient boosting machine algorithm.

It is challenging to apply traditional mutational scanning to voltage-gated sodium channels (Nas) and functionally annotate the large number of coding variants in these genes. Using a cytosine base editor and a pooled viability assay, we screen a library of 368 guide RNAs (gRNAs) tiling Na1.2 to identify more than 100 gRNAs that change Na1.2 function. We sequence base edits made by a subset of these gRNAs to confirm specific variants that drive changes in channel function. Electrophysiological characterization of these channel variants validates the screen results and provides functional mechanisms of channel perturbation. Most of the changes caused by these gRNAs are classifiable as loss of function along with two missense mutations that lead to gain of function in Na1.2 channels. This two-tiered strategy to functionally characterize ion channel protein variants at scale identifies a large set of loss-of-function mutations in Na1.2.

Scanning mutagenesis of the voltage-gated sodium channel Na1.2 using base editing.

Microstate analysis is a promising technique for analyzing high-density electroencephalographic data, but there are multiple questions about methodological best practices. Between and within individuals, microstates can differ both in terms of characteristic topographies and temporal dynamics, which leads to analytic challenges as the measurement of microstate dynamics is dependent on assumptions about their topographies. Here we focus on the analysis of group differences, using simulations seeded on real data from healthy control subjects to compare approaches that derive separate sets of maps within subgroups versus a single set of maps applied uniformly to the entire dataset. In the absence of true group differences in either microstate maps or temporal metrics, we found that using separate subgroup maps resulted in substantially inflated type I error rates. On the other hand, when groups truly differed in their microstate maps, analyses based on a single set of maps confounded topographic effects with differences in other derived metrics. We propose an approach to alleviate both classes of bias, based on a paired analysis of all subgroup maps. We illustrate the qualitative and quantitative impact of these issues in real data by comparing waking versus non-rapid eye movement sleep microstates. Overall, our results suggest that even subtle chance differences in microstate topography can have profound effects on derived microstate metrics and that future studies using microstate analysis should take steps to mitigate this large source of error.

A Potential Source of Bias in Group-Level EEG Microstate Analysis.

Multiple facets of sleep neurophysiology, including electroencephalography (EEG) metrics such as non-rapid eye movement (NREM) spindles and slow oscillations (SO), are altered in individuals with schizophrenia (SCZ). However, beyond group-level analyses which treat all patients as a unitary set, the extent to which NREM deficits vary among patients is unclear, as are their relationships to other sources of heterogeneity including clinical factors, illness duration and ageing, cognitive profiles and medication regimens. Using newly collected high density sleep EEG data on 103 individuals with SCZ and 68 controls, we first sought to replicate our previously reported (Kozhemiako et. al, 2022) group-level mean differences between patients and controls (original =130). Then in the combined sample (=301 including 175 patients), we characterized patient-to-patient variability in NREM neurophysiology.

A spectrum of altered non-rapid eye movement sleep in schizophrenia.

Aberrant functional connectivity is a hallmark of schizophrenia. The precise nature and mechanism of dysconnectivity in schizophrenia remains unclear, but evidence suggests that dysconnectivity is different in wake versus sleep. Microstate analysis uses electroencephalography (EEG) to investigate large-scale patterns of coordinated brain activity by clustering EEG data into a small set of recurring spatial patterns, or microstates. We hypothesized that this technique would allow us to probe connectivity between brain networks at a fine temporal resolution and uncover previously unknown sleep-specific dysconnectivity.

Electroencephalographic Microstates During Sleep and Wake in Schizophrenia.

Multiple facets of sleep neurophysiology, including electroencephalography (EEG) metrics such as non-rapid eye movement (NREM) spindles and slow oscillations, are altered in individuals with schizophrenia (SCZ). However, beyond group-level analyses, the extent to which NREM deficits vary among patients is unclear, as are their relationships to other sources of heterogeneity including clinical factors, ageing, cognitive profiles and medication regimens. Using newly collected high-density sleep EEG data on 103 individuals with SCZ and 68 controls, we first sought to replicate our previously reported group-level differences between patients and controls (original N=130) during N2 stage. Then in the combined sample (N=301 including 175 patients), we characterized patient-to-patient variability. We replicated all group-level mean differences and confirmed the high accuracy of our predictive model (AUC=0.93 for diagnosis). Compared to controls, patients showed significantly increased between-individual variability across many (26%) sleep metrics. Although multiple clinical and cognitive factors were associated with NREM metrics, collectively they did not account for much of the general increase in patient-to-patient variability. Medication regimen was a greater contributor to variability. Some sleep metrics including fast spindle density showed exaggerated age-related effects in SCZ, and patients exhibited older predicted biological ages based on the sleep EEG; further, among patients, certain medications exacerbated these effects, in particular olanzapine. Collectively, our results point to a spectrum of N2 sleep deficits among SCZ patients that can be measured objectively and at scale, with relevance to both the etiological heterogeneity of SCZ as well as potential iatrogenic effects of antipsychotic medication.

Article	Year
Novel Fluorescence-Based High-Throughput FLIPR Assay Utilizing Membrane-Tethered Genetic Calcium Sensors to Identify T-Type Calcium Channel Modulators. ACS pharmacology & translational science\| PubMed ID: 35311021	2022
Non-rapid eye movement sleep and wake neurophysiology in schizophrenia. eLife\| PubMed ID: 35578829	2022
IntelliSleepScorer, a software package with a graphic user interface for automated sleep stage scoring in mice based on a light gradient boosting machine algorithm. Scientific reports\| PubMed ID: 36922536	2023
Scanning mutagenesis of the voltage-gated sodium channel Na1.2 using base editing. Cell reports\| PubMed ID: 37267104	2023
A Potential Source of Bias in Group-Level EEG Microstate Analysis. Brain topography\| PubMed ID: 37548801	2024
A spectrum of altered non-rapid eye movement sleep in schizophrenia. bioRxiv : the preprint server for biology\| PubMed ID: 38234726	2023
Electroencephalographic Microstates During Sleep and Wake in Schizophrenia. Biological psychiatry global open science\| PubMed ID: 39296796	2024
A spectrum of altered non-rapid eye movement sleep in schizophrenia. Sleep\| PubMed ID: 39297495	2024

Lei A. Wang

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