We thank Kerena Yan and Jingwen Hu for manually scoring sleep stages and Eunah and Soonwiik for the recordings.

This study used data collected from in vivo experiments in mice. No human experiments were involved in the study. All the experiments with animals were approved by the Institutional Animal Care and Use Committee at the Broad Institute. All experiments were performed in accordance with relevant guidelines and regulations. The ARRIVE guidelines are not applicable to this study because the focus of this study is to develop machine learning models rather than comparing different treatment groups.
1. Data preparation
NOTE: Data compatibility: the recorded data can have any sampling rate higher than 40 Hz. There is no need to bandpass filter the signal because the software bandpass filters the EEG and EMG signals at the first step. The LightGBM models were developed and tested using data from mice. No evidence regarding the performance of the LightGBM models in other types of lab animals is available. The recording electrodes need to be placed at the frontal and the parietal cortex, or either place if only one EEG channel is recorded.
<ol>
	<li>EDF/EDF+ format arrangement and requirement 
	NOTE: The software used in this study only reads EDF/EDF+ files using the MNE-Python package. The standard EDF/EDF+ specification needs to be applied to generate the EDF/EDF+ files. In addition to the standard specification, ensure that the EDF/EDF+ annotations are encoded in UTF-8. Otherwise, the software application will crash.
	<ol>
		<li>Convert another file (non-EDF/EDF+ file) format into EDF/EDF+ format with online free tools. 
		NOTE: There is no requirement for the apparatus filter when obtaining EEG and EMG signals. As long as users sample their EEG and EMG data at a frequency of 40 Hz or higher, the software will function correctly. This is because, in the initial preprocessing step, the signals undergo bandpass filtering between 1 Hz and 40 Hz. This bandpass filtering is integrated into the software&#39;s preprocessing pipeline, eliminating users needing to perform any additional signal processing.</li>
	</ol>
	</li>
	<li>There are two models inside the software for scoring. One is LightGBM-2EEG, and the other is LightGBM-1EEG. The LightGBM-2EEG model is designated for recording data that has 2 EEG channels and 1 EMG channel. Perform the following steps depending on the model.
	<ol>
		<li>Organize data files for LightGBM-2EEG specifically in the following order: 1) EEG channel recorded in the parietal area; 2) EEG channel recorded in the frontal area; 3) EMG channel. The LightGBM-1EEG is designated for data that only contains 1 EEG channel (electrode placement on either parietal or frontal area) and 1 EMG channel.</li>
		<li>Organize the channels in EDF/EDF+ files for LightGBM-1EEG in the following order: 1) EEG channel; 2) EMG channel.</li>
	</ol>
	</li>
</ol>
2. Downloading IntelliSleepScorer for Windows, Mac, and Linux users
<ol>
	<li>For Windows users, a Windows Executable for the software is available using PyInstaller. Find the download link on the Pan group research page&#160;https://sites.broadinstitute.org/pan-lab/resources. For MacOS or Linux users, use the source code on the GitHub repository https://github.com/broadinstitute/IntelliSleepScorer to launch the software.&#160;</li>
	<li>Access two recorded example data to test the program saved as EDF files by downloading them through the GitHub repository.</li>
	<li>The source code repository does not include the models folder due to size limits. Instead, download models.zip, unzip it, and copy the models folder inside the repository for the program to run. Otherwise, the software will crash due to missing model files.</li>
</ol>
3. Workflow and Program launch and operation
<ol>
	<li>Launch IntelliSleepScorer
	<ol>
		<li>To launch the software in Windows, double-click IntelliSleepScorer.exe located in the root folder. To launch the software in MacOS or Linux, open a terminal emulator, change the directory to the root folder of the software, and then launch the software using the command: python3 IntelliSleepScorer.py.</li>
	</ol>
	</li>
	<li>Once the software opens, click Select EDF/EDF+ File(s) to select the intended file(s) to score. If files were selected by mistake, click the Clear button to clear the selected file list. 
	NOTE: By default, the software encodes the sleep stages as Wake:1, NREM:2, and REM:3 in the output score files. The default epoch length is set at 10 s. The current version (v1.2) of the GUI allows users to change stage encodings or epoch lengths to 4 s, 10 s, or 20 s with the dropdown menu.</li>
	<li>Select the desired epoch length. Use the provided dropdown menu to select the intended epoch length among options of 4 s, 10 s, and 20 s for sleep stage scoring.</li>
	<li>Select the model that is to be used for sleep scoring. LightGBM-2EEG is intended for data files with two EEG channels and one EMG channel, while the LightGBM-1EEG is designed for data with one EEG channel and one EMG channel.</li>
	<li>Before running the sleep stage prediction, include the additional SHAP calculation that helps explain the sleep stage prediction results. To process the SHAP calculation, check the Run/Plot SHAP checkbox. The SHAP calculation requires around 5-10 min to process.</li>
	<li>Click Score All Files. The model automatically scores all the EDF/EDF+ files and calculates the global and epoch SHAP values to interpret the scoring decisions in the list if that is chosen to do so. 
	NOTE: During the scoring process, the model generates the following files and saves them to the same folder where the EDF/EDF+ files are located. The model uses these files to plot the global SHAP values and epoch SHAP values. 
	 
	&#34;EDF/EDF+ file name}_{model_name}_features.csv&#34;; this file stores all the extracted feature values. 
	&#34;EDF/EDF+ file name}_{model_name}_scores.csv&#34;; this file stores the predicted sleep stages. 
	&#34;EDF/EDF+ file name}_{model_name}_rs_100hz.npy&#34;; this file stores a copy of the resampled/downsampled signals (100hz). To improve the visualization speed, the model uses the downsampled signal instead of the original signal when plotting the signal. 
	&#34;&#8203;EDF/EDF+ file name}_{model_name}explainer. pickle&#34;; &#34;{EDF/EDF+ file name}{model_name}shap_500samples.pickle&#34;; &#34;{EDF/EDF+ file name}{model_name}_indicies_500samples.npy&#34;;</li>
	<li>After finishing the sleep scoring process, click on the Visualize the Selected File option to visualize the EEG/EMG signals and a hypnogram time-aligned with the signals.
	<ol>
		<li>Score the selected file again before visualization if the epoch length is changed.</li>
	</ol>
	</li>
</ol>
4. Navigating the scored results
<ol>
	<li>Click the provided Navigation buttons to move forward and backward to see different epoch data.</li>
	<li>If SHAP calculation is chosen to be performed, view both the global and epoch-level SHAP values. Right-click on an epoch to plot the epoch-level SHAP values. 
	NOTE: It will take a few seconds to update the epoch-level SHAP plot. Figure 1 shows the GUI page overview after running the prediction for Example-1 EDF/EDF+ file with 1_LightBGM-2EEG model.</li>
</ol>
5. Interpretation of the scored sleep stages hypnogram
NOTE: There are 4 rows in the hypnogram (Figure 2). The top row is the predicted results. The bottom 3 rows are raw data of 2 EEG and 1 EMG channels, respectively. On the top row, orange suggests the Wake stage, blue suggests the NREM stage, and red suggests the REM stage in each epoch.
<ol>
	<li>To change the number of the epochs to display, click on the menu box on the right of Select Number of Epochs to Display and choose a desired value. In Figure 2, 100 epochs were chosen. Therefore, only 100 epochs are displayed in the sleep stage prediction plot. Select a smaller number in the dropdown menu to zoom into the plot.</li>
	<li>The pink transparent bar on the left of Figure 2 indicates the current location of the epoch. Left-click on any place on the hypnogram to switch to another epoch, or simply click on Go to Epoch and enter a specific number of epochs to be observed. Right-click the selected epoch to generate its epoch SHAP plot if the user has enabled the SHAP function.</li>
</ol>
6. Manual correction of the predicted sleep stages on GUI (Optional)
NOTE: if no anomaly is observed or extremely high accuracy is not required for REM stage prediction, manual verification is not needed.
<ol>
	<li>Left-click on an epoch in the sleep stage prediction plot (top plot) to select a specific epoch. The model predicted Stage of Selected Epoch is shown on the right of the text. To manually change the predicted stage on that epoch, click on the widget and select a new stage from the Wake, NREM, and REM options in the dropdown menu.</li>
	<li>The user-corrected stages are marked with dashed lines on top of the original plot (Figure 3). Close the GUI, and a new file with corrected prediction results will be automatically generated in the same folder.
	<ol>
		<li>To open a saved scored file on the GUI again, ensure that the epoch length setting and the selected model match the one used when the EDF file was initially processed to be able to reopen it. All the previously modified/scored information will be quickly loaded.</li>
	</ol>
	</li>
</ol>

Automated Sleep Stage Scoring Tool for Rodents Using LightGBM

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+sleep+and+wakefulness.">Control of sleep and wakefulness.</a> Physiol Rev. 92 (3), 1087-1187 (2012).</li><li>Lacroix, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+sleep+scoring+in+mice+reveals+human-like+stages.">Improved sleep scoring in mice reveals human-like stages.</a> BioRxiv. 489005, (2018).</li><li>Rayan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+scoring+in+rodents:+Criteria,+automatic+approaches+and+outstanding+issues.">Sleep scoring in rodents: Criteria, automatic approaches and outstanding issues.</a> Eur J Neurosci. 59 (4), 526-553 (2024).</li><li>Yamabe, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mc-sleepnet:+Large-scale+sleep+stage+scoring+in+mice+by+deep+neural+networks.">Mc-sleepnet: Large-scale sleep stage scoring in mice by deep neural networks.</a> Sci Rep. 9 (1), 15793 (2019).</li><li>Katsuki, F., Spratt, T. J., Brown, R. E., Basheer, R., Uygun, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep-deep-learner+is+taught+sleep-wake+scoring+by+the+end-user+to+complete+each+record+in+their+style.">Sleep-deep-learner is taught sleep-wake scoring by the end-user to complete each record in their style.</a> Sleep Adv. 5 (1), zpae022 (2024).</li><li>Allocca, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+'somnivore',+a+machine+learning+algorithm+for+automated+scoring+and+analysis+of+polysomnography+data.">Validation of 'somnivore', a machine learning algorithm for automated scoring and analysis of polysomnography data.</a> Front Neurosci. 13, 207 (2019).</li><li>Jha, P. K., Valekunja, U. K., Reddy, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slumbernet:+Deep+learning+classification+of+sleep+stages+using+residual+neural+networks.">Slumbernet: Deep learning classification of sleep stages using residual neural networks.</a> Sci Rep. 14 (1), 4797 (2024).</li><li>Barger, Z., Frye, C. G., Liu, D., Dan, Y., Bouchard, K. 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The authors declare no conflict of interest.

This paper presents how to use the IntelliSleepScorer (v1.2) graphic user interface to automatically score the sleep stages of mice and how to leverage SHAP values/plots to better understand the sleep stage scores generated by the model.
An important consideration when using the software is data compatibility. The in-house data used in this study was limited to electrodes placed in the frontal and parietal regions. In the independent dataset from Miladinovic and colleagues11<...

Sleep stage scoring in rodents is the procedure to identify the three stages: non-rapid eye movement sleep (NREM), rapid eye movement sleep (REM), and wake2. In rodents, NREM is characterized by reduced muscle activity, slow and regular breathing, decreased heart rate, and low-frequency oscillations of the brain waves. REM in rodents, similar to humans, shows muscle atonia, EEG activation, and rapid eye movements, although the occurrence of vivid dreaming is less clear in rodents compared to humans2,3. The &#34;wake&#34; state in rodents is marked by desynchronized brain activity with high-frequency, low-amplitude waves, increased muscle tone, and active behavior, such as grooming and exploration4. These three stages can be identified by inspecting electroencephalogram (EEG) and electromyogram (EMG) signals5.
The automatic sleep stage scoring models in rodents are in great need. First, manual sleep stage scoring by human experts is labor-intensive and time-consuming. Secondly, sleep patterns in rodents differ from those in humans and have more fragmented episodes of NREM and REM interspaced by waking, around 10 min, in contrast to 60-120 min in humans6. Therefore, identifying these brief periods during manual scoring is challenging. There have been many attempts since the 60s to develop an automatic scoring system of rodent sleep data7. Although many automated rodent sleep scoring methods exist, their performances vary8,9,10,11,12,13,14,15,16,17,18. Importantly, most high-performing models with high generalizability are not publicly available (some need special requests from developers) or are not cost-free for sleep researchers.
Therefore, to fill the current technology gap, we developed a machine learning-based model using a large dataset of 5776 h of EEG and EMG signals from 519 recordings across 124 mice with the LightGBM algorithm1. The lightGBM uses a gradient-boosting approach to construct decision trees19. In Wang et al., 2023, the LightGBM model (consisting of over 8000 decision trees) achieved an overall accuracy of 95.2% and a Cohen&#39;s kappa of 0.91, which outperformed two widely used baseline models such as the logistic regression model (accuracy = 93.3%) and the random forest model (accuracy = 94.3%, kappa = 0.89). The overall performance of the model also displayed a similar performance to that of human experts. Most importantly, the model has been proved to have generalizability and not overfitted to the original training data1: 1) It performed well (accuracy &#62; 89%) on two other publicly available independent datasets, from Miladinovic and colleagues11, with different sampling frequencies and epoch lengths; 2) The performance of the model is not impacted by the light/dark cycle of mice; 3) A modified LightGBM model performed well on data containing only one EEG and one EMG electrode with kappa &#8805; 0.89; 4) Both wildtype and mutant mice were used for the testing and the performances of the model were both accurate. This suggests the model can score sleep stages for mice with different genetic backgrounds.
In order to make this model accessible to sleep researchers who may not have coding expertise, we developed IntelliSleepScorer, a user-friendly software tool with a visually intuitive interface. The software can fully automate the sleep-scoring procedure in mice. It produces interactive visualizations of the signals, hypnogram, and Shapley Additive exPlanations (SHAP) values from an European data format (EDF)/EDF+ file input. The SHAP value approach, based on cooperative game theory, enhances the interpretability of machine learning models20. The model offers both global and epoch-level SHAP values, revealing how different feature values contribute to the scoring decision of the model overall and for each epoch. This advanced program significantly reduces the time and effort required for sleep stage scoring in mice while ensuring that downstream analysis can rely on highly accurate results. In this manuscript, we present step-by-step usage of IntelliSleepScorer (v1.2) with several updates upon version 1.0, including an option to run SHAP analysis separately from sleep pattern prediction, an user adjustable epoch length for sleep stage scoring, and a sleep stage manual correction feature integrated within the GUI.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Canonical Unbuntu 18.04</td>
			<td>Canonical</td>
			<td>https://releases.ubuntu.com/18.04/</td>
			<td>Supporting Operating System for the software IntelliSleep Scorer: Windows, Mac, or Linux</td>
		</tr>
		<tr>
			<td>Intel Core i7-8550U CPU @ 1.80 GHz 1.99 GHz; RAM: 24 GB&#160;</td>
			<td>Intel Corp</td>
			<td>https://www.intel.com/content/www/us/en/products/details/processors/core-ultra.html</td>
			<td>Hardware requirment for the software: Both Inte Core listed here have been used to process the data. It takes around 10 min to process 12 h of recording sampled at 1000 Hz for both hardwares. Any similar or superior hardware would yield comparable or better performance.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Intel Core i7-10610U CPU @1.80 GHz 2.30 GHz; RAM: 16 GB</td>
			<td>Intel Corp</td>
			<td>https://www.intel.com/content/www/us/en/products/details/processors/core-ultra.html</td>
			<td>Hardware requirment for the software: Both Inte Core listed here have been used to process the data. It takes around 10 min to process 12 h of recording sampled at 1000 Hz for both hardwares. Any similar or superior hardware would yield comparable or better performance.&#160;&#160;</td>
		</tr>
		<tr>
			<td>LightGBM</td>
			<td>Microsoft</td>
			<td>https://lightgbm.readthedocs.io/en/latest/index.html</td>
			<td>Machine learning-based algorithm that was used to train the software.&#160;</td>
		</tr>
		<tr>
			<td>MacBook Pro</td>
			<td>Apple</td>
			<td>https://www.apple.com/in/macbook-pro/</td>
			<td>Supporting Operating System for the software IntelliSleep Scorer: Windows, Mac, or Linux</td>
		</tr>
		<tr>
			<td>Windows</td>
			<td>Microsoft</td>
			<td>https://www.microsoft.com/en-in/windows/?r=1</td>
			<td>Supporting Operating System for the software IntelliSleep Scorer: Windows, Mac, or Linux</td>
		</tr>
	</tbody>
</table>

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 wake. Sleep stage scoring is crucial for studying sleep stage-specific measures and effects. 
Sleep patterns in rodents differ from those in humans, characterized by shorter episodes of NREM and REM interspaced by waking, and traditional manual sleep stage scoring by human experts is time-consuming. To address this issue, previous studies have used machine learning-based approaches to develop algorithms to automatically categorize sleep stages, but high-performing models with great generalizability are often not publicly available/cost-free nor user-friendly for non-trained sleep researchers. 
Therefore, we developed a machine learning-based LightGBM algorithm trained with a large dataset. To make the model available to sleep researchers without coding experience, a software tool named IntelliSleepScorer (v1.2- newest version) was developed based on the model, which features an easy-to-use graphic user interface. In this manuscript, we present step-by-step instructions for using the software to demonstrate a convenient and effective automatic sleep stage scoring tool in mice for sleep researchers.

There are three plots (only the top plot if SHAP values were not run) generated in the GUI after sleep stage scoring: the top plot presents EEG and EMG channels with a hypnogram of sleep stage prediction. The middle plot presents epoch SHAP values. The bottom plot presents Global SHAP values (Figure 1).
There are 4 types of data presented in the sleep stage prediction hypnogram plot (Figure 2). The top row is the predicted results. Th...

Author Spotlight: IntelliSleepScorer &#8212; A High-Accuracy, Accessible GUI Software for Automated Sleep Stage Scoring in Mice and its Application in Psychiatric Research

We present a software package with a graphic user interface for researchers without coding experience to score sleep stages in mice with a simple download and operation.

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 ...

intellisleepscorer-software-package-with-graphic-user-interface-for

Research

JoVE Journal

Neuroscience

358 Views.  Broad Institute of MIT and Harvard. We present a software package with a graphic user interface for researchers without coding experience to score sleep stages in mice with a simple download and operation. 

Video: Author Spotlight: IntelliSleepScorer — A High-Accuracy, Accessible GUI Software for Automated Sleep Stage Scoring in Mice and its Application in Psychiatric Research

A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously published phenotype assessments in several disease models, including spinocerebellar ataxias, Huntington s disease and spinobulbar muscular atrophy. Measures include hind limb clasping, ledge test, gait and kyphosis. Each measure is recorded on a scale of 0-3, with a combined total of 0-12 for all four measures. The results effectively discriminate between affected and non-affected individuals, while also quantifying the temporal progression of neurodegenerative disease phenotypes. Measures may be analyzed individually or combined into a composite phenotype score for greater statistical power. The ideal combination of the four described measures will depend upon the disorder in question. We present an example of the protocol used to assess disease severity in a transgenic mouse model of spinocerebellar ataxia type 7 (SCA7).
Albert R. La Spada and Gwenn A. Garden contributed to this manuscript equally.

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebellar ataxia. Measures include hind limb clasping, ledge test, gait and kyphosis. This protocol effectively discriminates between affected and non-affected individuals, and detects the progression of affected individuals over time.

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously ...

Automated Interactive Video Playback for Studies of Animal Communication

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, parameter-based computer animation offers the opportunity to independently manipulate any number of behavioral, morphological, or spectral characteristics in the context of realistic, moving images of animals on screen. A major limitation of conventional playback, however, is that the visual stimulus lacks the ability to interact with the live animal. Borrowing from video-game technology, we have created an automated, interactive system for video playback that controls animations in response to real-time signals from a video tracking system. We demonstrated this method by conducting mate-choice trials on female swordtail fish, Xiphophorus birchmanni. Females were given a simultaneous choice between a courting male conspecific and a courting male heterospecific (X. malinche) on opposite sides of an aquarium. The virtual male stimulus was programmed to track the horizontal position of the female, as courting males do in the wild. Mate-choice trials on wild-caught X. birchmanni females were used to validate the prototype's ability to effectively generate a realistic visual stimulus.

Video playback is a widely used technique in animal behavior. We created and evaluated a program that applies rules-based, interactive playback of 3-D computer animations in response to real-time, automated data on subject behavior.

Video playback is a widely-used technique for the controlled manipulation and presentation of visual signals in animal communication. In particular, ...

VisualEyes: A Modular Software System for Oculomotor Experimentation

Eye movement studies have provided a strong foundation forming an understanding of how the brain acquires visual information in both the normal and dysfunctional brain.1 However, development of a platform to stimulate and store eye movements can require substantial programming, time and costs. Many systems do not offer the flexibility to program numerous stimuli for a variety of experimental needs. However, the VisualEyes System has a flexible architecture, allowing the operator to choose any background and foreground stimulus, program one or two screens for tandem or opposing eye movements and stimulate the left and right eye independently. This system can significantly reduce the programming development time needed to conduct an oculomotor study. The VisualEyes System will be discussed in three parts: 1) the oculomotor recording device to acquire eye movement responses, 2) the VisualEyes software written in LabView, to generate an array of stimuli and store responses as text files and 3) offline data analysis. Eye movements can be recorded by several types of instrumentation such as: a limbus tracking system, a sclera search coil, or a video image system. Typical eye movement stimuli such as saccadic steps, vergent ramps and vergent steps with the corresponding responses will be shown. In this video report, we demonstrate the flexibility of a system to create numerous visual stimuli and record eye movements that can be utilized by basic scientists and clinicians to study healthy as well as clinical populations.

Neural control and cognitive processes can be studied through eye movements. The VisualEyes software allows an operator to program stimuli on two computer screens independently using a simple, custom scripting language. The system can stimulate tandem eye movements (saccades and smooth pursuit) or opposing eye movements (vergence) or any combination.

Eye movement studies have provided a strong foundation forming an understanding of how the brain acquires visual information in both the normal and ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

A Human-machine-interface Integrating Low-cost Sensors with a Neuromuscular Electrical Stimulation System for Post-stroke Balance Rehabilitation

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing delivery of oxygen and nutrients. About half of the stroke survivors are left with some degree of disability. Innovative methodologies for restorative neurorehabilitation are urgently required to reduce long-term disability. The ability of the nervous system to reorganize its structure, function and connections as a response to intrinsic or extrinsic stimuli is called neuroplasticity. Neuroplasticity is involved in post-stroke functional disturbances, but also in rehabilitation. Beneficial neuroplastic changes may be facilitated with non-invasive electrotherapy, such as neuromuscular electrical stimulation (NMES) and sensory electrical stimulation (SES). NMES involves coordinated electrical stimulation of motor nerves and muscles to activate them with continuous short pulses of electrical current while SES involves stimulation of sensory nerves with electrical current resulting in sensations that vary from barely perceivable to highly unpleasant. Here, active cortical participation in rehabilitation procedures may be facilitated by driving the non-invasive electrotherapy with biosignals (electromyogram (EMG), electroencephalogram (EEG), electrooculogram (EOG)) that represent simultaneous active perception and volitional effort. To achieve this in a resource-poor setting, e.g., in low- and middle-income countries, we present a low-cost human-machine-interface (HMI) by leveraging recent advances in off-the-shelf video game sensor technology. In this paper, we discuss the open-source software interface that integrates low-cost off-the-shelf sensors for visual-auditory biofeedback with non-invasive electrotherapy to assist postural control during balance rehabilitation. We demonstrate the proof-of-concept on healthy volunteers.

A novel low-cost human-machine interface for interactive post-stroke balance rehabilitation system is presented in this article. The system integrates off-the-shelf low-cost sensors towards volitionally driven electrotherapy paradigm. The proof-of-concept software interface is demonstrated on healthy volunteers.

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing ...

Polygraphic Recording Procedure for Measuring Sleep in Mice

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the homeodynamics and circuitry of sleep-wake regulation. In many laboratories, a cable-based sleep recording system is used to monitor the EEG and EMG in freely behaving mice in combination with computer software for automatic scoring of the vigilance states on the basis of power spectrum analysis of EEG data. A description of this system is detailed herein. Steel screws are implanted over the frontal cortical area and the parietal area of 1 hemisphere for monitoring EEG signals. In addition, EMG activity is monitored by the bilateral placement of wires in both neck muscles. Non-rapid eye movement (Non-REM; NREM) sleep is characterized by large, slow brain waves with delta activity below 4 Hz in the EEG, whereas a shift from low-frequency delta activity to a rapid low-voltage EEG in the theta range between 6 and 10 Hz can be observed at the transition from NREM to REM sleep. By contrast, wakefulness is identified by low- to moderate-voltage brain waves in the EEG trace and significant EMG activity.

The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the ...

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has proven to be an invaluable model organism in helping to tackle this challenge, from inspiring technological approaches,&#160;building the human brain connectome, to actually shedding light on the specific molecular drivers of basic functional patterns. The bulk of the behavioral studies in C. elegans have been performed on solid substrates. In liquid, animals exhibit behavioral patterns that include movement at a range of speeds in 3D, as well as partial body movements, such as a posterior curl without anterior shape change, which introduce new challenges for quantitation. The steps of a simple procedure, and use of a software that enables high-resolution analysis of C. elegans swim behavior, are presented here. The software, named CeleST, uses a specialized computer program that tracks multiple animals simultaneously and provides novel measures of C. elegans locomotion in liquid (swimming). The measures are mostly grounded in animal posture and based on mathematics used in computer vision and pattern recognition, without computational requirements for threshold cut-offs. The software tool can be used to both assess overall swimming prowess in hundreds of animals from combined small batch trials and to reveal novel phenotypes even in well-characterized genetic mutants. The preparation of specimens for analysis with CeleST is simple and low-tech, enabling wide adaptation by the scientific community. Use of the computational approach described here should therefore contribute to the greater understanding of behavior and behavioral circuits in the C. elegans model.

Automated Analysis of C. elegans Swim Behavior Using CeleST Software

An efficient and simple methodology for computer-based analysis of nematode swimming behavior in liquid is described. The method requires little to no investment for C. elegans laboratories. The hardware used is standard, and the computer software for the behavioral analysis (CeleST) is an open source one.

Dissecting the neuronal and neuromuscular circuits that regulate behavior remains a major challenge in biology. The nematode Caenorhabditis elegans has ...

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...