Name: noninvasive, high-throughput determination of sleep duration in rodents
Uploaded: 2018-04-18T15:00:00
Description: Watch this Scientific Journal Video about Noninvasive, High-throughput Determination of Sleep Duration in Rodents at JoVE.com

The authors would like to acknowledge the NIH Fellows Editorial Board for their editorial assistance. This research was funded by the Intramural Research Program of the NIMH (ZIA MH00889). RMS was also supported by a FRAXA Postdoctoral Fellowship.

All the procedures were approved by the National Institute of Mental Health Animal Care and Use Committee and performed in accordance with the National Institutes of Health Guidelines on the Care and Use of Animals.
1. Setting up the Sleep Detection Units
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	<li>Purchase the desired number of units and software.</li>
	<li>Follow the instructions to set up the monitoring systems.
	<ol>
		<li>Align a detector opposite to an emitter. Make sure the infrared beams are facing inwards and are al...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+sleep+restriction+during+development+can+lead+to+long-lasting+behavioral+effects.">Chronic sleep restriction during development can lead to long-lasting behavioral effects.</a> Physiology &amp; behavior. 155, 208-217 (2015).</li><li>Moretti, P., Bouwknecht, J. A., Teague, R., Paylor, R., Zoghbi, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormalities+of+social+interactions+and+home-cage+behavior+in+a+mouse+model+of+Rett+syndrome.">Abnormalities of social interactions and home-cage behavior in a mouse model of Rett syndrome.</a> Human molecular genetics. 14, 205-220 (2005).</li><li>Guzman, M. 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Here, we present a noninvasive, high-throughput method for determination of sleep duration based on activity monitoring in the home-cage. This method of assessment of total sleep time has been validated against EEG studies3. Activity-based home-cage monitoring is simple, noninvasive, and applicable to population studies in large numbers of animals. It is limited in that it cannot give detailed information about sleep (such as sleep bout duration and sleep stages).
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Sleep has important functions for the restoration of the body and brain following the daily burden of wakefulness1. It has been shown that sleep plays a role in memory retention and general brain plasticity1. The EEG is the gold standard to detect sleep2. In rodents, EEG monitoring requires surgical implantation of electrodes affixed to a head-mount, after which the animal needs a period of time to recover2. After the recovery, the animal is attached to the recording device and is given another period of habituation2. Because of these necessary periods of recovery and habituation, EEG is time consuming and laborious and cannot be reasonably performed on a large scale. Additionally, the surgical procedure of electrode implantation carries an inherent risk to the animal. Finally, the data analysis for scoring sleep in EEG studies is also very laborious. An alternative, non-invasive, high-throughput method of sleep monitoring would greatly aid rodent sleep research.
An activity-based home-cage monitoring system used to detect sleep addresses the limitations of EEG studies. The simple premise is that an inactive animal is likely a sleeping animal. It has been shown that 40 s of continuous inactivity (binned in 10 s epochs) is a reliable measure of sleep as measured with an EEG (shown to have 88-94% agreement)3. Home-cage monitoring systems can be used to study large groups of animals with minimal setup time. We have shown that it takes animals approximately one day to habituate to individual housing in the home-cage monitoring system4 in contrast to the weeks of recovery needed for EEG studies2. In addition, some setups can also detect physiological parameters such as core body temperature, heart rate, activity, and feeding. Temperature and heart rate are determined from the implantation of a small transmitter. These parameters can provide more information about the mouse and may be used in parallel with the sleep recording to further add to our understanding of sleep and how it is affected.
While it is a powerful tool, there are some limitations to the types of data that can be acquired from activity-based home-cage monitoring. EEG studies can differentiate between REM and non-REM sleep, which may be important for a deeper understanding of sleep architecture. Activity-based home-cage monitoring systems can only provide data for total sleep duration. In addition, although the output for activity-based home-cage monitoring gives information about sleep bout duration, we cannot accurately assess bout duration because of the inherent limitation of 40 s intervals3. Despite these limitations, home-cage monitoring of sleep duration provides an important biological measure that may influence many downstream factors including the animal&#39;s health and behavior5.
Activity-based home-cage monitoring has been used to detect sleep in many studies indicating its versatility. We cite a sample of these studies4,6,7,8,9,10,11,12. In addition to the method presented, there are other methods of detecting sleep via activity-based monitoring, each containing its own limitations13,14. Some of these studies examine long periods of uninterrupted sleep (72 h) while some examine sleep in blocks of 24 h. In this study, we present sleep analysis for each 24 h period after the response to daily intraperitoneal (IP) injections and to periodic cage changes in a mouse model of fragile X syndrome (Fmr1 KO mice). We chose Fmr1 KO mice because they have reduced sleep4 and are hypothesized to be hyper-reactive to sensory information15. Our data highlight the ability to detect changes in sleep patterns in response to a stressful event. This method is ideal for obtaining general information about sleep in large cohorts of mice. The method can be useful for understanding the effects of specific genetic alterations on sleep, the effects of pharmacological treatments, or responses to events, such as a stressor. In addition, the method provides a simple means of screening for a response before initiating more involved studies.

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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Comprehensive Lab Animal Monitoring System (CLAMS)</td>
			<td style="width:71px;">Columbus Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Equipment and software to analyze sleep duration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Captisol Research Grade</td>
			<td style="width:71px;">Captisol</td>
			<td style="width:119px;">RC-0C7-100</td>
			<td>Captisol for dissolving hydrophobic compounds</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">30 G BD Needle 1/2 inch</td>
			<td style="width:71px;">BD</td>
			<td align="right" style="width:119px;">305106</td>
			<td>Needle for injections</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD Disposable Syringes</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">14-823-30</td>
			<td>Syringes for injections</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">B6.129P2-Fmr1tm1Cgr/J</td>
			<td style="width:71px;">Jackson Labs</td>
			<td align="right" style="width:119px;">3025</td>
			<td>Fmr1 KO mice</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Super Mouse 750 Mouse Cage</td>
			<td style="width:71px;">Lab Products, Inc.&#160;</td>
			<td style="width:119px;"></td>
			<td>Homecages for the mice</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SANI-Chips Bedding</td>
			<td style="width:71px;">PJ Murphys</td>
			<td style="width:119px;"></td>
			<td>Bedding for the mice</td>
		</tr>
	</tbody>
</table>

noninvasive, high-throughput determination of sleep duration in rodents

Traditionally, sleep is monitored by an electroencephalogram (EEG). EEG studies in rodents require surgical implantation of the electrodes followed by a long recovery period. To perform an EEG recording, the animal is connected to a receiver, creating an unnatural tether to the head-mount. EEG monitoring is time consuming, carries risk to the animal, and is not a completely natural setting for the measurement of sleep. Alternative methods to detect sleep, particularly in a high-throughput fashion, would greatly advance the field of sleep research. Here, we describe a validated method for detecting sleep via activity-based home-cage monitoring. Previous studies have shown that sleep assessed via this method has a high degree of agreement with sleep defined by traditional EEG-based measures. Whereas this method is validated for total sleep time, it is important to note that sleep bout duration should be assessed by an EEG which has better temporal resolution. The EEG can also differentiate rapid eye movement (REM) and non-REM sleep, giving more detail about the exact nature of sleep. Nevertheless, activity-based sleep determination can be used to analyze multiple days of undisturbed sleep and to assess sleep as a response to an acute event (like stress). Here, we show the power of this system to detect the response of mice to daily intraperitoneal injections.

To determine the effect of daily injections on sleep and whether animals habituate to the injections, we performed daily IP injections for 14 consecutive days at 9:00 AM (light cycle began at 6:00 AM) and recorded sleep duration in 12 Fmr1 KO C57Bl/6J mice. We used a within subjects&#39; design, injecting each animal with normal saline for 4 consecutive days (Days 1-4) and then 30% cyclodextrin for the following ten consecutive days (Days 5-14). Cyclodextrin was selected because ...

Watch this Scientific Journal Video about Noninvasive, High-throughput Determination of Sleep Duration in Rodents at JoVE.com

Noninvasive, High-throughput Determination of Sleep Duration in Rodents

We describe a high-throughput method of measuring sleep by means of activity-based home-cage monitoring. This method offers advantages over traditional EEG-based methods. It is well validated for the determination of total sleep duration and can be a powerful tool to monitor sleep in rodent models of human disease.

Traditionally, sleep is monitored by an electroencephalogram (EEG). EEG studies in rodents require surgical implantation of the electrodes followed by a ...

The overall goal of this experiment is to record total sleep duration in a high throughput and non-invasive manner. This method could help answer key questions in the sleep field such as total sleep duration. The main advantage of this technique is that it is high throughput, non-invasive, and easily executable.Demonstrating the procedure will be Abigail Lemons, a post-baccalaureate from the laboratory. To begin the procedure, align a detector opposite to an emitter. Make sure the infrared beams are facing inwards and are aligned at the same height.Next, use the provided screws to position the detectors and emitters at the desired height on the metal stand. This height should be adjusted so that the bedding of the cage is below the level of the infrared beams, but the beam is at the proper height to detect the animals'activity. This creates an internal area of 27 centimeters by 32 centimeters.Then, connect to each detector with each emitter. Connect the emitter to the provided hub linked to the receiver. Perform this for both the X and the Y planes.Repeat this for all setups. Afterward, connect the receiver to a computer with the provided USB hub. To set up the software, click file, then open experiment configuration to open the default experiment configuration.Then, click experiment, properties. Subsequently click the scan tab. Change the scan rate to 10 seconds.Next, click the activity tab. Change the activity sampling rate to 10 seconds. Then, click file, save experiment configuration as, to save these configuration settings for future experiments.In this step singly house the mice in clean cages that are 31 centimeters long and 16.5 centimeters wide. To prevent the mice from building up the bedding and obstructing the beams, use bedding at a depth of 3 millimeters, and do not provide additional nesting material. Provide the mice with access to food and water ad libitum by means of a wire feeder that rests on the top of the cage out of the way of the beams.If necessary, refill the food and change the water bottles when cages are changed every three to five days throughout the duration of the study. Then, align the mouse cage inside the infrared beam setup ensuring that it rests in approximately the middle of the beans for full coverage. Now, open the default configuration file.Click file, open experiment configuration, to open the desired or default experiment configuration. Next, click experiment, then setup. Designate the location to save the data file, as well as the location to save the backup file.Then, designate the animal ID to each sleep chamber, and enter the weight of the animal if desired. If a chamber is not being used in the experiment, unchecking the box will deactivate that chamber. Once the identification information is entered, click done.Click file, save experiment as, to save the current experiment configuration under the desired file name. Afterward, click experiment, run, to start the recording. Wait for a 10 second epoch to make sure that all chambers are picking up activity.As the animals have just been injected, it is highly likely that the animals will be moving enough to be detected by the infrared beams. The following day, at the time of injection, click experiment, then stop, to end the recording. Afterward, click file, export, generate subject CSV's to collect the raw data for each mouse.Subsequently, click file, export, and sleep analysis to export the sleep file for each experiment by opening the raw. CDTA data file. Under detection parameters activity source, ensure that both the X-axis and Y-axis boxes are checked.Next, under sleep threshold epochs, ensure that four epochs are selected. Under sleep threshold activity threshold, ensure that 0 counts are selected, and under light-dark cycle, check the appropriate time for the light-dark cycle. Under analysis window, select the desired time for analysis.In the case of the injection studies, leave the day as default. Set the start time as ExpSTART, and the duration as 24:00 for 24 hours. Save the configuration to save time for exporting data, Then, click update.Click generate CSV file, and save the file to the desired location. To analyze the data for each recording session, open the sleep file. To assign the subject ID, open the individual CSV file for each chamber to determine the subject ID.Record the TOT sleep, hours, minutes, and seconds, for both the light and the dark phases for all animals.Check the individual subject CSV file for inconsistencies indicating the failure of recording. If high beam counts are detected in one plane, but no counts are observed on the other plane, this indicates beam failure. This figure shows the mice received daily IP injections of either saline or 30%cyclodextrin at 9 a.m.in the light phase. Boxes around the arrows indicate a cage change. Post hoc T-tests suggest that sleep duration differed in the light phase on day one from other days, indicating the habituation to both the sleep setup and the IP injections.The sleep was reduced by cage changes on days six, nine, and 13, compared to other days. Sleep duration following cyclodextrin injections was relatively stable across the days when cages were not changed, indicating that the mice habituated to the IP cyclodextrin injections. Once mastered, setup can be performed in under an hour and analysis can also performed very rapidly.Following this procedure, other methods such as EEG can be performed in order to answer additional questions like sleep fragmentation or sleep stages.

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