This research was funded by the Intramural Research Program of the NIMH (ZIA MH00889). RMS was also supported by a FRAXA Postdoctoral Fellowship.

NOTE: 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. Sleep Deprivation Setup
<ol>
	<li>Use soft paintbrushes to gently poke mice when asleep.
	<ol>
		<li>Make sure the paintbrush can fit through the bars of the mouse cage feeder to ensure that mice have access to food and water ad libitum during sleep deprivation....

<ol>
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Here we describe a method for sleep deprivation, gentle handling, that can be used in both rodent pups and in adults. Gentle handing requires researchers to observe mice for the duration of a sleep deprivation period and gently prod the animals whenever they are inactive or twitching to prevent sleep. Prior sleep deprivation studies have verified by means of simultaneous electroencephalogram (EEG) recordings that gentle handling prevents both REM and non-REM (NREM) sleep10. While gentle handling i...

Sleep plays a critical role in neuronal plasticity and synapse formation and elimination during brain development1,2. Specifically, rapid eye movement (REM) sleep is essential for forming stable synaptic circuits through both strengthening of specific synapses and synaptic pruning2. Pharmacological sleep deprivation early in life leads to many physiological and behavioral deficits3,4. In addition to pharmacological sleep deprivation, other forms of sleep deprivation such as constant shaking, forced locomotion, or presentation of an aversive stimulus have been associated with depression-like symptoms during adulthood, different neural activation patterns, and changes in sleep time and sleep continuity during a recovery period following deprivation5,6,7. When sleep is chronically restricted in mice for over five weeks during development, behavioral deficits were observed following a month of sleep recovery8. Taken together, these studies suggest that disruption of sleep in the neonatal rodent can affect later sleep patterns, brain function, and behavior. Highlighting the importance of sleep for normal brain development in humans, many patients with neurodevelopmental disorders, including Autism Spectrum Disorders, Tuberous Sclerosis Complex, Fragile X Syndrome, and Attention-Deficit/Hyperactivity Disorder, report abnormalities in sleep patterns in addition to a variety of behavioral deficits1.
Given the number of neurodevelopmental disorders that are associated with abnormal sleep patterns, it is important to understand how lack of sleep affects brain function and behavior. However, despite the data that support the importance of sleep during development, most methodologies that restrict or deprive animals of sleep focus on adults. These techniques include a variety of tests that require forced locomotor activity (e.g., constant treadmill), constant disruption (e.g., rotating sweeper bar or continual novel object presentation), or disturbing the animal at the onset of REM sleep (e.g., platform over water)9,10. Although these methods have effectively shown the problems associated with sleep deprivation in adults, none of these methods are appropriate for rodent pups because of their limited mobility, high sleep drive, and the adverse effects of stress early in life. Due to the importance of sleep during development, it is critical to understand how restricted sleep in neonatal pups affects future behavior during adulthood.
One technique to restrict sleep that can be used in rodent pups is gentle handling. Gentle handling involves the investigator directly interacting with the rodents any time sleep behavior is observed. Investigators can use their hands, a paintbrush, or presentation of novel objects to physically disrupt sleep in neonatal rodents8,10,11,12,13,14. Sleep can be behaviorally determined by lack of motor activity, myoclonic twitching, and eye closure (in older rodent pups). EEG validation confirms that disrupting sleep with gentle handling based on these behaviors reduces total sleep time by 91% in P12 rats14. Other techniques that have been used to deprive young mice include electric shock and presentation of unpleasant stimuli, but these techniques cannot be used for chronic sleep restriction and are more stressful than gentle handling7,15. Chronic sleep restriction can also be accomplished through a shaking protocol that can be used with or without EEG electrode implantation and associated computer software, but this protocol does not prevent all sleep and therefore is less controlled compared to gentle handling4,16. The ability to restrict or deprive mice of sleep for many days in a row without specific equipment, computer software, or electrode implantation is a benefit of the gentle handling technique. Chronic sleep restriction via gentle handling effectively limits sleep in neonatal rodents and produces a variety of behavioral changes following the sleep deprivation period8,11,12,17.
Here, we describe results in which chronic sleep deprivation for three hours per day from postnatal day 5 (P5) to P42 significantly affects sociability of mice following a 30-day recovery period in which sleep is not disturbed. These results highlight the long-term effects of chronic sleep restriction during development on future behavior.

<table>
	<tbody>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Jackson Labs</td>
			<td>000664</td>
			<td>Wildtype mice used</td>
		</tr>
		<tr>
			<td>Super Mouse 750 Mouse Cage</td>
			<td>Lab Products, Inc.&#160;</td>
			<td></td>
			<td>Cages for the mice</td>
		</tr>
		<tr>
			<td>SANI-Chips Bedding</td>
			<td>PJ Murphys</td>
			<td></td>
			<td>Bedding for the mice</td>
		</tr>
		<tr>
			<td>S&#38;S Bristle Brush Assortment Pack</td>
			<td>Staples</td>
			<td>Item # 13764, Model AB37100</td>
			<td>Paint brushes are used to gently poke mice</td>
		</tr>
	</tbody>
</table>

chronic sleep deprivation in mouse pups by means of gentle handling

Sleep is critical for proper development and neural plasticity. Moreover, abnormal sleep patterns are characteristic of many neurodevelopmental disorders. Studying how chronic sleep restriction during development can affect adult behavior may add to our understanding of the emergence of behavioral symptoms of neurodevelopmental disorders. While there are many methods that can be used to restrict sleep in rodents including forced locomotion, constant disruption, presentation of an aversive stimulus, or electric shock, many of these methods are very stressful and cannot be used in neonatal mice. Here, we describe gentle handling, a sleep deprivation technique that can be used chronically throughout development and into adulthood to achieve sleep restriction. Gentle handling involves close observation of the mice throughout the sleep deprivation period and requires the researcher to gently prod the animals whenever they are inactive or display behaviors associated with sleep. Coupled with EEG recordings, gentle handling could be used to selectively disrupt a specific phase of sleep such as rapid eye movement (REM) sleep. The technique of gentle handling is a powerful tool for the study of the effects of chronic sleep restriction even in neonatal mice that circumvents many of the more stressful procedures used for sleep deprivation.

To investigate the effects of chronic sleep restriction on behavior, we sleep restricted mice for three hours daily between 11:00 AM and 2:00 PM from P5 to P428. Following the sleep restriction, mice were left undisturbed for four weeks and allowed to recover from sleep restriction. Mice were tested for behavior following the four-week recovery period. The three-chamber social behavior test was used to assess sociability (preference for a novel mouse compared to a ...

Watch this Scientific Journal Video about Chronic Sleep Deprivation in Mouse Pups by Means of Gentle Handling at JoVE.com

Chronic Sleep Deprivation in Mouse Pups by Means of Gentle Handling

We describe a sleep deprivation technique known as gentle handling where investigators gently prod mice any time sleep behavior is observed. This method is a powerful tool that allows researchers to study the effects that chronic sleep restriction throughout development can have on future brain physiology and behavior.

Sleep is critical for proper development and neural plasticity. Moreover, abnormal sleep patterns are characteristic of many neurodevelopmental disorders. ...

This method can help answer key questions in the sleep field such as the affects of chronic sleep restriction during postnatal development. The main advantage of this technique is that it is less stressful than other sleep deprivation techniques and can easily be used throughout development and continue into adulthood. Begin by assigning each cage to either the sleep deprivation or control group.Ensure that mice have access to food and water ad libitum, and remove any food, bedding, or enrichment objects that might restrict the view of the mice. For the control group, remove the sire from the breeder cage on postnatal day five and leave the dam with the pups until weaning. Next, move mice assigned to the control group to the sleep deprivation room and gently prod the mice with a paintbrush continuously for 10 minutes a day for the duration of the experiment.After 10 minutes, return the mice to the animal holding room and do not disturb them. Begin by removing the sire from the breeder cage on postnatal day five and leave the dam with the pups until weaning. Move mice assigned to the sleep deprivation group to the sleep deprivation room daily during the light cycle where they will be monitored.During this time, gently prod the mouse with a paintbrush if suspected of being asleep until a response is observed. Alternatively, invert mice and push over onto their backs to disrupt sleep. Consider a mouse to be asleep if any of the following occur:twitching, inactivity, or if eyes are closed.Consider mice to be awake if they are moving around, trying to flip over after being on their backs, or grooming themselves. If the dam is covering the pups during the sleep deprivation, gently prod her away from the pups so the view of the pups is unrestricted. Continue with sleep deprivation protocol until the experiment endpoint.When the three-hour period of sleep deprivation is complete, put the bedding and any food that may have been removed back and put the lid back on the cage. Finally, return cages to the animal holding room until the next day of sleep deprivation. Following a one month recovery period, results indicated that in the sociability test, sleep restricted females spent more time sniffing the stranger mouse than control females and sleep restricted males had a tendency to spend more time sniffing the object when compared to control males.Further, in the social novelty phase of testing, sleep restricted female mice spent an increased amount of time sniffing the novel mouse than female controls. While attempting this procedure, it's important to remember to pay attention and promptly respond to pre-sleep behaviors, especially in neonatal mice where sleep need is high. Following this procedure, other methods like open field, rotarod, elevated plus maze, and social behavior testing can be performed in order to answer additional questions like the long-term behavioral affects of chronic sleep restriction during development.

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JoVE Journal

Behavior

9.9K visualizações.  National Institutes of Health. Este método pode ajudar a responder a perguntas-chave no campo do sono, como os efeitos da restrição crônica do sono durante o desenvolvimento pós-natal. A principal vantagem dessa técnica é que ela é menos estressante do que outras técnicas de privação do sono e pode ser facilmente usada durante todo o desenvolvimento e continuar até a idade adulta. Comece atribuindo cada gaiola à privação do sono ou ao grupo de controle.Certifique-se de que os ratos tenham acesso a ali...