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.</li>
		<li>Do not use paintbrushes for multiple cages to prevent the transfer of scents between cages. We suggest that you label each brush with its assigned cage and restrict its use to that cage.</li>
	</ol>
	</li>
	<li>Designate a room where all sleep deprivation will occur.</li>
</ol>
2. Animal Setup
<ol>
	<li>Randomly assign each cage to either sleep deprivation or control group.</li>
	<li>Give mice access to food and water ad libitum during the experiment.</li>
	<li>Make sure view of mice is not restricted by bedding, nest material, or enrichment objects in cage.
	<ol>
		<li>Remove extra bedding or enrichment objects that could restrict the view of the mice.</li>
	</ol>
	</li>
	<li>If food or water restricts view of mice, move mice to an area of cage where the view is not restricted.
	<ol>
		<li>If the food blocks a large portion of the cage, remove some food and place pellets at the bottom of the cage so that animals maintain access to food during the sleep deprivation period.</li>
	</ol>
	</li>
</ol>
3. Animal Conditions
NOTE: We chose to begin the three hour sleep deprivation period at 11 AM, when mice are in the middle of their inactive phase, but the time of day and duration of sleep deprivation can be chosen based on the desired experimental conditions.
<ol>
	<li>Control Group.
	<ol>
		<li>On P5, remove the sire from the breeder cage and leave the dam with the pups until weaning (P21).</li>
		<li>Move mice assigned to the control group to the sleep deprivation room and gently prod the mice with a paint brush continuously for 10 minutes a day for the duration of the experiment (P5 to P42).</li>
		<li>After these 10 minutes, return the mice to the animal holding room and do not disturb them</li>
	</ol>
	</li>
	<li>Sleep Deprivation Group.
	<ol>
		<li>On P5, remove the sire from the breeder cage and leave the dam with the pups until weaning (P21). 
		NOTE: Sleep deprivation prior to P5 may result in cannibalization of the pups. We did not observe significant cannibalization at P5 and later. 
		NOTE: In young pups, the drive to sleep is very high requiring almost constant stroking. As the mice develop, especially after weaning, the sleep pressure is lower and they do not need constant prodding.</li>
		<li>Continue with sleep deprivation every day until P42. 
		NOTE: Sleep deprivation protocols can vary in length and extend past P42 if desired.</li>
		<li>Move mice assigned to the sleep deprivation group to the sleep deprivation room daily during the light cycle where they will be monitored. The exact duration of sleep deprivation can be adjusted based on the specific research question. 
		NOTE: In neonatal mice sleep deprivation that lasts longer than 3 hours is difficult because of the increase in sleep pressure15.
		<ol>
			<li>During this time, gently prod the mice with the paintbrush any time that sleep is evident. Specifics of sleep deprivation are explained in the next section.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Sleep Deprivation
<ol>
	<li>Gently prod mouse with paintbrush if suspected of being asleep until a response is observed. Alternatively, invert mice and push over onto their backs to disrupt sleep.
	<ol>
		<li>Consider a mouse to be asleep if any of the following occur: inactivity, twitching (especially with pups), or if their eyes are closed (in mice older than P12).</li>
		<li>Consider mice to be awake if they are moving around, trying to flip over after being on their backs, or grooming themselves.</li>
	</ol>
	</li>
	<li>If dam is covering pups during sleep deprivation, gently prod her away from pups so the view of the pups is unrestricted.</li>
</ol>
5. After Sleep Deprivation
<ol>
	<li>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.</li>
	<li>Return cages to animal holding room until the next day of sleep deprivation.</li>
</ol>
6. Subsequent Experiments
<ol>
	<li>Depending on the goal of the sleep deprivation study, proceed with subsequent experiments.
	<ol>
		<li>Harvest serum from animals to determine corticosterone levels18.</li>
		<li>Do any of a number of behavioral tests, including Open field, RotaRod, Elevated Plus Maze, Social behavior testing, Marble Burying, etc.8,19,20 These behavior tests will also prevent the animals from sleeping so that should be factored in when determining how many days of continuous sleep deprivation to employ.</li>
	</ol>
	</li>
</ol>

<ol><li>Picchioni, D., Reith, R. M., Nadel, J. L., Smith, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sleep%2C+plasticity+and+the+pathophysiology+of+neurodevelopmental+disorders%3A+the+potential+roles+of+protein+synthesis+and+other+cellular+processes%5BTitle%5D)+AND+%22Brain+Sci%22%5BJournal%5D)">Sleep, plasticity and the pathophysiology of neurodevelopmental disorders: the potential roles of protein synthesis and other cellular processes.</a> Brain Sci. 4 (1), 150-201 (2014).</li>
<li>Li, W., Ma, L., Yang, G., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((REM+sleep+selectively+prunes+and+maintains+new+synapses+in+development+and+learning%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">REM sleep selectively prunes and maintains new synapses in development and learning.</a> Nat Neurosci. 20 (3), 427-437 (2017).</li>
<li>Peirano, P. D., Algarin, C. R. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/18575679">Sleep in brain development.</a> Biol Res. 40 (4), 471-478 (2007).</li>
<li>Mirmiran, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+experimental+suppression+of+active+%28REM%29+sleep+during+early+development+upon+adult+brain+and+behavior+in+the+rat%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">Effects of experimental suppression of active (REM) sleep during early development upon adult brain and behavior in the rat.</a> Brain Res. 283 (2-3), 277-286 (1983).</li>
<li>Feng, P., Ma, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Instrumental+REM+sleep+deprivation+in+neonates+leads+to+adult+depression-like+behaviors+in+rats%5BTitle%5D)+AND+%22Sleep%22%5BJournal%5D)">Instrumental REM sleep deprivation in neonates leads to adult depression-like behaviors in rats.</a> Sleep. 26 (8), 990-996 (2003).</li>
<li>Frank, M. G., Morrissette, R., Heller, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+sleep+deprivation+in+neonatal+rats%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Effects of sleep deprivation in neonatal rats.</a> Am J Physiol. 275 (1 Pt 2), R148-R157 (1998).</li>
<li>Todd, W. D., Gibson, J. L., Shaw, C. S., Blumberg, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brainstem+and+hypothalamic+regulation+of+sleep+pressure+and+rebound+in+newborn+rats%5BTitle%5D)+AND+%22Behav+Neurosci%22%5BJournal%5D)">Brainstem and hypothalamic regulation of sleep pressure and rebound in newborn rats.</a> Behav Neurosci. 124 (1), 69-78 (2010).</li>
<li>Sare, R. M., Levine, M., Hildreth, C., Picchioni, D., Smith, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+sleep+restriction+during+development+can+lead+to+long-lasting+behavioral+effects%5BTitle%5D)+AND+%22Physiol+Behav%22%5BJournal%5D)">Chronic sleep restriction during development can lead to long-lasting behavioral effects.</a> Physiol Behav. 155, 208-217 (2016).</li>
<li>Colavito, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experimental+sleep+deprivation+as+a+tool+to+test+memory+deficits+in+rodents%5BTitle%5D)+AND+%22Front+Syst+Neurosci%22%5BJournal%5D)">Experimental sleep deprivation as a tool to test memory deficits in rodents.</a> Front Syst Neurosci. 7, 106(2013).</li>
<li>Franken, P., Dijk, D. J., Tobler, I., Borbely, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sleep+deprivation+in+rats%3A+Effects+on+EEG+power+spectra%2C+vigilance+states%2C+and+cortical+temperature%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">Sleep deprivation in rats: Effects on EEG power spectra, vigilance states, and cortical temperature.</a> Am J Physiol. 261 (1 Pt 2), R198-R208 (1991).</li>
<li>Araujo, P., Coelho, C. A., Oliveira, M. G., Tufik, S., Andersen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neonatal+sleep+restriction+increases+nociceptive+sensitivity+in+adolescent+mice%5BTitle%5D)+AND+%22Pain+Physician%22%5BJournal%5D)">Neonatal sleep restriction increases nociceptive sensitivity in adolescent mice.</a> Pain Physician. 21 (2), E137-E148 (2018).</li>
<li>Araujo, P., Tufik, S., Anderson, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sleep+and+pain%3A+A+relationship+that+begins+in+early+life%5BTitle%5D)+AND+%22Pain+Physician%22%5BJournal%5D)">Sleep and pain: A relationship that begins in early life.</a> Pain Physician. 17 (6), E787-E798 (2014).</li>
<li>Hairston, I. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sleep+deprivation+effects+on+growth+factor+expression+in+neonatal+rats%3A+a+potential+role+for+BDNF+in+the+mediation+of+delta+power%5BTitle%5D)+AND+%22J+Neurophysiol%22%5BJournal%5D)">Sleep deprivation effects on growth factor expression in neonatal rats: a potential role for BDNF in the mediation of delta power.</a> J Neurophysiol. 91 (4), 1586-1595 (2004).</li>
<li>Hairston, I. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sleep+deprivation+elevates+plasma+corticosterone+levels+in+neonatal+rats%5BTitle%5D)+AND+%22Neurosci+Lett%22%5BJournal%5D)">Sleep deprivation elevates plasma corticosterone levels in neonatal rats.</a> Neurosci Lett. 315 (1-2), 29-32 (2001).</li>
<li>Blumberg, M. S., Middlemis-Brown, J. E., Johnson, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sleep+homeostasis+in+infant+rats%5BTitle%5D)+AND+%22Behav+Neurosci%22%5BJournal%5D)">Sleep homeostasis in infant rats.</a> Behav Neurosci. 118 (6), 1253-1261 (2004).</li>
<li>Feng, P., Vogel, G. W., Obermeyer, W., Kinney, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+instrumental+method+for+long-term+continuous+REM+sleep+deprivation+of+neonatal+rats%5BTitle%5D)+AND+%22Sleep%22%5BJournal%5D)">An instrumental method for long-term continuous REM sleep deprivation of neonatal rats.</a> Sleep. 23 (2), 175-183 (2000).</li>
<li>Xu, Z. Q., Gao, C. Y., Fang, C. Q., Zhou, H. D., Jiang, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mechanism+and+characterization+of+learning+and+memory+impairment+in+sleep-deprived+mice%5BTitle%5D)+AND+%22Cell+Biochem+Biophys%22%5BJournal%5D)">The mechanism and characterization of learning and memory impairment in sleep-deprived mice.</a> Cell Biochem Biophys. 58 (3), 137-140 (2010).</li>
<li>Barriga, C., Martin, M. I., Tabla, R., Ortega, E., Rodriguez, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circadian+rhythm+of+melatonin%2C+corticosterone+and+phagocytosis%3A+Effect+of+stress%5BTitle%5D)+AND+%22J+Pineal+Res%22%5BJournal%5D)">Circadian rhythm of melatonin, corticosterone and phagocytosis: Effect of stress.</a> J Pineal Res. 30 (3), 180-187 (2001).</li>
<li>Nadler, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+apparatus+for+quantitation+of+social+approach+behaviors+in+mice%5BTitle%5D)+AND+%22Genes+Brain+Behav%22%5BJournal%5D)">Automated apparatus for quantitation of social approach behaviors in mice.</a> Genes Brain Behav. 3 (5), 303-314 (2004).</li>
<li>Thomas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Marble+burying+reflects+a+repetitive+and+perseverative+behavior+more+than+novelty-induced+anxiety%5BTitle%5D)+AND+%22Psychopharmacology+%28Berl%29%22%5BJournal%5D)">Marble burying reflects a repetitive and perseverative behavior more than novelty-induced anxiety.</a> Psychopharmacology (Berl). 204 (2), 361-373 (2009).</li>
<li>Karlsson, K. A., Blumberg, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+union+of+the+state%3A+Myoclonic+twitching+is+coupled+with+nuchal+muscle+atonia+in+infant+rats%5BTitle%5D)+AND+%22Behav+Neurosci%22%5BJournal%5D)">The union of the state: Myoclonic twitching is coupled with nuchal muscle atonia in infant rats.</a> Behav Neurosci. 116 (5), 912-917 (2002).</li>
</ol>

The authors have nothing to disclose.

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 is one of the only sleep deprivation methods that can be used on young rodent pups, there are some limitations to its usage.
First, gentle handling relies heavily on the experimenter to be able to both recognize and promptly respond to pre-sleep behaviors. When depriving sleep in rodent pups, the pups must be almost constantly prodded or inverted to assure that they are awake for the entire sleep restriction period. Recognition of twitching in young pups is critical to properly prevent sleep21. Second, it should be acknowledged that sleep deprivation that occurs before weaning, when the pups are still with their mother, could affect the mother&#39;s sleep as a secondary effect; however, the extent of this effect has yet to be explored. Third, it is important to recognize that any sleep deprivation process will be inherently stressful, and it is not possible to separate the effects of sleep deprivation from the stress that is caused by the procedure. However, using the gentle handling protocol described here attempts to minimize the stress of sleep deprivation compared to other sleep deprivation protocols. And finally, whereas gentle handling is a useful technique for chronic neonatal sleep deprivation, it is a burdensome procedure for researchers as it is time consuming and requires constant attention. Longer sleep deprivation protocols are also difficult to perform in neonatal mice due to the increases in sleep pressure over time15.
Despite these limitations, gentle handing is a powerful tool to study the effects of chronic sleep restriction throughout development and into adulthood and how sleep restriction can affect both behavior and brain physiology. Most importantly, this method allows researchers to continue to explore the effects that sleep restriction at an early age can have on the development of the brain both immediately following sleep restriction and later in life.

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.&amp;nbsp;</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&amp;amp;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 novel object) and social novelty (preference for a novel mouse compared to a familiar mouse). In the sociability test, sleep restricted females spent more time sniffing the stranger mouse than control females (p &#60; 0.05) (Figure 1A) and sleep-restricted males had a tendency to spend more time sniffing the object when compared to control males (p = 0.077) (Figure 1A). In the social novelty phase of testing, sleep-restricted female mice spent an increased the amount of time sniffing the novel mouse than female controls (p &#60; 0.01) (Figure 1B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58150/58150fig1.jpg" /> 
Figure 1: Chronic sleep restriction alters social behavior following a 4-week sleep recovery period. (A) Sociability test: Time spent sniffing reveals that male mice that were sleep restricted spent more time sniffing the novel object compared to the controls males (p = 0.077). Sleep-restricted female mice spent more time sniffing the stranger mouse compared to control females (*p &#60; 0.05). Sleep-restricted female mice also tended to spend more time sniffing the object compared to control females (p = 0.065). (B) Social Novelty test: Time spent sniffing the novel or familiar mouse revealed that sleep restricted female mice spent statistically significant more time sniffing the novel mouse compared to female mice that were not sleep restricted (**p &#60; 0.01). Bars represent the means &#177; SEM in 11 control male, 13 sleep-restricted male, 22 control female, and 17 sleep-restricted female mice. Data were analyzed by means of post hoct-tests following an ANOVA. This figure has been modified with permission from Sare et al. 20168. <a href="https://www.jove.com/files/ftp_upload/58150/58150fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</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. ...

chronic-sleep-deprivation-in-mouse-pups-by-means-of-gentle-handling

Nanyang Technological University

Research

JoVE Journal

Behavior

10.0K Views.  National Institutes of Health. 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.

Video: Chronic Sleep Deprivation in Mouse Pups by Means of Gentle Handling

Using Chronic Social Stress to Model Postpartum Depression in Lactating Rodents

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals and humans. However, many animal models of depression were developed in males and are not applicable or effective in studies of postpartum females. Recent studies have reported significant effects of chronic social stress during lactation, an ethologically relevant and effective stressor, on maternal behavior, growth, and behavioral neuroendocrinology. This manuscript will describe this chronic social stress paradigm using repeated exposure of a lactating dam to a novel male intruder, and the assessment of the behavioral, physiological, and neuroendocrine effects of this model. Chronic social stress (CSS) is a valuable model for studying the effects of stress on the behavior and physiology of the dam as well as her offspring and future generations. The exposure of pups to CSS can also be used as an early life stress that has long term effects on behavior, physiology, and neuroendocrinology.

This article describes the use of chronic resident intruder social stress as an ethologically relevant paradigm to model postpartum depression and anxiety in lactating rodents.

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals ...

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.

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 ...

A Chronic Sleep Fragmentation Model using Vibrating Orbital Rotor to Induce Cognitive Deficit and Anxiety-Like Behavior in Young Wild-Type Mice

Sleep disturbance is generally common in populations as a chronic disease or a complained event. Chronic sleep disturbance is proposed to be closely linked to the pathogenesis of diseases, especially neurodegenerative diseases. We recently found that 2 months of sleep fragmentation initiated Alzheimer&#8217;s disease (AD)-like behavioral and pathological changes in young wild-type mice. Herein, we present a standardized protocol to achieve chronic sleep fragmentation (CSF). Briefly, CSF was induced by an orbital rotor vibrating at 110 rpm and operating with a repetitive cycle of 10 s-on, 110 s-off, during light-ON phase (8:00 AM&#8211;8:00 PM) continuously for up to 2 months. Impairments of spatial learning and memory, anxiety-like but not depression-like behavior in mice as consequences of CSF modeling, were evaluated with Morris water maze (MWM), Novel object recognition (NOR), Open field test (OFT) and Forced swimming test (FST). In comparison with other sleep manipulations, this protocol minimizes the handling labors and maximizes the modeling efficiency. It produces stable phenotypes in young wild-type mice and can be potentially generated for a variety of research purposes.

Presented here is a protocol for chronic sleep fragmentation (CSF) model achieved by an electrically controlled orbital rotor, which could induce confirmed cognitive deficit and anxiety-like behavior in young wild-type mice. This model can be applied to explore the pathogenesis of chronic sleep disturbance and related disorders.

Sleep disturbance is generally common in populations as a chronic disease or a complained event. Chronic sleep disturbance is proposed to be closely ...

Using a Murine Model of Psychosocial Stress in Pregnancy as a Translationally Relevant Paradigm for Psychiatric Disorders in Mothers and Infants

The peripartum period is considered a sensitive period where adverse maternal exposures can result in long-term negative consequences for both mother and offspring, including the development of neuropsychiatric disorders. Risk factors linked to the emergence of affective dysregulation in the maternal-infant dyad have been extensively studied. Exposure to psychosocial stress during pregnancy has consistently emerged as one of the strongest predictors. Several rodent models have been created to explore this association; however, these models rely on the use of physical stressors or a limited number of psychosocial stressors presented in a repetitive fashion, which do not accurately capture the type, intensity, and frequency of stressors experienced by women. To overcome these limitations, a chronic psychosocial stress (CGS) paradigm was generated that employs various psychosocial insults of different intensity presented in an unpredictable fashion. The manuscript describes this novel CGS paradigm where pregnant female mice, from gestational day 6.5 to 17.5, are exposed to various stressors during the day and overnight. Day stressors, two per day separated by a 2 h break, range from exposure to foreign objects or predator odor to frequent changes in bedding, removal of bedding, and cage tilting. Overnight stressors include continuous light exposure, changing cage mates, or wetting bedding. We have previously shown that exposure to CGS results in the development of maternal neuroendocrine and behavioral abnormalities, including increased stress reactivity, the emergence of fragmented maternal care patterns, anhedonia, and anxiety-related behaviors, core features of women suffering from perinatal mood and anxiety disorders. This CGS model, therefore, becomes a unique tool that can be used to elucidate molecular defects underlying maternal affective dysregulation, as well as trans-placental mechanisms that impact fetal neurodevelopment and result in negative long-term behavioral consequences in the offspring.

The chronic psychosocial stress (CGS) paradigm employs clinically relevant stressors during pregnancy in mice to model psychiatric disorders of mothers and infants. Here, we provide a step-by-step procedure of applying the CGS paradigm and downstream assessments to validate this model.

The peripartum period is considered a sensitive period where adverse maternal exposures can result in long-term negative consequences for both mother and ...

Handling Techniques to Reduce Stress in Mice

Laboratory animals are subjected to multiple manipulations by scientists or animal care providers. The stress this causes can have profound effects on&#160;animal well-being and can also be a confounding factor for experimental variables such as anxiety measures. Over the years, handling techniques that minimize handling-related stress have been developed with a particular focus on rats, and little attention to mice. However, it has been shown that mice can be habituated to manipulations using handling techniques. Habituating mice to handling reduces stress, facilitates routine handling, improves animal wellbeing, decreases data variability, and improves experimental reliability. Despite beneficial effects of handling, the tail-pick up approach, which is particularly stressful, is still widely used. This paper provides a detailed description and demonstration of a newly developed mouse-handling technique intended to minimize the stress experienced by the animal during human interaction. This manual technique is performed over 3 days (3D-handling technique) and focuses on the animal&#39;s capacity to habituate to the experimenter. This study also shows the effect of previously established tunnel handling techniques (using a polycarbonate tunnel) and the tail-pick up technique. Specifically studied are their effects on anxiety-like behaviors, using behavioral tests (Elevated-Plus Maze and Novelty Suppressed Feeding), voluntary interaction with experimenters and physiological measurement (corticosterone levels). The 3D-handling technique and the tunnel handling technique reduced anxiety-like phenotypes. In the first experiment, using 6-month-old male mice, the 3D-handling technique significantly improved experimenter interaction. In the second experiment, using 2.5-month-old female, it reduced corticosterone levels. As such, the 3D-handling is a useful approach in scenarios where interaction with the experimenter is required or preferred, or where tunnel handling may not be possible during the experiment.

This paper describes a handling technique in mice, the 3D-handling technique, which facilitates routine handling by reducing anxiety-like behaviors and presents details on two existing related techniques (tunnel and tail handling).

Laboratory animals are subjected to multiple manipulations by scientists or animal care providers. The stress this causes can have profound effects ...

Modeling Early Life Trauma in Mice to Study PTSD and Social Avoidance Behavior

Chronic Social Defeat Stress in Early Adolescent Male Mice

Physical abuse and trauma in childhood is reported in as many as 1 in 7 children and is a major risk factor for the development of psychiatric diseases, such as Post-Traumatic Stress Disorder (PTSD), in adolescence and adulthood. Hallmark behavioral symptoms of PTSD include avoidance of cues and contexts associated with trauma. The ability to model long-lasting changes in the brain due to early life trauma is critical to determine potential circuit and molecular targets for the modulation of resultant symptoms. This manuscript describes a protocol for modeling early life physical and psychological trauma in early adolescent male mice that produces socially avoidant behavior. Adolescent male mice are exposed to repeated aggressive encounters followed by overnight housing, which provides an added dimension of psychological stress, with an adult male aggressor every day for 10 days. Repeated social defeat paired with overnight housing by the aggressor in early adolescence produces robust social avoidance behavior that lasts throughout adulthood. Social avoidance can be readily quantified in early adolescents, adolescents, and adults using open field social interaction testing. Early adolescent social defeat robustly produces more than 60% susceptible animals in adulthood.

This protocol details how to perform 10 day chronic social defeat stress in early adolescent male mice to produce long-lasting social avoidance in adulthood in more than 60% of mice.

Physical abuse and trauma in childhood is reported in as many as 1 in 7 children and is a major risk factor for the development of psychiatric diseases, ...

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the age of 50, yet it is the least understood and studied chronic pelvic pain disorder. A significant subset of patients with chronic pelvic pain report having experienced early life stress or abuse, which can markedly affect the functioning and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Mast cell activation, which has been shown to be increased in both urine and expressed prostatic secretions of CP/CPPS patients, is partially regulated by downstream activation of the HPA axis. Neonatal maternal separation (NMS) has been used for over two decades to study the outcomes of early life stress in rodent models, including changes in the HPA axis and visceral sensitivity. Here we provide a detailed protocol for using NMS as a preclinical model of CP/CPPS in male C57BL/6 mice. We describe the methodology for performing NMS, assessing perigenital mechanical allodynia, and histological evidence of mast cell activation. We also provide evidence that early psychological stress can have long-lasting effects on the male urogenital system in mice.

We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm &#8211; neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the ...

Medicine

Adolescent Social Stress Paradigm in C57BL&#47;6 Mice for Behavioral Research

Social Defeat Stress Model for Adolescent C57BL&#47;6 Male and Female Mice

Social adversity in adolescence is prevalent and can negatively impact mental health trajectories. Modeling social stress in adolescent male and female rodents is needed to understand its effects on ongoing brain development and behavioral outcomes. The chronic social defeat stress paradigm (CSDS) has been widely used to model social stress in adult C57BL/6 male mice by leveraging on the aggressive behavior displayed by an adult male rodent to an intruder invading its territory. An advantage of this paradigm is that it allows to categorize defeated mice into resilient and susceptible groups based on their individual differences in social behavior 24 h after the last defeat session. Implementing this model in adolescent C57BL/6 mice has been challenging because adult or adolescent mice do not typically attack early adolescent male or female mice and because adolescence is a short period of life, encompassing discreet temporal windows of vulnerability. This limitation was overcome by adapting an accelerated version of the CSDS to be used for adolescent male and female mice. This 4-day stress paradigm with 2 physical attack sessions per day uses a C57BL/6 male adult to prime the CD-1 mouse for aggressiveness such that it readily attacks the male or female adolescent mouse. This model was termed accelerated social defeat stress (AcSD) for adolescent mice. Adolescent exposure to AcSD induces social avoidance 24 h later in both males and females, but only in a subset of defeated mice. This vulnerability occurs despite the number of attacks being consistent across sessions between resilient and susceptible groups. The AcSD model is short enough to allow exposure during discrete periods within adolescence, allows the segregation of mice according to the presence or absence of social avoidance behavior, and is the first model available to study social defeat stress in adolescent C57BL/6 female mice.

Author Spotlight: Understanding Adolescent Social Adversity Effects on Neurodevelopment in Mice

We developed an accelerated social defeat stress model for adolescent C57BL/6 mice, which works in both males and females and allows exposure during discrete adolescent periods. Exposure to this model induces social avoidance, but only in a subset of defeated male and female mice.

Social adversity in adolescence is prevalent and can negatively impact mental health trajectories. Modeling social stress in adolescent male and female ...

Neuroscience

Modeling Early-Life Adversity in Mice Using Limited Bedding and Nesting

Limited Bedding and Nesting as a Model for Early-Life Adversity in Mice

Early-life adversity (ELA), such as abuse, neglect, lack of resources, and an unpredictable home environment, is a known risk factor for developing neuropsychiatric disorders such as depression. Animal models for ELA have been used to study the impact of chronic stress on brain development, and typically rely on manipulating the quality and/or quantity of maternal care, as this is the major source of early-life experiences in mammals, including humans. Here, a detailed protocol for employing the Limited Bedding and Nesting (LBN) model in mice is provided. This model mimics a low-resource environment, which provokes fragmented and unpredictable patterns of maternal care during a critical developmental window (postnatal days 2-9) by limiting the amount of nesting materials given to the dam to build a nest for her pups and separating the mice from the bedding via a mesh platform in the cage. Representative data are provided to illustrate the changes in maternal behavior, as well as the diminished pup weights and long-term changes in basal corticosterone levels, that result from the LBN model. As adults, offspring reared&#160;in the LBN environment have been shown to exhibit an aberrant stress response, cognitive deficits, and anhedonia-like behavior. Therefore, this model is an important tool to define how the maturation of stress-sensitive brain circuits is altered by ELA and results in long-term behavioral changes that confer vulnerability to mental disorders.

Author Spotlight: Exploring Microglial Interactions with Stress-Response Circuitry Using the Limited Bedding and Nesting Model

This protocol describes an animal model for studying how early-life adversity, provoked by an impoverished environment and unpredictable maternal care during the early postnatal period, affects brain development and the future risk of mental disorders.

Early-life adversity (ELA), such as abuse, neglect, lack of resources, and an unpredictable home environment, is a known risk factor for developing ...

Establishing a Device for Sleep Deprivation in Mice

Circadian rhythm disruption refers to the desynchronization between the external environment or behavior and the endogenous molecular clock, which significantly impairs health. Sleep deprivation is one of the most common causes of circadian rhythm disruption. Various modalities (e.g., platforms on the water, gentle handling, sliding bar chambers, rotating drums, orbital shakers, etc.) have been reported for inducing sleep deprivation in mice to investigate its effects on health. The current study introduces an alternative method for sleep deprivation in mice. An automated rocker platform-based device was designed that is cost-effective and efficiently disrupts sleep in group-housed mice at adjustable time intervals. This device induces characteristic changes of sleep deprivation with minimal stress response. Consequently, this method may prove useful for investigators interested in studying the effects and underlying mechanisms of sleep deprivation on the pathogenesis of multiple diseases. Moreover, it offers a cost-effective solution, particularly when multiple sleep deprivation devices are required to run in parallel.

The present protocol outlines a method for setting up a cost-effective rocker platform-based device used for inducing sleep deprivation in mice. This device has proven to be effective in causing disruptions in electroencephalogram (EEG)-evidenced sleep patterns, as well as inducing metabolic and molecular changes associated with sleep deprivation.