The authors would like to thank the Neurocognitive Development Lab at the University of Maryland, College Park and the Somneuro Lab at the University of Massachusetts, Amherst for assistance with this project. Funding was provided by NIH (HD094758) and NSF (BCS 1749280) to TR and RS. Representative results were funded by NIH HL111695.

Prior to beginning any research procedures, written consent should be obtained from the parent and verbal consent should be obtained from the child for all study procedures.
NOTE: See Figure 2 for an overview of the procedures.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60200/60200fig2.jpg" /> 
Figure 2: Overview of protocol. Each square represents one day. <a href="https://www.jove.com/files/ftp_upload/60200/60200fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Nap promotion condition
<ol>
	<li>Ensure that the nap promotion condition is counterbalanced with the wake promotion condition across participants as discussed below.</li>
	<li>Schedule the nap promotion condition to begin approximately one hour before the child&#39;s typical nap period to allow time to apply PSG and to conduct the visuospatial memory task. Ensure that the time between immediate and delayed recall are the same between the wake promotion and nap promotion conditions.</li>
	<li>Explain the procedures for this session to children and parents using age-appropriate materials. 
	NOTE: Age appropriate materials include story books or short videos of another child undergoing the same procedures.</li>
	<li>Apply polysomnography equipment (see section 3).</li>
	<li>Conduct the encoding and the immediate memory assessment for the visuospatial memory task (see section 4).</li>
	<li>Have the child use the restroom and then initiate the child&#39;s typical pre-nap routine.
	<ol>
		<li>Allow the parent/caregiver to put the child to sleep as they usually would. Interfere as little as possible because most children fall asleep faster when provided with their normal routine.</li>
		<li>Allow the child to nap utilizing their typical nap location.</li>
		<li>Utilize nap promotion techniques, but only when necessary as these have proven less successful in the home if they deviate too far from the child&#39;s normal routine. 
		NOTE: Nap promotion techniques include using a weighted blanket, rubbing the participant&#39;s back, wrapping the child in a blanket (similar to swaddling), progressive muscle relaxation, and playing soothing music.</li>
	</ol>
	</li>
	<li>Ensure that the amount of time the child sleeps in the nap promotion condition matches the amount of time they play in the wake promotion condition. 
	NOTE: If the nap promotion condition is first, allow the child to wake up naturally and use this duration to set the duration of the awake session. If the wake promotion condition is first, use this duration to determine the length of the nap. If the nap exceeds this time, wake the child as naturally as possible by opening the door, walking around outside the bedroom, and gradually speaking louder.</li>
	<li>Conduct the delayed recall assessment for the visuospatial memory task, approximately 15&#8722;30 min after the child wakes to avoid sleep inertia.</li>
	<li>Collect child and experimenter rating for Visual Sleepiness Scale (VSS)26 and Visual Mood Scale (VMS)27.</li>
	<li>Remove PSG electrodes.</li>
</ol>
2. Wake promotion condition
<ol>
	<li>Ensure that the wake promotion condition is counterbalanced with the nap promotion condition discussed above.</li>
	<li>Schedule the wake promotion condition to begin approximately one hour before the child&#39;s typical nap period to equate time of day across conditions. Ensure that the time between immediate and delayed recall are approximate the same between the wake promotion and nap promotion conditions.</li>
	<li>Explain the procedures for this session to children and parents using age-appropriate materials.</li>
	<li>Apply PSG electrodes (see section 3) in order to equate the wake and nap promotion conditions. 
	NOTE: Although sleep is not expected, this equates conditions and can be used to verify the absence of sleep if in doubt.</li>
	<li>Conduct the encoding and the immediate memory assessment for the visuospatial memory task (see section 4).</li>
	<li>Have the child use the restroom and then proceed to the location that they typically nap.
	<ol>
		<li>Do not allow the child to nap, instead, have the child play quietly with non-stimulating toys in the same location as their typical nap. 
		NOTE: Acceptable non-stimulating toys include small sensorimotor toys such as wax sticks and age-appropriate interlocking plastic bricks.</li>
		<li>Have the child play for their typical nap length or for the time they slept during the nap promotion condition (see step 1.7 for additional information).</li>
		<li>Record any unusual activity such as talking, leaving the room, and playing with toys that are not provided.</li>
	</ol>
	</li>
	<li>Ensure that the amount of time the child sleeps in the nap promotion condition matches the amount of time they play in the wake promotion condition.</li>
	<li>Conduct the delayed recall assessment for the visuospatial memory task, approximately 15&#8722;30 min after the child is finished playing in order to keep delay time similar between conditions.</li>
	<li>Collect child and experimenter rating for VSS26 and VMS27.</li>
	<li>Remove PSG electrodes.</li>
</ol>
3. Polysomnography (PSG)
<ol>
	<li>Preparation
	<ol>
		<li>Facilitate PSG electrode application by having the child engage in a quiet activity such as reading a book, playing with playdough, eating a snack if they are hungry, or watching a short movie. 
		NOTE: If a movie is used, ensure that the movie is age appropriate but does not elicit rowdiness in the child (e.g., popular child-friendly animated films or shows).</li>
		<li>Accessibility to a parent or guardian is not required. However, for shy and tentative children ensure that trusted caregivers are available. 
		NOTE: For a small number of children, parents and guardians may be distracting instead of helpful. If this is the case, ask the parent if they would be willing to step out of the child&#39;s sight.</li>
	</ol>
	</li>
	<li>Collect head measurements.
	<ol>
		<li>Use a flexible tape measure and china marker to mark locations for subsequent electrode application.</li>
		<li>Measure the distance from the inion to nasion and place a mark at the halfway point. Measure the distance from preauricular notch in one ear to the preauricular notch in the other ear and place another mark at the halfway point. The intersection of these two marks is the &#34;Reference&#34; point (CZ).</li>
		<li>Measure 10% of the inion to nasion distance up from the inion. Then measure out 10% of the preauricular notch to preauricular notch measurement from this point on either side. Make two marks, one on each side (O3 and O4).</li>
		<li>Measure 20% of the preauricular notch to preauricular notch measurement from the reference point on either side of the head. Make two marks, one on each side (C3 and C4).</li>
		<li>Measure 20% of the inion to nasion distance up from the reference point. Then measure out 20% of the preauricular notch to preauricular notch measurement from this point on either side. Make two marks, one on each side (F3 and F4).</li>
	</ol>
	</li>
	<li>Prepare one electrode at a time for placement.
	<ol>
		<li>Clean each electrode location using an alcohol swab. Exfoliate using a slightly abrasive gel and then remove any residual cleaning material.</li>
		<li>Fill each electrode using electrode cream.
		<ol>
			<li>For electrodes placed where hair is present, apply an additional drop of electrode cream to a gauze square and place it on the back of the electrode.</li>
			<li>For electrodes placed on the face, use medical tape to adhere the electrode to the skin.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Place an electrode on each corresponding EEG, EOG, and EMG location.
	<ol>
		<li>Place an electrode on each marked location on the scalp (CZ, O3, O4, C3, C4, F3, and F4).</li>
		<li>Place one electrode on each mastoid (small bony process behind the ear) and one in the center of the forehead.</li>
		<li>Place one EOG electrode adjacent to each eye. Place one of these electrodes slightly superior to the outside of the right eye (termed ROC) and one to the outside and slightly inferior to the left eye (termed LOC).</li>
		<li>Place two EMG electrodes around the chin area. Place one electrode on the right cheek just above the smile line. Place the other on the left side just above where the chin meets the neck, adjacent to the esophagus. Find the second location by having the participant say the word &#34;milk&#34; out loud while feeling for the location where muscle contractions in the neck and chin are maximal.</li>
	</ol>
	</li>
	<li>Attach electrodes to the recording device and initiate recording.</li>
	<li>Record impedance readings for all electrodes. Ensure all electrodes pass the impedance test. 
	NOTE: Some devices may note a &#39;Pass&#39; or &#39;Fail&#39;, while other devices may give numeric values. In the latter, impedances under 25 k&#937; are acceptable. If an impedance fails or is too high, remove and replace the batteries. If this does not amend the issue, reapply that electrode.</li>
	<li>At the completion of each conditions, remove the PSG electrodes.
	<ol>
		<li>For electrodes applied in the hair, soak the location of the electrode with a water-based spray. Allow the spray to sit for about one minute then remove the electrode. 
		NOTE: Detangling hair spray is highly effective for the purpose of removing hair electrodes.</li>
		<li>For electrodes applied with tape, typically on the face and mastoids, use a cotton pad with baby oil applied to it to saturate the tape. When the tape is completely covered in baby oil, gently pull the tape up from the corners.</li>
	</ol>
	</li>
</ol>
4. Visuospatial memory task
<ol>
	<li>Administer nine to-be-remembered stimuli arranged in a 3 x 3 matrix to children younger than 44 months of age. Administer the 12 to-be-remembered stimuli arranged in a 3 x 4 matrix to children older than 44 months of age. 
	NOTE: If a child assigned to the 12-item matrix is too challenged, the 9-item matrix can be used. Likewise, if it is evident that the 9-item matrix is too easy, the 12-item matrix can be used to avoid ceiling effects. This is justified because within-subject accuracy is of the variable of interest and not raw scores. Stimuli are typically cartoon-like pictures of common images (e.g., bear, car, scissors) arranged in a matrix and presented on a laptop screen. There are two sets of stimuli. This allows the task to be counterbalanced across the two conditions (i.e., nap versus wake promotion) so that children do not receive the same pictures in both conditions.</li>
	<li>Administer the task in three phases: encoding, immediate recall, and delayed recall. For each phase allow the child to answer each question at their own pace. 
	NOTE: Typical durations are: 6 min for the encoding phase, 2 min for the immediate recall phase, and 2 min for the delayed recall phase.
	<ol>
		<li>In the encoding phase, direct the child to identify each image by name, then instruct the child to remember their location of each item on the grid. Following encoding, the cards are replaced with &#39;blank&#39; images and the child must then locate the position of each image until they reach an encoding score &#8805;75%. 
		NOTE: A threshold of 75% was chosen based on studies in young adults28,29,30 and reflects a point when learning is clearly reached but not at ceiling.
		<ol>
			<li>During this block, participants receive visual feedback from the task after each response. After the child selects an image location, reveal the associated image, informing the child whether that was the correct or incorrect location.</li>
			<li>Provide verbal feedback on performance to motivate the child but ensure that the amount of feedback is consistent across both conditions. When the child succeeds at locating an image use language like &#34;Great job, you got that one!&#34; When a child fails use language that highlights the child&#39;s effort (e.g., &#34;Whoops! Not quite but good try! Let&#39;s see if you can get the next one.&#34;).</li>
			<li>Provide children who are assigned to the 12-item matrix that cannot pass encoding after 4 rounds with an opportunity to stretch, do jumping jacks, and move for about 5 min. If the child still cannot pass encoding after an additional 2 rounds, restart encoding with the 9-item matrix.</li>
			<li>Provide children assigned to the 9-item matrix who receive an encoding score of 100% on the first round with the encoding task for the 12-item matrix. If they do not go through all the necessary steps to drop back to the 9-item matrix, use the 12-item matrix for the following two phases.</li>
		</ol>
		</li>
		<li>During the immediate recall phase, present the images again, one at a time, and ask the child to recall the corresponding location. Do not provide visual or verbal feedback, and only probe each item once. However, do provide feedback on effort (i.e., &#34;Good job giving your best effort&#34;).</li>
		<li>Conduct the delayed recall phase immediately after the wake or sleep condition. 
		NOTE: This phase is identical to the immediate recall phase.
		<ol>
			<li>At times children will become fussy during the delayed recall phase. If this happens, entice the child to complete the task with a prize or by offering more time to watch their movie during PSG removal. During this time do not allow the child to play with toys or engage in other tasks until the memory task is complete.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/60200/60200fig3.jpg" /> 
Figure 3: Examples of screen displays during the visuospatial memory task. <a href="https://www.jove.com/files/ftp_upload/60200/60200fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Actigraphy
<ol>
	<li>Program the activity watch. 
	NOTE: The activity watch is sampled at 32 Hz, with a sensitivity of &#60;0.01 g. Activity is stored in 15-s epochs.</li>
	<li>Provide each participant with a pre-programmed activity watch and instructions. Tell the parent that the watch should always be worn. Highlight that it is waterproof so there is no reason to remove the device.
	<ol>
		<li>Instruct the child to wear the watch on their non-dominant hand continuously.</li>
		<li>Instruct the parent to press the button on the side of the watch face every time their child attempts to sleep, and then again when they wake. 
		NOTE: This generates an event marker in the data which assists with scoring actigraphy.</li>
	</ol>
	</li>
	<li>Provide the parent with a sleep diary (similar to a log or spreadsheet) on which they can record sleep times and watch removal. 
	NOTE: This also assists with scoring actigraphy.
	<ol>
		<li>In the sleep diary, ask the parent to provide a complete log of all sleep for the number of days that the activity watch will be worn, including the time that the child goes to bed and when the child wakes up. The parent should provide this information for both naps and regular overnight sleep. Additionally, ask the parent to provide information about any time when the watch is removed.</li>
	</ol>
	</li>
</ol>
6. Data analysis
<ol>
	<li>Visuospatial memory task
	<ol>
		<li>Calculate accuracy for each recall phase as the percent of items recalled.</li>
		<li>Calculate change in recall over the nap and wake intervals as follows.
		<ol>
			<li>Calculate change in recallnap by subtracting immediate recall accuracy (before nap) from delayed recall accuracy (after nap).</li>
			<li>Calculate change in recallwake by subtracting immediate recall accuracy (before wake) from delayed recall accuracy (after wake).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>PSG
	<ol>
		<li>Characterize sleep stages in accordance with the standard scoring criteria (e.g., The AASM Manual for the Scoring of Sleep and Associated Events v. 2.5).</li>
		<li>Detect sleep spindles at C3 using specialized software by marking spindle onsets and offsets.</li>
		<li>Verify sleep stages and spindle onsets and offsets with second trained researcher. In the event the scorings are not concordant, have a third trained scorer make the consensus decision.</li>
		<li>Analyze spindle density using specialized software and an in-house MATLAB code based on previous studies31. In brief, filter EEG data from 0.5&#8722;35 Hz. Consider the maximum voltage between the identified spindle onset and offset the peak spindle amplitude. Use a fast Fourier transform of each spindle to identify the peak spectral frequency between 9&#8722;15 Hz24,32.</li>
	</ol>
	</li>
	<li>Actigraphy
	<ol>
		<li>Score activity watch data using specialized software following standardized protocols20. 
		NOTE: Multiple days and nights of data are required to ensure reliability of the data. At minimum, participants need at least three days and three nights of actigraphy data (days and nights do not need to be consecutive); however, 5 nights is preferable, particularly when these data are of primary interest33.</li>
		<li>Use sleep diary information and event markers (button presses) to verify sleep onset and offset. 
		NOTE: These two items must be within 20 min of each other in order to score the start and end of a rest interval.
		<ol>
			<li>If a participant is missing sleep diary information, event markers, or the diary and event markers are not within 20 min of each other, determine sleep onset and offset manually32: determine sleep onset by the first three minutes of continuous sleep33 and determine sleep offset by the last five minutes of continuous sleep34.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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S., Schneider, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timely+sleep+facilitates+declarative+memory+consolidation+in+infants.">Timely sleep facilitates declarative memory consolidation in infants.</a> Proceedings of the National Academy of Sciences. 112 (5), 1625-1629 (2015).</li><li>Kurdziel, L., Duclos, K., Spencer, R. M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+spindles+in+midday+naps+enhance+learning+in+preschool+children.">Sleep spindles in midday naps enhance learning in preschool children.</a> Proceedings of the National Academy of the Sciences of the United States of America. 110 (43), 17267-17272 (2013).</li><li>Kurdziel, L. B. F., Kent, J., Spencer, R. M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep-dependent+enhancement+of+emotional+memory+in+early+childhood.">Sleep-dependent enhancement of emotional memory in early childhood.</a> Scientific Reports. 8 (12609), 1-10 (2018).</li><li>Desrochers, P. C., Kurdziel, L. B. F., Spencer, R. M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+benefit+of+naps+on+motor+learning+in+preschool+children.">Delayed benefit of naps on motor learning in preschool children.</a> Experimental Brain Research. 234 (3), 763-772 (2016).</li><li>Maldonado, C. C., Bentley, A. J., Mitchell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pictorial+sleepiness+scale+based+on+cartoon+faces.">A pictorial sleepiness scale based on cartoon faces.</a> Sleep. 27 (3), 541-548 (2004).</li><li>Stern, R. A., Arruda, J. E., Hooper, C. R., Wolfner, G. D., Morey, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+analogue+mood+scales+to+measure+internal+mood+state+in+neurologically+impaired+patients:+Description+and+initial+validity+evidence.">Visual analogue mood scales to measure internal mood state in neurologically impaired patients: Description and initial validity evidence.</a> Aphasiology. 11 (1), 59-71 (1997).</li><li>Plihal, W., Born, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+early+and+late+nocturnal+sleep+on+indicators+of+procedural+and+declarative+memory.">Effects of early and late nocturnal sleep on indicators of procedural and declarative memory.</a> Journal of Cognitive Neuroscience. 9 (4), 534-547 (1997).</li><li>Donohue, K. C., Spencer, R. M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+re-exposure+to+environmental+sound+cues+during+sleep+does+not+improve+memory+for+semantically+unrelated+word+pairs.">Continuous re-exposure to environmental sound cues during sleep does not improve memory for semantically unrelated word pairs.</a> Journal of Cognitive Education and Psychology. 10 (2), 167-177 (2015).</li><li>Wilson, J. K., Baran, B., Pace-Schott, E. F., Ivry, R. B., Spencer, R. M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+modulates+word-pair+learning+but+not+motor+sequence+learning+in+healthy+older+adults.">Sleep modulates word-pair learning but not motor sequence learning in healthy older adults.</a> Neurobiology of Aging. 33 (5), 991-1000 (2012).</li><li>Wamsley, E. J., 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=Reduced+sleep+spindles+and+spindle+coherence+in+schizophrenia:+Mechanisms+of+impaired+memory+consolidation?.">Reduced sleep spindles and spindle coherence in schizophrenia: Mechanisms of impaired memory consolidation?.</a> Biological Psychiatry. 71 (2), 154-161 (2012).</li><li>M&#246;lle, M., Bergmann, T. O., Marshall, L., Born, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+slow+spindles+during+the+sleep+slow+oscillation:+Disparate+coalescence+and+engagement+in+memory+processing.">Fast and slow spindles during the sleep slow oscillation: Disparate coalescence and engagement in memory processing.</a> Sleep. 34 (10), 1411-1421 (2011).</li><li>Acebo, C., 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/wake+patterns+derived+from+activity+monitoring+and+maternal+report+for+healthy+1-+to+5-year-old+children.">Sleep/wake patterns derived from activity monitoring and maternal report for healthy 1- to 5-year-old children.</a> Sleep. 28 (12), 1568-1577 (2005).</li><li>Acebo, C., 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=Estimating+sleep+patterns+with+activity+monitoring+in+children+and+adolescents:+How+many+nights+are+necessary+for+reliable+measures?.">Estimating sleep patterns with activity monitoring in children and adolescents: How many nights are necessary for reliable measures?.</a> Sleep. 22 (1), 95-103 (1999).</li><li>Geiger, 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=The+sleep+EEG+as+a+marker+of+intellectual+ability+in+school+age+children.">The sleep EEG as a marker of intellectual ability in school age children.</a> Sleep. 34 (2), 181-189 (2011).</li><li>Wagner, U., Gais, S., Born, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emotional+memory+formation+is+enhanced+across+sleep+intervals+with+high+amounts+of+rapid+eye+movement+sleep.">Emotional memory formation is enhanced across sleep intervals with high amounts of rapid eye movement sleep.</a> Learning and Memory. 8, 112-119 (2001).</li><li>G&#243;mez, R. L., Bootzin, R. R., Nadel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naps+promote+abstraction+in+language-learning+infants.">Naps promote abstraction in language-learning infants.</a> Psychological Science. 17 (8), 670-674 (2006).</li><li>Konrad, C., Herbert, J. S., Schneider, S., Seehagen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gist+extraction+and+sleep+in+12-month-old+infants.">Gist extraction and sleep in 12-month-old infants.</a> Neurobiology of Learning and Memory. 134, 216-220 (2016).</li></ol>

The authors have nothing to disclose.

This article describes how to investigate sleep-dependent consolidation of declarative memory during naps in early childhood. Methods include behavioral assessment of memory across nap and awake conditions, actigraphy and parent-report to assess sleep cycles, and PSG to assess sleep architecture. This unique combination is critical for assessing memory, characterizing sleep cycles, and examining the neural mechanisms underlying the benefit of sleep on memory. Representative results indicate that learning and memory were ...

Given sleep&#39;s prevalence in our daily routine, it is important to understand its function. Studies with this objective require precise measurement of sleep. Polysomnography (PSG) is the gold-standard measure of sleep. PSG allows for objective, quantitative measurement of sleep with high temporal resolution and can be useful for both research and clinical purposes. PSG is a combination of physiological recordings. At minimum, a PSG montage includes the following measures: electroencephalography (EEG), electrooculography (EOG), and electromyography (EMG). These measures assess electrical potentials from the brain, the eyes, and muscles respectively, and allow for classification of sleep stages (see Figure 1). Other measures, such as electrocardiography (ECG), respiration, and pulse oximetry may be included to identify the presence of disordered sleep.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60200/60200fig1b.jpg" /> 
Figure 1: Example electrode placement and description of activity recorded via PSG. <a href="https://www.jove.com/files/ftp_upload/60200/60200fig1blarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
PSG allows sleep to be characterized into four distinct sleep stages: non-rapid eye movement (non-REM) stage 1 (nREM1; 4&#8722;7 Hz), non-REM stage 2 (nREM2; 12&#8722;15 Hz), and non-REM stage 3 (more commonly known as slow wave sleep [SWS]; 0.5&#8722;4 Hz), and rapid-eye movement (REM) sleep. nREM1 marks sleep onset, and is identified based on reduced muscle tone in the EMG recoding and mixed amplitude EEG oscillations relative to the alpha observed in resting wake. This is followed by nREM2, which can be distinguished by the presence of sleep spindles (short bursts of sigma frequency activity; 11&#8722;16 Hz) and K-complexes (single slow-waves that stand out from the surrounding activity) in the EEG. SWS is characterized by distinct slow-frequency high-amplitude EEG oscillations. REM sleep is characterized by fast low-amplitude oscillatory brain activity very similar to that observed during wake. However, what distinguishes REM sleep from wake is that it is also characterized by phasic rapid eye movements (hence the moniker REM) and muscle atonia. Over the course of a sleep bout, sleep stages are experienced cyclically, at a rate of about 90 min/cycle.
Sleep also follows the circadian rhythm, with sleep bouts taking place in 24-h cycles. Sleep timing and consistency may influence sleep function and are also important to assess. Although PSG is necessary to characterize sleep stages, it is time-consuming to apply and therefore not ideal for assessing multiple sleep bouts (e.g., multiple nights of sleep, naps and overnight sleep). For this, actigraphy is beneficial. Actigraphy uses a tri-axial accelerometer, typically on the wrist, to estimate sleep based on the absence of movement. Although actigraphy cannot be used to characterize sleep stages, it has been shown to be reliable at detecting sleep onset and wake onset (including sleep fragmentation or wake after sleep onset) in a range of populations from infants1 to older adults2. Both PSG and actigraphy are preferred methods over self/parent-report measures. Self/parent-report measures are easy to administer and relatively inexpensive, however, they are also subject to bias and non-compliance. Finally, it is worth noting that these methods can be used in combination to capitalize on the strengths of each. For example, PSG can be combined with actigraphy and/or self/parent-report to obtain both overnight sleep quality as well as verification of sleep quantities or sleep-wake cycles, especially over long durations (e.g., weeks or months).
One function of sleep that has garnered particular interest is sleep-dependent memory consolidation, the processing of memories that leaves them stronger and less vulnerable to interference3. Although memory consolidation can take place during wake in children4 and adults5, there is substantial evidence that consolidation is enhanced during sleep. Past research has provided behavioral examples of sleep-dependent memory consolidation by comparing changes in memory performance following an interval of sleep (e.g., 8 pm&#8722;8 am) to changes following an equivalent interval spent awake (e.g., 8 am&#8722;8 pm). In adults, memories are protected6 or even enhanced7 following an interval of sleep while memories typically decay over an equivalent interval of wake. Controls have been employed that dissociate performance changes from circadian influences8,9,10. For example, similar benefits of sleep are observed when comparing performance over a mid-day nap to an equivalent mid-day wake period9.
Although sleep was once thought to reflect a passive process, simply protecting memories from decay or interference, modern theories suggest sleep plays a more active role and actually promotes memory through reactivations11,12,13. Support for this comes from observed correlations between behavioral measures of memory consolidation over sleep (change in memory recall after sleep compared to before sleep) and specific aspects of sleep physiology. For many declarative memory tasks, memory consolidation is associated with aspects of non-REM sleep, specifically measures of SWS or sleep spindles found in nREM2 and SWS. If sleep&#39;s role was passive protection from interference, such a correlation would not be expected; rather a correlation between time asleep (regardless of sleep stage) and performance would be expected, as more time asleep would provide more protection from interference14.
Additional support for the active role of SWS in memory consolidation is evident in studies of targeted memory reactivation. In these studies, a memory is learned in the context of a perceptual cue, for instance an odor, and recall of the memory is greater following sleep if the cue is re-presented during sleep, SWS in particular15. Although the underlying mechanism is debated16,17, one prominent theory, systems consolidation theory, contends that memories encoded in the hippocampus are stabilized in the cortex though hippocampal-neocortical dialogue. Specifically, cortical slow waves and sleep spindles, occurring in conjunction with hippocampal ripples associated with memory reactivation, support the memory transfer3.
The role of sleep in memory consolidation during development is less clear. Early childhood is a period of particular interest as children begin to transition from a biphasic (consisting of a mid-day nap and an overnight sleep bout) to a monophasic sleep pattern. Recent research suggests that this transition may reflect brain maturation18. This argument is consistent with empirical data showing developmental changes in overnight sleep (i.e., topography of slow wave activity) mirrors that of cortical maturation19.
Although there are several behavioral demonstrations of overnight sleep-dependent consolidation in children20,21 and infants22, research on the neural underpinnings of memory consolidation with mid-day sleep are just beginning to be investigated. In ground-breaking work examining the role of mid-day naps on memory in preschool children, naps were shown to protect memories of recently learned information, whereas memory was reduced (by ~12%) when children stayed awake during the nap interval23. This &#34;nap benefit&#34; was greatest in children who napped habitually (i.e., 5 or more times per week as measured with actigraphy) regardless of their age. By recording PSG during the nap, the change in memory performance across the nap period was found to be specifically associated with sleep spindle density (the number of sleep spindles per minute of nREM), suggesting nap quality (not quantity) was a critical factor in promoting memory retention (see the representative results section).
This study highlights the significance of PSG in exploring relations between sleep and memory during development. It points to the importance of characterizing sleep macro- (sleep stages) and micro- (qualities of those stages such as frequencies and the presence of spindles) structures during naps for memory consolidation. It also highlights the importance of assessing sleep rhythms (characterizing children as habitual or non-habitual nappers). Although our work has characterized the function of naps in visuospatial learning (and more recently emotional24 and procedural25 learning), many questions remain. For instance, it will be important to examine other declarative memory tasks to assess the generalizability of these findings and to assess tasks used in preschool classrooms to understand specific parameters (e.g., amount of nap benefit relative to learning) for ecologically valid tasks. Additional work will also be necessary to understand when wake is sufficient for memory consolidation. Thus, our objective is to demystify the process of measuring sleep and sleep-dependent memory consolidation in children. We provide practical tips for examining the benefit of an afternoon nap on declarative memory in typically developing preschoolers (approximately 3 to 4 years of age) using a computerized visuospatial memory task as well as methods for assessing nap habituality using actigraphy, parent-report, and nap physiology using PSG. Although these methods were developed for preschool age children who nap with varying frequency, these methods could be adapted to any age group.

<table><tbody><tr><td>Actiwatch Spectrum Plus Starter Kit</td><td>Philips Respironics</td><td>1109516</td><td>Includes: Actiwatch Spectrum Plus Device, Actiware Software License, and manual</td></tr><tr><td>Actiware software&amp;nbsp;</td><td>Philips Respironics&amp;nbsp;</td><td>1114828</td><td>Alternatives may be available.&amp;nbsp;</td></tr><tr><td>Brain Analyzer</td><td>Brain Products</td><td>BV-BP-170-1000</td><td>Alternatives may be available.&amp;nbsp;</td></tr><tr><td>Dell Latitude 5580 Laptop</td><td>Dell</td><td>X5580T [210-AKJR]</td><td>Laptop for running MatLab, Actiware, and RemLogic as well as storing/uploading data</td></tr><tr><td>EC2 cream</td><td>Grass</td><td>12643</td><td>Possible alternatives include Ten20 paste and Lic2 electride cream</td></tr><tr><td>Embla REMLogic software&amp;nbsp;</td><td>Natus Medical Inc.</td><td>21475</td><td>Alternatives may be available.&amp;nbsp;</td></tr><tr><td>Embletta MPR PG Sys - XR - US</td><td>Natus Medical Inc.</td><td>12077</td><td>Embletta system for PSG recordings</td></tr><tr><td>Embletta MPR ST + Proxy Kit</td><td>Natus Medical Inc.</td><td>12696</td><td>Attachment to Embletta to record PSG sensors</td></tr><tr><td>Nuprep cleaning solution</td><td>Natus Medical Inc.</td><td>12643</td><td>Possible alternatives may be available.</td></tr><tr><td>Sleep Supplies Starter Kit for Embletta MPR ST/ST + Proxy</td><td>Natus Medical Inc.</td><td>12643</td><td>Started kit for sleeping including guaze, EC2 cream, NuPrep cleaning solution, cotton swabs and more.&amp;nbsp;</td></tr></tbody></table>

measuring neural mechanisms underlying sleep-dependent memory consolidation during naps in early childhood

Sleep is critical for daily functioning. One important function of sleep is the consolidation of memories, a process that makes them stronger and less vulnerable to interference. The neural mechanisms underlying the benefit of sleep for memory can be investigated using polysomnography (PSG). PSG is a combination of physiological recordings including signals from the brain (EEG), eyes (EOG), and muscles (EMG) that are used to classify sleep stages. In this protocol, we describe how PSG can be used in conjunction with behavioral memory assessments, actigraphy, and parent-report to examine sleep-dependent memory consolidation. The focus of this protocol is on early childhood, a period of significance as children transition from biphasic sleep (consisting of a nap and overnight sleep) to monophasic sleep (overnight sleep only). The effects of sleep on memory performance are measured using a visuospatial memory assessment across periods of sleep and wakeful-rest. A combination of actigraphy and parent report is used to assess sleep rhythms (i.e., characterizing children as habitual or non-habitual nappers). Finally, PSG is used to characterize sleep stages and qualities of those stages (such as frequencies and the presence of spindles) during naps. The advantage of using PSG is that it is the only tool currently available to assess sleep quality and sleep architecture, pointing to the relevant brain state that supports memory consolidation. The main limitations of PSG are the length of time it takes to prepare the recording montage and that recordings are typically taken over one sleep bought. These limitations can be overcome by engaging young participants in distracting tasks during application and combining PSG with actigraphy and self/parent-report measures to characterize sleep cycles. Together, this unique combination of methods allows for investigations into how naps support learning in preschool children.

Using the procedures described here, Kurdziel and colleagues23 examined sleep-dependent memory consolidation during naps in preschool children. Results showed children&#39;s recall accuracy on the visuospatial memory task after a nap was better than their recall accuracy after a similar period during which they remained awake (i.e., signifying a &#34;nap benefit&#34;, Figure 4). Moreover, those who spent the prior day in the wake condition did not ...

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Measuring Neural Mechanisms Underlying Sleep-Dependent Memory Consolidation During Naps in Early Childhood

This protocol describes methods used to examine neural mechanisms underlying sleep-dependent memory consolidation during naps in early childhood. It includes procedures for examining the effect of sleep on behavioral memory performance, as well as the application and recording of both polysomnography and actigraphy.

Sleep is critical for daily functioning. One important function of sleep is the consolidation of memories, a process that makes them stronger and less ...

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11.9K Views.  University of Maryland. This protocol describes methods used to examine neural mechanisms underlying sleep-dependent memory consolidation during naps in early childhood. It includes procedures for examining the effect of sleep on behavioral memory performance, as well as the application and recording of both polysomnography and actigraphy.

Video: Measuring Neural Mechanisms Underlying Sleep-Dependent Memory Consolidation During Naps in Early Childhood

Eye Tracking, Cortisol, and a Sleep vs. Wake Consolidation Delay: Combining Methods to Uncover an Interactive Effect of Sleep and Cortisol on Memory

Although rises in cortisol can benefit memory consolidation, as can sleep soon after encoding, there is currently a paucity of literature as to how these two factors may interact to influence consolidation. Here we present a protocol to examine the interactive influence of cortisol and sleep on memory consolidation, by combining three methods: eye tracking, salivary cortisol analysis, and behavioral memory testing across sleep and wake delays. To assess resting cortisol levels, participants gave a saliva sample before viewing negative and neutral objects within scenes. To measure overt attention, participants&#8217; eye gaze was tracked during encoding. To manipulate whether sleep occurred during the consolidation window, participants either encoded scenes in the evening, slept overnight, and took a recognition test the next morning, or encoded scenes in the morning and remained awake during a comparably long retention interval. Additional control groups were tested after a 20&#160;min&#160;delay in the morning or evening, to control for time-of-day effects. Together, results showed that there is a direct relation between resting cortisol at encoding and subsequent memory, only following a period of sleep. Through eye tracking, it was further determined that for negative stimuli, this beneficial effect of cortisol on subsequent memory may be due to cortisol strengthening the relation between where participants look during encoding and what they are later able to remember. Overall, results obtained by a combination of these methods uncovered an interactive effect of sleep and cortisol on memory consolidation.

We present a protocol used to discover an interactive effect between sleep and cortisol on memory consolidation, particularly for negative arousing images. Specifically, the experimental design utilizes eye tracking, salivary cortisol analysis, and behavioral memory testing &#8211; methods that can be used with both healthy and clinical participants.

Although rises in cortisol can benefit memory consolidation, as can sleep soon after encoding, there is currently a paucity of literature as to how these ...

Behavior

Examining Recall Memory in Infancy and Early Childhood Using the Elicited Imitation Paradigm

The ability to recall the past allows us to report on details of previous experiences, from the everyday to the significant. Because recall memory is commonly assessed using verbal report paradigms in adults, studying the development of this ability in preverbal infants and children proved challenging. Over the past 30 years, researchers have developed a non-verbal means of assessing recall memory known as the elicited or deferred imitation paradigm. In one variant of the procedure, participants are presented with novel three-dimensional stimuli for a brief baseline period before a researcher demonstrates a series of actions that culminate in an end- or goal-state. The participant is allowed to imitate the demonstrated actions immediately, after a delay, or both. Recall performance is then compared to baseline or to performance on novel control sequences presented at the same session; memory can be assessed for the individual target actions and the order in which they were completed. This procedure is an accepted analogue to the verbal report techniques used with adults, and it has served to establish a solid foundation of the nature of recall memory in infancy and early childhood. In addition, the elicited or deferred imitation procedure has been modified and adapted to answer questions relevant to other aspects of cognitive functioning. The broad utility and application of imitation paradigms is discussed, along with limitations of the approach and directions for future research.

The elicited imitation procedure was established to examine the development of recall memory in infancy and early childhood. This procedure has been widely used to establish a solid foundation of the nature of recall memory in infancy and early childhood.

The ability to recall the past allows us to report on details of previous experiences, from the everyday to the significant. Because recall memory is ...

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.

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. ...

Simultaneous Monitoring of Wireless Electrophysiology and Memory Behavioral Test as a Tool to Study Hippocampal Neurogenesis

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on animals and humans. Adult neurogenesis mechanism is also linked to memory and learning improvement. Traditionally, researchers used to assess learning and memory parameters in rodent models by behavioral tasks. Therefore, the simultaneous monitoring of behavioral changes and EEG is particularly interesting in correlating data between brain activity and task-related behaviors. However, most of the equipment required to perform both studies are either complex, expensive, or uses a wired setup network that hinders the natural animals&#8217; movement. In this study, EEG was recorded with a wireless electrophysiology device along with the execution of a novel object recognition task (NORT). The animal&#8217;s behavior was monitored simultaneously by a video tracking system. Both recordings were analyzed offline by their timestamps which were synched to link EEG signals with the animal&#8217;s actions. Subjects consist of adult Wistar rats after medium-term environmental enrichment treatment. Six skull screw electrodes were fixed in pairs on both hemispheres over frontal, central, and parietal regions and were referenced to an electrode located posterior of the nasal bone. NORT protocol consists of exposing the animal to two identical objects for 10 min. After 2 h and 24 h, one of the objects was replaced with a novel one. Exploration time for each object was monitored by a behavioral tracking software (BTS) and EEG data recording. The analysis of the EEG synced with behavioral data consists of estimations of alpha and beta relative band power and comparisons between novel object recognition versus familiar object exploration, between three experimental stages. In this manuscript, we have discussed electrodes manufacturing process, epidural electrodes implantation surgery, environmental enrichment protocol, NORT protocol, BTS setup, EEG &#8211; BTS coupling for simultaneous monitoring in real-time, and EEG data analysis based on automatic events detection.

The protocol presented here provides information on the simultaneous electroencephalography (EEG) and behavioral assessment in real-time. We have discussed all steps involved in this protocol as an attractive solution for researchers in many fields of neuroscience, particularly in learning and memory areas.

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on ...

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 ...

A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal care. Such a preference memory can last from hours, to days, even life-long, depending on training parameters. Early odor preference learning provides us with a model in which the critical changes for a natural form of learning occur in the olfactory circuitry. An additional feature that makes it a powerful tool for the analysis of memory processes is that early odor preference learning can be lateralized via single naris occlusion within the critical period. This is due to the lack of mature anterior commissural connections of the olfactory hemispheres at this early age. This work outlines behavioral protocols for lateralized odor learning using nose plugs. Acute, reversible naris occlusion minimizes tissue and neuronal damages associated with long-term occlusion and more aggressive methods such as cauterization. The lateralized odor learning model permits within-animal comparison, therefore greatly reducing variance compared to between-animal designs. This method has been used successfully to probe the circuit changes in the olfactory system produced by training. Future directions include exploring molecular underpinnings of odor memory using this lateralized learning model; and correlating physiological change with memory strength and durations.

This protocol introduces lateralized early odor preference learning in rats using acute single naris occlusion. Lateralized learning permits the examination of behavioral outcomes and underpinning biological mechanisms within the same animals, reducing variance induced by between-animal designs. This protocol can be used to investigate molecular mechanisms underpinning early odor learning.

Rat pups during a critical postnatal period (&#8804; 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal ...

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 ...

Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 (ChR2), is expressed by a virus vector in a particular type of neuronal cells in various Cre-driver mice. Activation of these opsins is triggered by application of light pulses which are delivered by laser or LED through optic cables, and the effect of activation is observed with very high time resolution. Experimenters are able to acutely stimulate neurons while monitoring behavior or another physiological outcome in mice. Optogenetics can enable useful strategies to evaluate function of neuronal circuits in the regulation of sleep/wakefulness states in mice. Here we describe a technique for examining the effect of optogenetic manipulation of neurons with a specific chemical identity during electroencephalogram (EEG) and electromyogram (EMG) monitoring to evaluate the sleep stage of mice. As an example, we describe manipulation of GABAergic neurons in the bed nucleus of the stria terminalis (BNST). Acute optogenetic excitation of these neurons triggers a rapid transition to wakefulness when applied during NREM sleep. Optogenetic manipulation along with EEG/EMG recording can be applied to decipher the neuronal circuits that regulate sleep/wakefulness states.

Here, we describe methods of optogenetic manipulation of particular types of neurons during monitoring of sleep/wakefulness states in mice, presenting our recent work on the bed nucleus of the stria terminalis as an example.

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 ...

Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. Adeno-associated viruses (AAVs) are increasingly used to improve understanding of brain circuits and their functions. The AAV-mediated manipulation of specific cell populations and/or of precise molecular components has been tremendously useful to identify new sleep regulatory circuits/molecules and key proteins contributing to the adverse effects of sleep loss. For instance, inhibiting activity of the filamentous actin-severing protein&#160;cofilin&#160;using AAV prevents sleep deprivation-induced memory impairment. Here, a protocol is&#160;described that combines the manipulation of cofilin function in a cerebral cortex area with the recording of ECoG activity to examine whether cortical cofilin modulates the wakefulness and sleep ECoG signals. AAV injection is performed during the same surgical procedure as the implantation of ECoG and electromyographic (EMG) electrodes in adult male and female mice. Mice are anesthetized, and their heads are shaved. After skin cleaning and incision, stereotaxic coordinates of the motor cortex are determined, and the skull is pierced at this location. A cannula prefilled with an AAV expressing cofilinS3D, an inactive form of cofilin, is slowly positioned in the cortical tissue. After AAV infusion, gold-covered screws (ECoG electrodes) are screwed through the skull and cemented to the skull with gold wires inserted in the neck muscles (EMG electrodes). The animals are allowed three weeks to recover and to ensure sufficient expression of cofilinS3D. The infected area and cell type are verified using immunohistochemistry, and the ECoG is analyzed using visual identification of vigilance states and spectral analysis. In summary, this combined methodological approach allows the investigation of the precise contribution of molecular components regulating neuronal morphology and connectivity to the regulation of synchronized cerebral cortex activity during wakefulness and sleep.

This article describes a protocol for the manipulation of molecular targets in the cerebral cortex using adeno-associated viruses and for monitoring the effects of this manipulation during wakefulness and sleep using electrocorticographic recordings.

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. ...

The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila

Sleep homeostasis, the increase in sleep observed following sleep loss, is one of the defining criteria used to identify sleep throughout the animal kingdom. As a consequence, sleep deprivation and sleep restriction are powerful tools that are commonly used to provide insight into sleep function. Nonetheless, sleep deprivation experiments are inherently problematic in that the deprivation stimulus itself may be the cause of observed changes in physiology and behavior. Accordingly, successful sleep deprivation techniques should keep animals awake and, ideally, result in a robust sleep rebound without also inducing a large number of unintended consequences. Here, we describe a sleep deprivation technique for Drosophila melanogaster. The Sleep Nullifying Apparatus (SNAP) administers a stimulus every 10s to induce negative geotaxis. Although the stimulus is predictable, the SNAP effectively prevents &#62;95% of nighttime sleep even in flies with high sleep drive. Importantly, the subsequent homeostatic response is very similar to that achieved using hand-deprivation. The timing and spacing of the stimuli can be modified to minimize sleep loss and thus examine non-specific effects of the stimulus on physiology and behavior. The SNAP can also be used for sleep restriction and to assess arousal thresholds. The SNAP is a powerful sleep disruption technique that can be used to better understand sleep function.

The Sleep Nullifying Apparatus: A Highly Efficient Method of Sleep Depriving Drosophila

Sleep deprivation is a powerful tool to investigate sleep function and regulation. We describe a protocol to sleep deprive Drosophila using the Sleep Nullifying Apparatus, and to determine the extent of rebound sleep induced by deprivation.

Sleep homeostasis, the increase in sleep observed following sleep loss, is one of the defining criteria used to identify sleep throughout the animal ...

Measuring Neural Mechanisms Underlying Sleep-Dependent Memory Consolidation During Naps in Early Childhood

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