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/60200/60...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+napping+on+cognitive+function+in+preschoolers.">The effects of napping on cognitive function in preschoolers.</a> Journal of Developmental &amp; Behavioral Pediatrics. 32 (2), 90-97 (2011).</li><li>Kurth, S., Ringli, M., Geiger, A., Lebourgeois, M., Jenni, O. G., Huber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-density+sleep+electroencephalogram+study.">High-density sleep electroencephalogram study.</a> Journal of Neuroscience. 30 (40), 13211-13219 (2010).</li><li>Backhaus, J., Hoeckesfeld, R., Born, J., Hohagen, F., Junghanns, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immediate+as+well+as+delayed+post+learning+sleep+but+not+wakefulness+enhances+declarative+memory+consolidation+in+children.">Immediate as well as delayed post learning sleep but not wakefulness enhances declarative memory consolidation in children.</a> Neurobiology of Learning and Memory. 89 (1), 76-80 (2008).</li><li>Wilhelm, I., Diekelmann, 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=Sleep+in+children+improves+memory+performance+on+declarative+but+not+procedural+tasks+TT+-+Bei+Kindern+verbessert+Schlaf+die+Ged&#228;chtnisleistung+f&#252;r+deklarative+aber+nicht+f&#252;r+prozedurale+Aufgaben.">Sleep in children improves memory performance on declarative but not procedural tasks TT - Bei Kindern verbessert Schlaf die Ged&#228;chtnisleistung f&#252;r deklarative aber nicht f&#252;r prozedurale Aufgaben.</a> Learning and Memory. 15 (5), 373-377 (2008).</li><li>Seehagen, S., Konrad, C., Herbert, J. 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>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</td>
			<td>Philips Respironics&#160;</td>
			<td>1114828</td>
			<td>Alternatives may be available.&#160;</td>
		</tr>
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			<td>Brain Analyzer</td>
			<td>Brain Products</td>
			<td>BV-BP-170-1000</td>
			<td>Alternatives may be available.&#160;</td>
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			<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>
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			<td>Embla REMLogic software&#160;</td>
			<td>Natus Medical Inc.</td>
			<td>21475</td>
			<td>Alternatives may be available.&#160;</td>
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			<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.&#160;</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 ...

Watch this Scientific Journal Video about Measuring Neural Mechanisms Underlying Sleep-Dependent Memory Consolidation During Naps in Early Childhood at JoVE.com

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 ...

This protocol allows for the measurement of sleep-dependent memory consolidation in children and provides a practical approach for examining the benefit of napping on declarative memory in typically developing children. Results from this technique will have implications for early childhood education, as they will provide empirically based recommendations for nap lengths in preschool classrooms. The main advantage of this method is that it can be used to assess both sleep distribution and sleep architecture, which provide data regarding brain states that support memory consolidation.Although these methods were developed for examining memory consolidation during naps in preschool-age children, they can be adapted for use with any age group and for overnight sleep bouts. Proper electrode site measurement and preparation are critical for achieving low impedances and high-quality data, which can be difficult in preschool children. Demonstrating the procedure with Tamara Allard will be Arcadia Ewell and Benjamin Weinberg, research assistants in the laboratory.After explaining the procedure to the child and parent using age-appropriate materials, have the child engage in a quiet activity. Using a flexible tape measure and china marker, measure the distance from the inion to nasion, and place a mark at the halfway point. Measure the distance from the preauricular notch on one ear to the preauricular notch on the other ear, and place a mark at the halfway point.The intersection of these two marks is the reference point, or CZ.Measure 10%of the inion to nasion distance up from the inion and 10%of the preauricular notch to preauricular notch measurement from this point on either side. Then make the O three marks on one side and the O four mark on the other. Measure out 20%of the preauricular notch to preauricular notch measurement from the reference point on either side of the head, and make the C three and C four marks on each side.Measure 20%of the inion to nasion distance up from the reference point, and measure out 20%of the preauricular notch to preauricular notch measurement from this point on either side. Then make the F three and F four marks on each side of this point. When all of the marks have been placed, use an alcohol swab to clean the first electrode location before using a slightly abrasive gel to exfoliate the skin.Remove any residual cleaning material, and fill an electrode with electrode cream. For electrodes being placed where hair is present, apply an additional drop of electrode cream to a gauze square, and place the gauze on the back of the electrode. Place the electroencephalography electrodes on each marked location on the scalp, on each mastoid, and in the center of the forehead.Place the right electrooculography electrode adjacent and slightly superior to the outside of the right eye, and place the left electrooculography electrode to the outside and slightly inferior to the left eye. Place one electromyography electrode on the right cheek, just above the smile line, and have the child say the word milk out loud while feeling for the location at which the muscle contractions in the neck and chin are maximal, to place a second electromyograph electrode on the left side, just above where the chin meets the neck, adjacent to the esophagus. When all of the electrodes have been placed, attach the electrodes to the recording device, and initiate the recording to ensure that all of the electrodes pass the impedance test.To administer the encoding phase of the visuospatial memory task, set out an age-appropriate number of to-be-remembered stimuli, and direct the child to identify each image by name. Instruct the child to remember the location of each image within the grid, and replace the cards with blank images. Then present the images one at a time, and ask the child to identify the previous location of the image within the grid, informing the child whether or not the correct location was indicated.After the child has reached a threshold of 75%correct image identification, administer the immediate recall phase. Present the images again one at a time, and ask the child to recall the corresponding location without providing visual or verbal feedback and probing each item only one time. At the conclusion of the immediate recall phase, have the child use the restroom before initiating the child's typical pre-nap routine.Allow the child to nap in their typical nap location. Then conduct the delayed recall phase for the visuospatial memory task as previously demonstrated for the immediate recall phase approximately 15 to 30 minutes after the child wakes to avoid sleep inertia. At the end of the delayed recall assessment, use water-based spray to remove the electrodes.For electrodes applied with tape, use a cotton pad soaked with baby oil to saturate the tape before gently pulling the tape up from the corners. In this representative experiment, children'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, signifying a nap benefit. Moreover, those who spent the prior day in the wake condition did not recover memories during overnight sleep.Of interest, the nap benefit was only significant in children who napped habitually. Moreover, performance on the visuospatial memory task was related to the data obtained from the PSG recordings during the nap. Specifically, the fewer sleep spindles recorded during the non-rapid eye movement stage two sleep was related to a better immediate recall accuracy.Importantly, there was a positive correlation between the change in recall after nap and sleep spindle density during non-rapid eye movement stage two sleep. No other measure of sleep physiology was determined to be related to memory performance. Although this paper focuses on PSG, a complete understanding of sleep-dependent memory consolidation will rely on proper administration of complementary techniques, such as actigraphy and parent report, to determine sleep cycles.To compare data obtained from the nap and wake promotion conditions, it is important to keep the conditions as similar as possible in terms of their duration and interfering, distracting content. This technique can enhance our understanding of the relationship between sleep and memory during early childhood, a period of developmental significance as children transition from biphasic to monophasic sleep patterns.

measuring-neural-mechanisms-underlying-sleep-dependent-memory

Research

JoVE Journal

Neuroscience

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Investigating the Neural Mechanisms of Aware and Unaware Fear Memory with fMRI

Pavlovian fear conditioning is often used in combination with functional magnetic resonance imaging (fMRI) in humans to investigate the neural substrates of associative learning 1-5. In these studies, it is important to provide behavioral evidence of conditioning to verify that differences in brain activity are learning-related and correlated with human behavior. 
Fear conditioning studies often monitor autonomic responses (e.g. skin conductance response; SCR) as an index of learning and memory 6-8. In addition, other behavioral measures can provide valuable information about the learning process and/or other cognitive functions that influence conditioning. For example, the impact unconditioned stimulus (UCS) expectancies have on the expression of the conditioned response (CR) and unconditioned response (UCR) has been a topic of interest in several recent studies 9-14. SCR and UCS expectancy measures have recently been used in conjunction with fMRI to investigate the neural substrates of aware and unaware fear learning and memory processes 15. Although these cognitive processes can be evaluated to some degree following the conditioning session, post-conditioning assessments cannot measure expectations on a trial-to-trial basis and are susceptible to interference and forgetting, as well as other factors that may distort results 16,17 .
Monitoring autonomic and behavioral responses simultaneously with fMRI provides a mechanism by which the neural substrates that mediate complex relationships between cognitive processes and behavioral/autonomic responses can be assessed. However, monitoring autonomic and behavioral responses in the MRI environment poses a number of practical problems. Specifically, 1) standard behavioral and physiological monitoring equipment is constructed of ferrous material that cannot be safely used near the MRI scanner, 2) when this equipment is placed outside of the MRI scanning chamber, the cables projecting to the subject can carry RF noise that produces artifacts in brain images, 3) artifacts can be produced within the skin conductance signal by switching gradients during scanning, 4) the fMRI signal produced by the motor demands of behavioral responses may need to be distinguished from activity related to the cognitive processes of interest. Each of these issues can be resolved with modifications to the setup of physiological monitoring equipment and additional data analysis procedures. Here we present a methodology to simultaneously monitor autonomic and behavioral responses during fMRI, and demonstrate the use of these methods to investigate aware and unaware memory processes during fear conditioning.

A methodology to investigate the neural mechanisms that support aware and unaware memory processes during fear conditioning is described. This method monitors blood oxygen level dependent (BOLD) functional magnetic resonance imaging, skin conductance response, and unconditioned stimulus expectancy during Pavlovian fear conditioning to assess the neural correlates of distinct memory processes.

Pavlovian fear conditioning is often used in combination with functional magnetic resonance imaging (fMRI) in humans to investigate the neural substrates ...

Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents the return of fear. Pavlovian fear-conditioning paradigms are commonly employed to create a controlled, novel fear association in the laboratory. After pairing an innocuous stimulus (conditioned stimulus, CS) with an aversive outcome (unconditioned stimulus, US) we can elicit a fear response (conditioned response, or CR) by presenting just the stimulus alone1,2 . Once fear is acquired, it can be diminished using extinction training, whereby the conditioned stimulus is repeatedly presented without the aversive outcome until fear is no longer expressed3. This inhibitory learning creates a new, safe representation for the CS, which competes for expression with the original fear memory4. Although extinction is effective at inhibiting fear, it is not permanent. Fear can spontaneously recover with the passage of time. Exposure to stress or returning to the context of initial learning can also cause fear to resurface3,4.
Our protocol addresses the transient nature of extinction by targeting the reconsolidation window to modify emotional memory in a more permanent manner. Ample evidence suggests that reactivating a consolidated memory returns it to a labile state, during which the memory is again susceptible to interference5-9. This window of opportunity appears to open shortly after reactivation and close approximately 6hrs later5,11,16, although this may vary depending on the strength and age of the memory15. By allowing new information to incorporate into the original memory trace, this memory may be updated as it reconsolidates10,11. Studies involving non-human animals have successfully blocked the expression of fear memory by introducing pharmacological manipulations within the reconsolidation window, however, most agents used are either toxic to humans or show equivocal effects when used in human studies12-14. Our protocol addresses these challenges by offering an effective, yet non-invasive, behavioral manipulation that is safe for humans.
By prompting fear memory retrieval prior to extinction, we essentially trigger the reconsolidation process, allowing new safety information (i.e., extinction) to be incorporated while the fear memory is still susceptible to interference. A recent study employing this behavioral manipulation in rats has successfully blocked fear memory using these temporal parameters11. Additional studies in humans have demonstrated that introducing new information after the retrieval of previously consolidated motor16, episodic17, or declarative18 memories leads to interference with the original memory trace14. We outline below a novel protocol used to block fear recovery in humans.

Conditioned fear can be diminished through an inhibitory process called extinction, but can resurface under conditions such as the passage of time or exposure to stress. Our protocol presents a novel way of preventing fear recovery by introducing extinction during the reconsolidation window (the re-storage phase of a reactivated memory).

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents ...

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

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 ...

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or manipulations; a powerful tool in elucidating underlying mechanisms and circuitry. While the technique of microdialysis has been used for decades to measure nearly any analyte of interest in the brain, this technique is limited in temporal resolution. Alternatively, fast scan cyclic voltammetry is both temporally precise and extremely sensitive; however, because this technically difficult method relies on the electroactivity of the analyte of interest, the possibility to detect nonelectroactive substances (e.g., the neurotransmitter glutamate) is eliminated. This paper details the use of a turn-key system that combines fixed-potential amperometry and enzymatic biosensing to measure both electroactive and nonelectroactive neurotransmitters with temporal precision. The pairing of these two powerful techniques allows for the measurement of both tonic and phasic neurotransmission with relative ease, and permits recording of multiple neurotransmitters simultaneously. The aim of this manuscript is to demonstrate the process of measuring dopamine and glutamate neurotransmission in vivo using a naturally rewarding behavior (i.e., sexual behavior) in female hamsters, with the ultimate goal of displaying the technical feasibility of this assay for examining other behaviors and experimental paradigms.

Measuring In Vivo Changes in Extracellular Neurotransmitters During Naturally Rewarding Behaviors in Female Syrian Hamsters

This paper details the use of fixed-potential amperometric recordings using carbon fiber electrodes and enzymatic biosensor technology to measure the release of dopamine and glutamate with high temporal resolution during natural rewarding behavior in the female hamster.

The ability to measure neurotransmitter release on a rapid time scale allows patterns of neurotransmission to be linked to specific behaviors or ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...