This study was supported by grant BCS-0963581 from the National Science Foundation (to EAK and JDP). The authors thank Christine Cox for her helpful discussion of the data, as well as Halle Zucker, John Morris, Christopher Stare, Sondra Corgan, and Maite Balda for their assistance with data collection. Please address correspondence to Kelly A. Bennion (kelly.bennion@bc.edu; 140 Commonwealth Avenue, Boston College Psychology, McGuinn 300, Chestnut Hill, MA 02467).

1. Participant Screening and Preparation for the Experiment
<ol>
	<li>Recruit participants who are native English speakers with normal or corrected-to-normal vision. They should be free of neurological, psychiatric, and sleep disorders, and may not be taking any medications affecting the central nervous system or sleep architecture. Be sure to recruit a similar balance of male and female participants within the desired age range of interest (e.g., 18 to 35 years old), keeping in mind that changes in sleep architecture can occur as early as the mid-30s&#160;16.</li>
	<li>To most accurately assess the effects of sleep on cognition, have participants maintain a regular sleep schedule and restrict their alcohol consumption leading up to the study. Ask participants to sleep for at least 7&#160;hr&#160;a night and be in bed by 2:00 am for the five nights preceding the study. Also ensure that they restrict their alcohol consumption to a maximum of 2&#160;drinks during the 5&#160;days preceding the study, with absolutely no alcohol consumption the day before or day of the study.</li>
	<li>During scheduling, ensure that participants will comply with the guidelines related to the salivary cortisol sample (see also Section 4): They must refrain from physical activity, eating, drinking (anything besides water), smoking, and brushing their teeth for the 2&#160;hr&#160;prior to encoding. They must also refrain from drinking water for at least 15 min prior to encoding.</li>
	<li>Scheduling participants. If full random assignment is not possible, ensure that participants do not differ in age, scores on the Morningness-Eveningness Questionnaire17 (MEQ), Beck Depression Inventory18 (BDI), Beck Anxiety Inventory19 (BAI), and the amount of sleep obtained on the night before retrieval.</li>
</ol>
2. Conditions and Experimental Design
<ol>
	<li>Schedule Sleep participants such that the encoding session occurs in the evening (7:00-10:00 pm) and the retrieval session occurs 12 hr later, following a full night of sleep in the laboratory. Schedule Wake participants such that the encoding session occurs in the morning (7:00-10:00 am) and the retrieval session occurs 12 hr later following a full day of wakefulness; ensure that they do not nap between sessions.</li>
	<li>Include Morning and Evening Short Delay conditions (those that have only a 20-min delay between encoding and retrieval, as compared to a 12-hr delay for the Sleep and Wake groups) in the experimental design to minimize the concern that any differences found between the Sleep and Wake groups are due to the time of testing (morning vs. evening) rather than due to the sleep occurring during the consolidation delay. These Short Delay conditions can also be thought of as &#8220;circadian control&#8221; conditions.
	<ol>
		<li>Arrange for participants in the Morning Short Delay condition to encode the stimuli between 7:00-10:00 am, and for participants in the Evening Short Delay condition to encode the stimuli between 7:00-10:00 pm. Test the participants 20 min after encoding.</li>
	</ol>
	</li>
</ol>
3. Stimuli Construction
<ol>
	<li>Stimuli construction for encoding. Select stimuli based on the specific experimental question. This protocol focuses on the effects of sleep and cortisol on emotional memory, and as such, the visual stimuli during encoding are scenes composed of either a negative object or a neutral object placed on a neutral background.
	<ol>
		<li>Ensure that all emotional stimuli have either been previously rated for valence and arousal20,21, or that they are rated by participants upon completing the study using a Likert scale from 1 to 7. Negative objects should be rated as highly arousing and low in valence (e.g., arousal: 5-7; valence &#60; 3 on 7-point scales with high values indicating high arousal and high positivity, respectively), and neutral objects should be rated as non-arousing and neutral in valence (e.g., arousal &#60; 4; valence: 3-5).</li>
		<li>Randomization and design. Randomly intermix the negative and neutral scenes between blocks (if applicable). The present study employs 2&#160;blocks lasting approximately 10&#160;min&#160;each, allowing participants to have a short break to rest their eyes from the eye tracker in between. Breaks may be helpful every 10-15 min for most young adult participants, but if testing a different population (e.g., children), more frequent breaks may be required.</li>
	</ol>
	</li>
	<li>Stimuli construction for retrieval. Select stimuli based on the specific experimental question. Here, memory for the objects is the focus, and as such, participants are presented with objects and backgrounds (half of which were presented during encoding, and half new) separately during retrieval.</li>
</ol>
4. Cortisol Procedure
<ol>
	<li>Ensure that participants have followed all requirements in 1.3: No physical activity, eating, drinking (anything besides water), smoking, and brushing their teeth for the 2&#160;hr&#160;prior to encoding, as well as no water for at least 15 min prior to encoding.</li>
	<li>Immediately prior to encoding, instruct participants to rinse their mouths with approximately 1 ounce of water. Remind them not to swallow the water, as to avoid sample dilution.</li>
	<li>Have participants salivate on an oral swab (see Materials) for 2 min.</li>
	<li>Upon having participants place the oral swab in the swab storage tube, store the swabs at the approximate temperature of 0 &#186;F until analyzed.</li>
</ol>
5. Eye Tracking/Encoding Procedure
<ol>
	<li>Eye tracking procedure. The eye tracker used here tracks participants&#8217; left eye gaze patterns at 500 Hz (see Materials). Alternative trackers can be used; in order to most accurately assess attention during encoding, follow the instructions of the specific eye tracker used.
	<ol>
		<li>First, ask participants to sit with their chin on the chinrest and forehead up against a bar. Make adjustments to the chair height and chinrest as needed, ensuring that the center of the screen aligns with participants&#8217; eyes.</li>
		<li>Ensure that the eye tracker is accurately tracking participants&#8217; gaze within 1&#176;&#160;of accuracy by having each participant complete a calibration task. Ideally, a 9- or 17-point calibration would be used, depending on the system, but a 3- or 5-point calibration may also be sufficient.</li>
		<li>First, ask participants to follow a black dot with their eyes as it moves to different points on the screen and to fixate on it when it stops.</li>
		<li>Once the eye tracker is calibrated accurately, ask participants if they are ready to begin the task, and then press the &#8220;record&#8221; button.</li>
	</ol>
	</li>
	<li>Encoding procedure. Ask participants to perform a task that is likely to lead to deep encoding, such as having them indicate via mouse click&#160;whether they would approach or back away from the scene (e.g., left = approach; right = back away) if they were to encounter it in real life10. See Figure 2 for a visual depiction of the encoding procedure.
	<ol>
		<li>Allow participants to have a short self-determined break (e.g., ~10-60 sec) between blocks so that they may sit back from the eye tracker and rest their eyes prior to continuing. Ask them to indicate when they are ready to continue.</li>
	</ol>
	</li>
	<li>Analysis of eye gaze data. To measure participants&#8217; attention to certain parts of the scene, use software to draw Areas of Interest22 (AOIs) around those parts.
	<ol>
		<li>After drawing the AOIs, calculate the proportion of time participants look at the AOI relative to the rest of the scene. Alternatively, count the number of saccades that participants make to that AOI within a certain time frame.</li>
	</ol>
	</li>
</ol>
6. Study-test Delay
<ol>
	<li>Ensure that the delay length between encoding and retrieval for the Sleep and Wake conditions is equal (e.g., 12 hr), as well as the delay length for the 2&#160;control conditions (e.g., 20 min).
	<ol>
		<li>For the Sleep participants, ensure that the 12&#160;hr delay includes approximately 8&#160;hr&#160;of sleep. Conversely, ensure that the Wake participants do not sleep or nap during this interval.</li>
		<li>Ask the Morning and Evening Short Delay participants to remain in the laboratory during their 20&#160;min delay. Tell them that they may do whatever they please during this time, provided that they do not nap.</li>
	</ol>
	</li>
</ol>
7. Recognition Procedure
<ol>
	<li>Following the delay period, give participants a memory test.
	<ol>
		<li>Ask participants to indicate whether the displayed stimulus is &#8220;old&#8221; (included in a previously studied scene), or &#8220;new&#8221; (not previously studied) by pressing corresponding keys on a keyboard (e.g., &#8220;1&#8221; = old; &#8220;2&#8221; = new).</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

This experimental design provided the first evidence that the beneficial effects of pre-encoding cortisol on memory are significant only when sleep occurs during the consolidation period. Only by both measuring cortisol levels and manipulating whether the consolidation interval included sleep was it possible to determine that sleep and cortisol have an interactive effect on memory. This design was critical in determining that, in the prior studies that have tied pre-learning cortisol to facilitated memory for negative stimuli1,5, these effects of cortisol may manifest because of the sleep occurring during the consolidation interval.
This finding is made even more interesting considering that the sleep versus wake design necessitated testing participants in the two groups at different times of day. Cortisol follows a circadian rhythm26, with the highest cortisol levels in the early morning, and the lowest in the evening. As such, the Sleep participants were all within a narrow range of relatively low cortisol levels, having encoded in the evening, while the Wake participants had higher and more variable cortisol levels, having encoded in the morning. By using this design, it appears that even small differences in relatively low cortisol levels are sufficient to influence memory consolidation over a period of sleep. At the same time, however, the diurnal variation in cortisol also complicates the design. Future work may consider how to control for the effects of the diurnal variation of cortisol in different ways. One such way would be to replicate the present findings using an afternoon nap paradigm. Because cortisol levels between a Nap and Wake condition would be statistically equivalent, circadian fluctuations in cortisol in such a paradigm would not be a concern.
While many questions remain about how cortisol interacts with sleep to enhance emotional memory consolidation, the current work provides evidence that the facilitative effect of pre-encoding cortisol on emotional memory following delays of at least 24 hr&#160;including sleep1,5,27 may be due to interactions between cortisol and sleep-dependent consolidation processes. Without a design in which one condition&#8217;s delay includes sleep and the other does not, it would not be possible to isolate whether sleep is necessary for this effect to be observed. Furthermore, by assessing participants&#8217; eye gaze during encoding, it was determined that this facilitative effect of cortisol on emotional memory may be due to cortisol modulating the relation between attention to emotional information at encoding and the subsequent consolidation of that information during sleep: With elevated cortisol, there is a stronger relation between what participants look at during encoding and what they later remember. It is possible that this is because elevated cortisol at encoding &#8216;tags&#8217; this emotional information as important to remember, which leads sleep to selectively strengthen that salient information during the consolidation interval.
This unique combination of methods &#8211; a sleep versus wake design, a measurement of resting salivary cortisol values, an assessment of eye gaze during encoding, and an administration of a behavioral recognition memory test &#8211; led not only to uncovering an interactive effect of cortisol and sleep on consolidation, but also to determining a potential attentional mechanism behind this effect. Overall, this study can be used as an example of how to combine methodologies that are typically used independently in order to achieve a better understanding of complex interactions between variables that affect cognition.

The ability to consolidate information is dependent on countless factors, with sleep and the stress hormone cortisol being two of the most influential variables. Prior research has shown that elevated cortisol levels, either induced through exogenous cortisol administration or psychosocial stress, often are associated with a selective enhancement of memory for emotional relative to neutral stimuli1-7. Sleep has been shown to have a similar selective effect on emotional memory8,9: When participants are presented with scenes composed of a negative or neutral object placed on a neutral background, sleep unbinds the scenes, preserving memory for the negative objects, while allowing memory for the less salient scene components (neutral objects and neutral backgrounds) to decay10-12.
While these two literatures focus on the independent effects of cortisol and sleep on emotional memory, it is possible that cortisol and sleep have an interactive effect. Interestingly, because studies investigating the effects of cortisol on memory include a retention delay of at least 24 hr, which necessarily includes a night of sleep, it is impossible to determine whether sleep is necessary for cortisol to have a facilitative effect on consolidation. Similarly, while research has shown that sleep preferentially benefits memory for emotional information, it is possible that these effects may be intensified in individuals with higher cortisol levels during encoding.
For these reasons, it is important to understand not only how sleep and cortisol independently support memory formation, but also to investigate whether sleep and cortisol interact to support memory formation. After all, there are important links between sleep and cortisol in normal aging13, and forms of psychopathology that are associated with memory deficits14,15. Only by using a combination of methodologies is it possible to understand the complex interactions between sleep and cortisol, and their effects on memory consolidation. Specifically, by combining the method of salivary cortisol collection with a sleep versus wake design, it is possible to determine whether there is an interactive effect of these two variables on consolidation. Also valuable is that through the use of eye tracking to measure eye gaze during encoding, it is possible to elucidate a potential attentional mechanism underlying this effect.

<table border="0" cellpadding="0" cellspacing="0" width="634">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Salimetrics oral swabs (SOS)</td>
			<td style="width:105px;">Salimetrics</td>
			<td style="width:119px;">5001.02</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Swab storage tubes (SST)</td>
			<td style="width:105px;">Salimetrics</td>
			<td style="width:119px;">5001.05</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:243px;">Eye-tracker</td>
			<td style="width:105px;">SensoMotoric Instruments (SMI)</td>
			<td style="width:119px;">iView X Hi-Speed</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

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.

Effects of Sleep and Cortisol on Memory for Emotional and Neutral Stimuli
The first hypothesis addressed is that elevated cortisol during encoding will facilitate memory for emotional more than neutral stimuli, and that this effect is dependent on sleep occurring between encoding and retrieval. Figure 4A plots the effect of cortisol on memory for negative objects. Standardized levels of cortisol (x-axis) and memory for negative objects (y-axis) were directly related in the Sleep group (in red) but not the Wake group (in gray). The Group (Sleep vs. Wake) by Cortisol interaction was significant [t(41) = 2.23, &#946; = 2.92, p = 0.031]: Higher cortisol at encoding predicted memory for negative objects if participants slept between encoding and retrieval [t(24) = 2.31, &#946; = 0.43, p = 0.031], but not if they stayed awake [t(16) = 0.40, &#946; = 0.10, p = 0.70; see Figure 4A]. These significant effects were not due to gender, menstrual cycle, or critically, time of day, which was determined by running additional analyses with the Morning and Evening Short Delay groups. For neutral memory (see Figure 4B), a similar, but weaker pattern was observed. There was a marginally significant relation between cortisol levels prior to encoding (x-axis) and memory for neutral objects (y-axis) in the Sleep group [in blue; t(24) = 1.76, &#946; = 0.34, p = .092], but not the Wake group [in gray; t(16) = 0.98, &#946; = 0.25, p = 0.34].&#160;The interaction between Cortisol and Group was marginally significant [t(41) = 1.95, &#946; = 2.55, p = 0.059].
Effects of Sleep and Cortisol on the Interaction between Attention during Encoding and Consolidation
It was hypothesized that this beneficial effect of cortisol on emotional memory may be partly due to cortisol&#8217;s ability to &#8216;tag&#8217; information as important to remember at the time of encoding, leading to the subsequent prioritization of that information during sleep. This &#8220;emotional tagging&#8221; concept suggests that encoding arousing stimuli activates neural mechanisms, leading to long-term plasticity in the synapses marked by the tag23-25. It is possible that elevated cortisol during encoding helps to set these tags, leading to the selective preservation of this information during consolidation. To investigate this possibility, the eye-tracking data were analyzed to determine whether higher cortisol increases the likelihood that sleep-based consolidation processes preferentially strengthen memory for the information that receives the most attention during encoding. First, the proportion of time each participant spent looking at each object within each scene (i.e., the AOI) relative to the total scene viewing time was calculated. The scenes were then sorted on a post-hoc basis, using each participant&#8217;s recognition data to sort the scenes into those for which the participant later remembered the object and those for which the participant later forgot the object. Lastly, a score was computed to reflect the difference in looking time between subsequently remembered and subsequently forgotten objects (See Figure 5). For example, if participants looked at the objects they subsequently remembered for an average of 75% of the time that the scene was on the screen, and looked at the objects they subsequently forgot for an average of 65% of the time that the scene was on the screen, their difference in looking time score would be 10%.
Similar to the analyses conducted on the effects of sleep and cortisol on memory (Figure 4), a linear regression was used to test the effects of sleep and cortisol on this difference in looking time at encoding as a function of later memory. For negative objects (see Figure 6A), resting cortisol (x-axis) marginally predicted the difference in looking time at encoding as a function of later memory (y-axis) in the Sleep group [in red; t(23) = 1.869, &#946; = 0.37, p = 0.075] but not the Wake group [in gray; t(16) = 0.168, &#946; = 0.043, p = 0.87]. The interaction between Cortisol and Group was significant [t(40) = -2.04, &#946; = -2.99, p = 0.049], and critically, this significant effect was not due to gender, menstrual cycle, or time of day. For neutral objects (see Figure 6B), there was no effect of cortisol in the Sleep group (in blue) nor the Wake group (in gray), and the interaction between Cortisol and Group was not significant.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51500/51500fig1highres.jpg" width="500" /> 
Figure 1. Visual depiction of the procedure described in this video report, separated by Group. This figure displays the four groups of participants (Sleep, Wake, Morning Short Delay, and Evening Short Delay), as well as the timing of the pre-encoding cortisol sample, encoding, and retrieval for each group.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51500/51500fig2highres.jpg" width="500" /> 
Figure 2. Visual depiction of stimuli used during encoding. This figure shows that each scene was composed of either a negative or a neutral object placed in front of a neutral background. It also shows that participants viewed these scenes for three seconds each, during which time they indicated whether they would approach or back away from the scene if they encountered it during real life.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51500/51500fig3highres.jpg" width="500" /> 
Figure 3. Visual depiction of stimuli used during retrieval. This figure shows that participants were presented with objects and backgrounds (separately) during the recognition memory test. These objects and backgrounds were either previously presented during encoding (&#8220;old&#8221;) or had never before been seen in the context of the experiment (&#8220;new&#8221;).
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51500/51500fig4highres.jpg" width="500" /> 
Figure 4. Effect of cortisol on memory for negative and neutral objects. A plots the effect of standardized cortisol levels on memory for negative objects. B plots the effect of standardized cortisol levels on memory for neutral objects. Legend: Sleep [red diamonds (neg), blue diamonds (neu)], Wake [gray squares], Sleep Linear Fit [red line (neg), blue line (neu)], Wake Linear Fit [gray line].
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51500/51500fig5highres.jpg" width="500" /> 
Figure 5. Visual depiction of the encoding (left) and retrieval (right) procedure as related to the dependent variable (difference in looking time during encoding as a function of later memory) assessed in eye-tracking analyses. This figure shows how the dependent variable in eye-tracking analyses (the difference in looking time during encoding as a function of later memory) was calculated. Particularly, this score reflects the proportion of time during encoding that participants looked at objects that they later remembered (&#8220;hits&#8221;) minus the proportion of time during encoding that participants looked at objects that they later forgot (&#8220;misses&#8221;).
<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/51500/51500fig6highres.jpg" width="500" /> 
Figure 6. Effect of cortisol on the difference in looking time at encoding as a function of later memory for negative and neutral objects. Looking time was calculated as the proportion of total scene viewing time that participants spent looking at the object within the scene.&#160;A score was then computed to reflect the difference in looking time between subsequently remembered and subsequently forgotten objects, and a linear regression was used to test the effects of cortisol and sleep on this score. A plots the effect for negative objects, while B plots the effect for neutral objects. Legend: Sleep [red diamonds (neg), blue diamonds (neu)], Wake [gray squares], Sleep Linear Fit [red line (neg), blue line (neu)], Wake Linear Fit [gray line].

Watch this Scientific Journal Video about Eye Tracking, Cortisol, and a Sleep vs. Wake Consolidation Delay: Combining Methods to Uncover an Interactive Effect of Sleep and Cortisol on Memory at JoVE.c

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

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 ...

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26.8K Views.  Boston College. 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 – methods that can be used with both healthy and clinical participants.

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

Manipulation of Epileptiform Electrocorticograms (ECoGs) and Sleep in Rats and Mice by Acupuncture

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little research to reveal the possible mechanisms behind these effects. To investigate the effect of acupuncture on epilepsy and sleep, several issues need to be addressed. The first is to identify the acupoints, which correspond between humans, rats, and mice. Furthermore, the depth of insertion of the acupuncture needle, the degree of needle twist in manual needle acupuncture, and the stimulation parameters for electroacupuncture (EA) need to be determined. To evaluate the effects of acupuncture on epilepsy and sleep, a feasible model of epilepsy in rodents is required. We administer pilocarpine into the left central nucleus of the amygdala (CeA) to simulate focal temporal lobe epilepsy (TLE) in rats. Intraperitoneal (IP) injection of pilocarpine induces generalized epilepsy and status epilepticus (SE) in rats. Five IP injections of pentylenetetrazol (PTZ) with a one-day interval between each injection successfully induces spontaneous generalized epilepsy in mice. Recordings of electrocorticograms (ECoGs), electromyograms (EMGs), brain temperature, and locomotor activity are used for sleep analysis in rats, while ECoGs, EMGs, and locomotor activity are employed for sleep analysis in mice. ECoG electrodes are implanted into the frontal, parietal, and contralateral occipital cortices, and a thermistor is implanted above the cerebral cortex by stereotactic surgery. EMG electrodes are implanted into the neck muscles, and an infrared detector determines locomotor activity. The criteria for categorizing vigilance stages, including wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep are based on information from ECoGs, EMGs, brain temperature, and locomotor activity. Detailed classification criteria are stated in the text.

Manipulation of Epileptiform Electrocorticograms ECoGs and Sleep in Rats and Mice by Acupuncture

This paper demonstrates the performance of acupuncture, epilepsy models, and the analysis of sleep in rodents. The acupuncture procedure and the identification of acupoints are described. Pilocarpine or pentylenetetrazol (PTZ) is used to induce epilepsy. Electrocorticogram (ECoG), electromyogram (EMG), brain temperature, and locomotor activity recordings are employed for sleep analysis.

Ancient Chinese literature has documented that acupuncture possesses efficient therapeutic effects on epilepsy and insomnia. There is, however, little ...

Noninvasive, High-throughput Determination of Sleep Duration in Rodents

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

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

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

A High-performance Liquid Chromatography Measurement of Kynurenine and Kynurenic Acid: Relating Biochemistry to Cognition and Sleep in Rats

The kynurenine pathway (KP) of tryptophan degradation has been implicated in psychiatric disorders. Specifically, the astrocyte-derived metabolite kynurenic acid (KYNA), an antagonist at both N-methyl-d-aspartate (NMDA) and &#945;7 nicotinic acetylcholine (&#945;7nACh) receptors, has been implicated in cognitive processes in health and disease. As KYNA levels are elevated in the brains of patients with schizophrenia, a malfunction at the glutamatergic and cholinergic receptors is believed to be causally related to cognitive dysfunction, a core domain of the psychopathology of the illness. KYNA may play a pathophysiologically significant role in individuals with schizophrenia. It is possible to elevate endogenous KYNA in the rodent brain by treating animals with the direct bioprecursor kynurenine, and preclinical studies in rats have demonstrated that acute elevations in KYNA may impact their learning and memory processes. The current protocol describes this experimental approach in detail and combines a) a biochemical analysis of blood kynurenine levels and brain KYNA formation (using high-performance liquid chromatography), b) behavioral testing to probe the hippocampal-dependent contextual memory (passive avoidance paradigm), and c) an assessment of sleep-wake behavior [telemetric recordings combining electroencephalogram (EEG) and electromyogram (EMG) signals] in rats. Taken together, a relationship between elevated KYNA, sleep, and cognition is studied, and this protocol describes in detail an experimental approach to understanding function outcomes of kynurenine elevation and KYNA formation in vivo in rats. Results obtained through variations of this protocol will test the hypothesis that the KP and KYNA serve pivotal roles in modulating sleep and cognition in health and disease states.

Alterations in the kynurenine pathway (KP) neuroactive metabolites are implicated in psychiatric illnesses. Investigating the functional outcomes of an altered kynurenine pathway metabolism in vivo in rodents may help elucidate novel therapeutic approaches. The current protocol combines biochemical and behavioral approaches to investigate the impact of an acute kynurenine challenge in rats.

The kynurenine pathway (KP) of tryptophan degradation has been implicated in psychiatric disorders. Specifically, the astrocyte-derived metabolite ...

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. ...

Eye-tracking to Distinguish Comprehension-based and Oculomotor-based Regressive Eye Movements During Reading

Regressive eye movements are eye movements that move backwards through the text and comprise approximately 10-25% of eye movements during reading. As such, understanding the causes and mechanisms of regressions plays an important role in understanding eye movement behavior. Inhibition of return (IOR) is an oculomotor effect that results in increased latency to return attention to a previously attended target versus a target that was not previously attended. Thus, IOR may affect regressions. This paper describes how to design materials to distinguish between regressions caused by comprehension-related and oculomotor processes; the latter is subject to IOR. The method allows researchers to identify IOR and control the causes of regressions. While the method requires tightly controlled materials and large numbers of participants and materials, it allows researchers to distinguish and control the types of regressions that occur in their reading studies.

The method was designed to investigate the role of inhibition of return (IOR) in regressive eye movements during reading. The focus is on differentiating between regressions triggered as a result of comprehension difficulty versus those triggered from oculomotor error, including the role of IOR in the two types of regressions.

Regressive eye movements are eye movements that move backwards through the text and comprise approximately 10-25% of eye movements during reading. As ...

Automated Measurements of Sleep and Locomotor Activity in Mexican Cavefish

Across phyla, sleep is characterized by highly conserved behavioral characteristics that include elevated arousal threshold, rebound following sleep deprivation, and consolidated periods of behavioral immobility. The Mexican cavefish, Astyanax mexicanus (A. mexicanus), is a model for studying trait evolution in response to environmental perturbation. A. mexicanus exist as in eyed surface-dwelling forms and multiple blind cave-dwelling populations that have robust morphological and behavioral differences. Sleep loss has occurred in multiple, independently-evolved cavefish populations. This protocol describes a methodology for quantifying sleep and locomotor activity in A. mexicanus cave and surface fish. A cost-effective video monitoring system allows for behavioral imaging of individually-housed larval or adult fish for periods of a week or longer. The system can be applied to fish aged 4 days post fertilization through adulthood. The approach can also be adapted for measuring the effects of social interactions on sleep by recording multiple fish in a single arena. Following behavioral recordings, data is analyzed using automated tracking software and sleep analysis is processed using customized scripts that quantify multiple sleep variables including duration, bout length, and bout number. This system can be applied to measure sleep, circadian behavior, and locomotor activity in almost any fish species including zebrafish and sticklebacks.

This protocol details the methodology for quantifying locomotor behavior and sleep in the Mexican cavefish. Previous analyses are extended to measure these behaviors in socially-housed fish. This system can be widely applied to study sleep and activity in other fish species.

Across phyla, sleep is characterized by highly conserved behavioral characteristics that include elevated arousal threshold, rebound following sleep ...

Collecting Sleep, Circadian, Fatigue, and Performance Data in Complex Operational Environments

Sleep loss and circadian misalignment contribute to a meaningful proportion of operational accidents and incidents. Countermeasures and work scheduling designs aimed at mitigating fatigue are typically evaluated in controlled laboratory environments, but the effectiveness of translating such strategies to operational environments can be challenging to assess. This manuscript summarizes an approach for collecting sleep, circadian, fatigue, and performance data in a complex operational environment. We studied 44 airline pilots over 34 days while they flew a fixed schedule, which included a baseline data collection with 5 days of mid-morning flights, four early flights, four high-workload mid-day flights, and four late flights that landed after midnight. Each work block was separated by 3&#8211;4 days of rest. To assess sleep, participants wore a wrist-worn research-validated activity monitor continuously and completed daily sleep diaries. To assess the circadian phase, pilots were asked to collect all urine produced in four or eight hourly bins during the 24 h after each duty block for the assessment of 6-sulfatoxymelatonin (aMT6s), which is a biomarker of the circadian rhythm. To assess subjective fatigue and objective performance, participants were provided with a touch-screen device used to complete the Samn-Perelli Fatigue Scale and Psychomotor Vigilance Task (PVT) during and after each flight, and at waketime, mid-day, and bedtime. Using these methods, it was found that sleep duration was reduced during early starts and late finishes relative to baseline. Circadian phase shifted according to duty schedule, but there was a wide range in the aMT6s peak between individuals on each schedule. PVT performance was worse on the early, high-workload, and late schedules relative to baseline. Overall, the combination of these methods was practical and effective for assessing the influence of sleep loss and circadian phase on fatigue and performance in a complex operational environment.

Sleep loss and circadian misalignment contribute to numerous operational accidents and incidents. The effectiveness of countermeasures and work scheduling designs aimed at mitigating fatigue can be challenging to evaluate in operational environments. This manuscript summarizes an approach for collecting sleep, circadian, fatigue, and performance data in complex operational environments.

Sleep loss and circadian misalignment contribute to a meaningful proportion of operational accidents and incidents. Countermeasures and work scheduling ...

Providing Meaningful Environmental Enrichment and Measuring Saliva Cortisol in Pigs Housed on Slatted Flooring

As pork is the most consumed meat worldwide, the welfare of animals in the swine industry has increasingly become a major public concern, which imposes a substantial pressure coming from customers, legislators and other stakeholders, to make management changes to improve the well-being of these animals. Several studies have demonstrated that providing environmental enrichment to pigs allows them to express their natural behavior, such as rooting and exploring, as well as nesting prior to farrowing, and is associated with reduced stress and improved production and welfare. However, many considerations should be taken into account when providing environmental enrichment, such as the type of floor, drainage and sewage systems, the pigs&#39; stage in life, the material, as well as its hanging method, height and location within the pen. The objectives of this paper are (1) to give methodologic information on how to provide a relatively simple and practical meaningful environmental enrichment for pigs which are housed on slatted floors during the different stages of their life, and (2) to demonstrate how to collect saliva samples for the measurement of cortisol concentrations, as a biomarker for acute stress. Protocols include information regarding the use of jute, cotton ropes, straw in racks, as well as chewable silicone sticks devices as environmental enrichment in pens of farrowing and lactation, weaners and finishers. In addition, the use of cotton rope for a non-invasive saliva samples collection for cortisol concentrations analysis is detailed. The protocols provided are relevant for professionals aiming to improve and monitor animal welfare, in both research and industrial swine farming.

This protocol demonstrates how to provide practical meaningful environmental enrichment for pigs which are housed on slatted flooring during the different stages of their lives, and how to collect saliva samples in a non-invasive manner for the measurement of cortisol concentrations, as a biomarker for acute stress.

As pork is the most consumed meat worldwide, the welfare of animals in the swine industry has increasingly become a major public concern, which imposes a ...

Loneliness Assuaged: Eye-Tracking an Audience Watching Barrage Videos

Researchers usually theorize media exposure based on assumptions of legacy media. However, a new interactive video viewing format, in this case barrage video where viewers&#8217; comments are overlaid over visual content, challenges past perspectives. This study proposes an activation and match satisfaction model to study viewing behaviors of lonely people and to challenge previous claims. It presents a protocol to examine the mechanism of how loners use barrage videos by combining eye tracking and self-report measures. Eye tracking documents the audience&#39;s conscious and subconscious watching behaviors in real time and allows for inference of the amount of allocated cognitive resources in response to rational and emotional content. The self-report gauges the amount of satisfaction obtained. Overall, results from the measures supported an activation and match satisfaction model regarding loners and their barrage video viewing behaviors. Implications are discussed.

The study proposes an activation-match model to study how loneliness is mitigated when a lonely audience watches barrage videos of rational and emotional appeals. The protocol uses eye tracking to document duration and fixation, accounting for the degree of satisfaction when emotional needs are appeased by content and barrage.

Researchers usually theorize media exposure based on assumptions of legacy media. However, a new interactive video viewing format, in this case barrage ...

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 ...