Name: polygraphic recording procedure for measuring sleep in mice
Uploaded: 2016-01-25T19:00:00
Description: Watch this Scientific Journal Video about Polygraphic Recording Procedure for Measuring Sleep in Mice at JoVE.com

We thank Dr. Larry D. Frye for editorial help with this manuscript. This work was supported by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research 24300129 (to M.L.), 25890005 (to Y.O.) and 26640025 (to Y.T.), the National Agriculture and Food Research Organization (to Y.U.), the World Premier International Research Center Initiative (WPI) from the Ministry of Education, Culture, Sports, Science, and Technology (to Y.O., Y.T., Y.U. and M.L.) and the Nestl&#233; Nutrition Council, Japan (to M.L.).

Ethics Statement: Procedures involving animal subjects have been approved by the Institutional Animal Experiment Committee at the University of Tsukuba.
1. Preparation of Electrodes and Cables for EEG/EMG Recordings
<ol>
	<li>Prepare EEG/EMG recording electrode according to the following procedure. 
	Note: The electrode is disposable and can be used only for 1 animal. Plan carefully the wiring configuration for all connectors. Place marks on the connectors for the correct orientation.
	...

<ol>
	<li>Berger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#220;ber+das+Elektrenkephalogramm+des+Menschen.">&#220;ber das Elektrenkephalogramm des Menschen.</a> Arch. Psych. 87 (1), 527-570 (1929).</li><li>Tobler, I., Deboer, T., Fischer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+and+sleep+regulation+in+normal+and+prion+protein-deficient+mice.">Sleep and sleep regulation in normal and prion protein-deficient mice.</a> J. Neurosci. 17 (5), 1869-1879 (1997).</li><li>Kohtoh, S., 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=Algorithm+for+sleep+scoring+in+experimental+animals+based+on+fast+Fourier+transform+power+spectrum+analysis+of+the+electroencephalogram.">Algorithm for sleep scoring in experimental animals based on fast Fourier transform power spectrum analysis of the electroencephalogram.</a> Sleep Biol. Rhythm. 6 (3), 163-171 (2008).</li><li>Rosenbaum, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Warum m&#252;ssen wir schlafen? : eine neue Theorie des Schlafes. , (1892).</li><li>Kubota, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kuniomi+Ishimori+and+the+first+discovery+of+sleep-inducing+substances+in+the+brain.">Kuniomi Ishimori and the first discovery of sleep-inducing substances in the brain.</a> Neurosci. Res. 6 (6), 497-518 (1989).</li><li>Ishimori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=True+cause+of+sleep:+a+hypnogenic+substance+as+evidenced+in+the+brain+of+sleep-deprived+animals.">True cause of sleep: a hypnogenic substance as evidenced in the brain of sleep-deprived animals.</a> Tokyo Igakkai Zasshi. 23, 429-457 (1909).</li><li>Legendre, R., Pieron, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recherches+sur+le+besoin+de+sommeil+cons&#233;cutif+&#224;+une+veille+prolong&#233;e.">Recherches sur le besoin de sommeil cons&#233;cutif &#224; une veille prolong&#233;e.</a> Z. Allegem. Physiol. 14, 235-262 (1913).</li><li>Inou&#233;, S., Honda, K., Komoda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+as+neuronal+detoxification+and+restitution.">Sleep as neuronal detoxification and restitution.</a> Behav. Brain. Res. 69 (1-2), 91-96 (1995).</li><li>Urade, Y., Hayaishi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostaglandin+D2+and+sleep/wake+regulation.">Prostaglandin D2 and sleep/wake regulation.</a> Sleep Med. Rev. 15 (6), 411-418 (2011).</li><li>Ueno, R., Ishikawa, Y., Nakayama, T., Hayaishi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostaglandin+D2+induces+sleep+when+microinjected+into+the+preoptic+area+of+conscious+rats.">Prostaglandin D2 induces sleep when microinjected into the preoptic area of conscious rats.</a> Biochem. Biophys. Res. Commun. 109 (2), 576-582 (1982).</li><li>Krueger, J. M., Walter, J., Dinarello, C. A., Wolff, S. M., Chedid, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep-promoting+effects+of+endogenous+pyrogen+(interleukin-1).">Sleep-promoting effects of endogenous pyrogen (interleukin-1).</a> Am. J. Physiol. 246 (6 Pt 2), R994-R999 (1984).</li><li>Porkka-Heiskanen, T., 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=Adenosine:+a+mediator+of+the+sleep-inducing+effects+of+prolonged+wakefulness.">Adenosine: a mediator of the sleep-inducing effects of prolonged wakefulness.</a> Science. 276 (5316), 1265-1268 (1997).</li><li>Garcia-Garcia, F., Acosta-Pena, E., Venebra-Munoz, A., Murillo-Rodriguez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep-inducing+factors.">Sleep-inducing factors.</a> CNS Neurol. Disord. Drug. Targets. 8 (4), 235-244 (2009).</li><li>Huitron-Resendiz, S., 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=Urotensin+II+modulates+rapid+eye+movement+sleep+through+activation+of+brainstem+cholinergic+neurons.">Urotensin II modulates rapid eye movement sleep through activation of brainstem cholinergic neurons.</a> J. Neurosci. 25 (23), 5465-5474 (2005).</li><li>Wilkins, R. H., Brody, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encephalitis+lethargica.">Encephalitis lethargica.</a> Arch. Neurol. 18 (3), 324-328 (1968).</li><li>von Economo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Die+encephalitis+lethargica.">Die encephalitis lethargica.</a> Wien. Klin. Wochenschr. 30, 581-585 (1917).</li><li>Moruzzi, G., Magoun, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+stem+reticular+formation+and+activation+of+the+EEG.">Brain stem reticular formation and activation of the EEG.</a> Electroencephalogr. Clin. Neurophysiol. 1 (4), 455-473 (1949).</li><li>Saper, C. B., Fuller, P. M., Pedersen, N. P., Lu, J., Scammell, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleep+state+switching.">Sleep state switching.</a> Neuron. 68 (6), 1023-1042 (2010).</li><li>Jones, B. E., Krnjevic , K., L, D. e. s. c. a. r. r. i. e. s., S, M. i. r. c. e. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Progress in Brain Research. 145, 157-169 (2004).</li><li>Saper, C. B., Scammell, T. E., Lu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+regulation+of+sleep+and+circadian+rhythms.">Hypothalamic regulation of sleep and circadian rhythms.</a> Nature. 437 (7063), 1257-1263 (2005).</li><li>Fort, P., Bassetti, C. L., Luppi, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternating+vigilance+states:+new+insights+regarding+neuronal+networks+and+mechanisms.">Alternating vigilance states: new insights regarding neuronal networks and mechanisms.</a> Eur. J. Neurosci. 29 (9), 1741-1753 (2009).</li><li>Lu, J., Sherman, D., Devor, M., Saper, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+putative+flip-flop+switch+for+control+of+REM+sleep.">A putative flip-flop switch for control of REM sleep.</a> Nature. 441 (7093), 589-594 (2006).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The mouse brain in stereotaxic coordinates. , (2001).</li><li>Lazarus, M., 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=Arousal+effect+of+caffeine+depends+on+adenosine+A2A+receptors+in+the+shell+of+the+nucleus+accumbens.">Arousal effect of caffeine depends on adenosine A2A receptors in the shell of the nucleus accumbens.</a> J. Neurosci. 31 (27), 10067-10075 (2011).</li><li>Huang, Z. L., 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=Adenosine+A2A,+but+not+A1,+receptors+mediate+the+arousal+effect+of+caffeine.">Adenosine A2A, but not A1, receptors mediate the arousal effect of caffeine.</a> Nat. Neurosci. 8 (7), 858-859 (2005).</li><li>Qu, W. M., Huang, Z. L., Xu, X. H., Matsumoto, N., Urade, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+D1+and+D2+receptors+are+essential+for+the+arousal+effect+of+modafinil.">Dopaminergic D1 and D2 receptors are essential for the arousal effect of modafinil.</a> J. Neurosci. 28 (34), 8462-8469 (2008).</li><li>Huang, Z. L., 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=Arousal+effect+of+orexin+A+depends+on+activation+of+the+histaminergic+system.">Arousal effect of orexin A depends on activation of the histaminergic system.</a> Proc. Natl. Acad. Sci. USA. 98 (17), 9965-9970 (2001).</li><li>Xu, Q., 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=A+mouse+model+mimicking+human+first+night+effect+for+the+evaluation+of+hypnotics.">A mouse model mimicking human first night effect for the evaluation of hypnotics.</a> Pharmacol. Biochem. Behav. 116, 129-136 (2014).</li><li>Cho, S., 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=Marine+polyphenol+phlorotannins+promote+non-rapid+eye+movement+sleep+in+mice+via+the+benzodiazepine+site+of+the+GABAA+receptor.">Marine polyphenol phlorotannins promote non-rapid eye movement sleep in mice via the benzodiazepine site of the GABAA receptor.</a> Psychopharmacol. 231 (14), 2825-2837 (2014).</li><li>Liu, Y. Y., 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=Piromelatine+exerts+antinociceptive+effect+via+melatonin,+opioid,+and+5HT1A+receptors+and+hypnotic+effect+via+melatonin+receptors+in+a+mouse+model+of+neuropathic+pain.">Piromelatine exerts antinociceptive effect via melatonin, opioid, and 5HT1A receptors and hypnotic effect via melatonin receptors in a mouse model of neuropathic pain.</a> Psychopharmacol. 231 (20), 3973-3985 (2014).</li><li>Qu, W. M., 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=Lipocalin-type+prostaglandin+D+synthase+produces+prostaglandin+D2+involved+in+regulation+of+physiological+sleep.">Lipocalin-type prostaglandin D synthase produces prostaglandin D2 involved in regulation of physiological sleep.</a> Proc. Natl. Acad. Sci. USA. 103 (47), 17949-17954 (2006).</li></ol>

This protocol describes a set-up for EEG/EMG recordings that allows the assessment of sleep and wakefulness under low-noise, cost-effective, and high-throughput conditions. Due to the small size of the EEG/EMG electrode head assembly, this system can be combined with other implants for intra-brain experiments, including optogenetics (optical fiber implantation) or, in conjunction with simultaneous cannula implantation, microinfusion of drugs into the mouse brain.31 Moreover, the design of the electrode head as...

Technical advances have often precipitated quantum leaps in the understanding of neurobiological processes. For example, Hans Berger&#39;s discovery in 1929 that electrical potentials recorded from the human scalp took the form of sinusoidal waves, the frequency of which was directly related to the level of wakefulness of the subject, led to rapid advances in the understanding of sleep-wake regulation, in both animals and humans alike.1 To this day the electroencephlogram (EEG), in conjunction with the electromyogram (EMG), i.e., electrical activity produced by skeletal muscles, represents the data &#34;backbone&#34; of nearly every experimental and clinical assessment that seeks to correlate behavior and physiology with the activity of cortical neurons in behaving animals, including humans. In most basic sleep research laboratories these EEG recordings are performed by using a cable-based system (Figure 1) wherein acquired data is subjected off-line to pattern and spectrum analysis [e.g., applying a fast Fourier transform (FFT) algorithm] to determine the vigilance state of the subject being recorded.2, 3 Sleep consists of rapid-eye movement (REM) and non-REM (NREM) sleep. REM sleep is characterized by a rapid low-voltage EEG, random eye movement, and muscle atonia, a state in which the muscles are effectively paralyzed. REM sleep is also known as paradoxical sleep, because the brain activity resembles that of wakefulness, whereas the body is largely disconnected from the brain and appears to be in deep sleep. By contrast, motor neurons are stimulated during NREM sleep but there is no eye movement. Human NREM sleep can be divided into 4 stages, whereby stage 4 is called deep sleep or slow-wave sleep and is identified by large, slow brain waves with delta activity between 0.5 - 4 Hz in the EEG. On the other hand, a subdivision between phases of NREM sleep in smaller animals, like rats and mice, has not been established, mostly because they do not have long consolidated periods of sleep as seen in humans.
Over the years, and on the basis of EEG interpretation, several models of sleep-wake regulation, both circuit- and humoral-based, have been proposed. The neural and cellular basis of the need for sleep or, alternatively, &#34;sleep drive,&#34; remains unresolved, but has been conceptualized as a homeostatic pressure that builds during the waking period and is dissipated by sleep. One theory is that endogenous somnogenic factors accumulate during wakefulness and that their gradual accumulation is the underpinning of sleep homeostatic pressure. While the first formal hypothesis that sleep is regulated by humoral factors has been credited to Rosenbaum&#39;s work published in 18924, it was Ishimori5, 6 and Pieron7 who independently, and over 100 years ago, demonstrated the existence of sleep-promoting chemicals. Both researchers proposed, and indeed proved, that hypnogenic substances or &#39;hypnotoxins&#39; were present in the cerebral spinal fluid (CSF) of sleep-deprived dogs.8 Over the past century several additional putative hypnogenic substances implicated in the sleep homeostatic process have been identified (for review, see ref. 9), including prostaglandin (PG) D2,10 cytokines,11 adenosine,12 anandamide,13 and the urotensin II peptide.14
Experimental work by Economo15, 16, Moruzzi and Magoun17, and others in the early and mid-20th century produced findings that inspired circuit-based theories of sleep and wakefulness and, to a certain degree, overshadowed the then prevailing humoral theory of sleep. To date, several &#34;circuit models&#34; have been proposed, each informed by data of varying quality and quantity (for review, see ref. 18). One model, for example, proposes that slow-wave sleep is generated through adenosine-mediated inhibition of acetylcholine release from cholinergic neurons in the basal forebrain, an area mainly consisiting of the nucleus of the horizontal limb of the diagonal band of Broca and the substantia inominata.19 Another popular model of sleep/wake regulation describes a flip-flop switch mechanism on the basis of mutually inhibitory interactions between sleep-inducing neurons in the ventrolateral preoptic area and wake-inducing neurons in the hypothalamus and brain stem.18, 20, 21 Moreover, for the switching in and out of REM sleep, a similar reciprocally inhibitory interaction has been proposed for areas in the brain stem, that is the ventral periaqueductal gray, lateral pontine tegmentum, and sublaterodorsal nucleus.22 Collectively, these models have proven valuable heuristics and afforded important interpretative frameworks for studies in sleep research; however, a yet fuller understanding of the molecular mechanisms and circuits regulating the sleep-wake cycle will require a more complete knowledge of its components. The system for polygraphic recording detailed below should aid in this goal.

<table border="0" cellpadding="0" cellspacing="0" width="729">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">4-pin header</td>
			<td style="width:143px;">Hirose</td>
			<td style="width:167px;">A3B-4PA-2DSA(71)</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Ampicillin</td>
			<td style="width:143px;">Meiji Seika</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Analog-to-digital converter</td>
			<td style="width:143px;">Contec</td>
			<td style="width:167px;">AD16-16U(PCIEV)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Caffeine</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:167px;">C0750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Carbide cutter</td>
			<td style="width:143px;">Minitor</td>
			<td style="width:167px;">B1055</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Crimp housing</td>
			<td style="width:143px;">Hirose</td>
			<td style="width:167px;">DF11-4DS-2C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Crimp socket</td>
			<td style="width:143px;">Hirose</td>
			<td style="width:167px;">DF11-30SC</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dental cement (Toughron Rebase)</td>
			<td style="width:143px;">Miki Chemical Product</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Epoxy adhesive</td>
			<td style="width:143px;">Konishi</td>
			<td style="width:167px;">#16351</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">FFC/FPC connector</td>
			<td style="width:143px;">Honda Tsushin Kogyo</td>
			<td style="width:167px;">FFC-10BMEP1(B)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Flat cable</td>
			<td style="width:143px;">Hitachi Cable</td>
			<td style="width:167px;">20528-ST LF</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Instant glue (Aron Alpha A)</td>
			<td style="width:143px;">Toagosei</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Meloxicam</td>
			<td style="width:143px;">Boehringer Ingelheim</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pentobarbital</td>
			<td style="width:143px;">Kyoritsu Seiyaku</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Signal amplifier</td>
			<td style="width:143px;">Biotex</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sleep recording chamber</td>
			<td style="width:143px;">APL</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SleepSign software</td>
			<td style="width:143px;">Kissei Comtec</td>
			<td style="width:167px;">N/A</td>
			<td>for EEG/EMG recording/analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Slip ring</td>
			<td style="width:143px;">Biotex</td>
			<td style="width:167px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stainless steel screw</td>
			<td style="width:143px;">Yamazaki</td>
			<td style="width:167px;">N/A</td>
			<td>&#966;1.0&#215;2.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stainless steel wire</td>
			<td style="width:143px;">Cooner Wire</td>
			<td style="width:167px;">AS633</td>
			<td></td>
		</tr>
	</tbody>
</table>

polygraphic recording procedure for measuring sleep in mice

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the homeodynamics and circuitry of sleep-wake regulation. In many laboratories, a cable-based sleep recording system is used to monitor the EEG and EMG in freely behaving mice in combination with computer software for automatic scoring of the vigilance states on the basis of power spectrum analysis of EEG data. A description of this system is detailed herein. Steel screws are implanted over the frontal cortical area and the parietal area of 1 hemisphere for monitoring EEG signals. In addition, EMG activity is monitored by the bilateral placement of wires in both neck muscles. Non-rapid eye movement (Non-REM; NREM) sleep is characterized by large, slow brain waves with delta activity below 4 Hz in the EEG, whereas a shift from low-frequency delta activity to a rapid low-voltage EEG in the theta range between 6 and 10 Hz can be observed at the transition from NREM to REM sleep. By contrast, wakefulness is identified by low- to moderate-voltage brain waves in the EEG trace and significant EMG activity.

Figure 1B illustrates examples of the mouse EEG in the different vigilance states. As shown in Table 1, epochs are classified as NREM sleep if the EEG shows large, slow brain waves with a delta rhythm below 4 Hz and the EMG has only a weak or no signal. Epochs are classified as REM sleep if the EEG shows rapid low-voltage brain waves in the theta range between 6 and 10 Hz and the EMG shows low amplitude. Other epochs should be classified as wakefulness (<...

Watch this Scientific Journal Video about Polygraphic Recording Procedure for Measuring Sleep in Mice at JoVE.com

Polygraphic Recording Procedure for Measuring Sleep in Mice

The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the ...

The overall goal of this polygraphic recording procedure is to determine the vigilant state of mice and measure the time spent in wakefulness, rapid eye movement sleep or non-rapid eye movement sleep. This method can help answer key questions in nearly every neuroscience field that seeks to correlate behavior and physiology with the activity of cortical neurons in behaving animals. The main advantage of this technique is that it can record a lot of nodes, electroencephalogram and electromyogram and can be combined with other brain implants such as optic fibers and infusion cannulas.Visual demonstration of this method is very important because the implantation of the electrode needs to be performed properly and the quality of the electrode is extremely critical to judge the physical states. Demonstrating the procedure will be Yohko Takata, a postdoc from my laboratory. Before starting, sterilize all the surgical tools in a hot bead sterilizer.Once the mouse is confirmed as anesthetized by a toe pinch, shave off the hair on the head and neck. Next, secure the house to the stereotaxic frame and fix the head between the two ear bars. Then, apply ophthalmic ointment to the eyes to prevent dryness.Proceed with cleansing the shaved skin with alcohol and iodine based scrubs. Then, cut along the midline with a scalpel to expose the skull and secure the skin to the side using clips to keep the surgical area open. Using a carbide cutter with a 0.8 millimeter drill, make two holes.Put one hole over the frontal cortical area. Put the second hole over the parietal area of the right hemisphere. Next, using a jeweler's screwdriver, screw stainless steel EEG recording screws into the holes.Insert the screws with just two to two and a half turns to obtain epidural positioning over the cortex. The screws should not wiggle once inserted. This is very important.Now, fix the electrode assembly with the pins upward to the skull. The electrodes must be attached straight on. Use a cotton swab to help secure the electrode's position and glue the electrode down using cyanoacrylate followed by dental cement.Next, make small holes in the trapezius and insert the EMG electrode into those holes. Then, suture the skin with 0.1 millimeter diameter silk thread. Now, remove the mouse from the stereotaxic frame and place it on a heat pad.Give the mouse intraperitoneal injections of ampicillin and meloxicam and monitor the mouse until it regains sternal recumbency. Then, house the mouse alone. One week after implanting the electrodes, transfer the mouse to an experimental cage in a soundproof recording chamber.Secure the EEG/EMG electrode assembly on the mouse to a recording cable connected to a slip ring. Connect the AD converter cable to an EEG/EMG signal filter amplifier connected to the slip ring. Use an AD converter to convert the signal to a 128 hertz digital signal and, finally, record the signal to a computer.Habituate the mouse for two to three days in the recording chamber. If the EEG/EMG recording includes drug administrations, gently handle the mouse on each day as those there was a drug administration being given. Subsequently, start the EEG/EMG recording software.To begin, click on the Data file information tab and click the box next to the File name. Enter a file name and click Save. Then, from the Recording condition tab, select all the EEG/EMG channels which need to be recorded.Also, select the Sampling frequency under this tab. Now, in the Channel information tab, check if the selected channels are displayed properly. Once confirmed, start the record by first selecting the Timer setting tab.Then, click monitor to display EEG and EMG. If the signals are displayed correctly, set the clock time for the beginning and end of the recording in the Main Timer area. Then, click the Monitor button to start the recording.Now, record the signals under baseline conditions and different treatment conditions over several days. When the experiment is finished, euthanize the mouse with pentobarbital. Start the software for EEG/EMG analysis.Open the EEG/EMG raw data, which is a KCD file. Click the Sleep tab and set the Epoch time to 10 seconds. Then, select Multi-screening to automatically score all ten second epochs into three stages on the basis of the amplitudes of the EEG and the EMG and based on the power spectral analysis of the EEG.Next, click on the FFT condition for EEG tab. There, set the parameters for the power spectral analysis. Use 256 datum points corresponding to every two seconds of EEG, use the hamming window function, and use an average of five spectra per epoch.Then click Ok.Now, click Start Screening to begin the automatic screening. Then, open the stored data, which is saved in a RAF file format and check the results of the automatic screening. As needed, correct the results if they do not meet the standard criteria.To make a correction, click and hold the left mouse button on an incorrectly scored epoch and drag the cursor across the string of incorrectly scored epochs. Release the left mouse button and select the correct behavioral state in the popup window. Under baseline conditions, the mice exhibited a clear circadian sleep-wake rhythm consistent with their nocturnal nature.During the 12 hour light period, the mice averaged 6.7 hours of NREM sleep and 0.9 hours of REM sleep. Whereas, during their 12 hour dark period, wakefulness was predominant. Other measurements were also recorded.These included the sleep epoch distribution, their mean durations and the stage transitions for each vigilant state. The EEG power spectrums for NREM and REM sleep show a strong EEG power density in the frequency range of 0.5 to four hertz for NREM sleep compared to a power density from six to 10 hertz for REM sleep. To assess the impact of caffeine on sleep, C57 black 6 mice were treated with a vehicle on one day and with caffeine on the next day.Intraperitoneal injections of caffeine were given early in the light cycle. As expected, the recording revealed the caffeine increased the amount of wakefulness in the mice. A three-fold increase in wakefulness occurred for three hours after the injection.Once mastered, the electrode implantation procedure can be done in less than 20 minutes. Scoring and correcting the EEG data for a 24 hour period can be done in about 30 minutes. After watching this video, you should have a good understanding of how to set up recording of electroencephalogram and electromyogram in mice and how to assess if and how long an animal is awake or asleep.

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Measuring Motor Coordination in Mice

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such as emotionality or cognition can be assessed, however, it is vital to determine whether the motor capabilities of e.g. a mutant or lesioned mouse allow such an assessment. Performance on a maze task requiring strength and coordination, such as the Morris water maze, might well be impaired in a mouse by motor, rather than cognitive, impairments, so it is essential to selectively dissect the latter from the former. For example, sensorimotor impairments caused by NMDA antagonists have been shown to impair water maze performance2. 
Motor coordination has traditionally been assessed in mice and rats by the rotarod test, in which the animal is placed on a horizontal rod that rotates about its long axis; the animal must walk forwards to remain upright and not fall off. Both set speed and accelerating versions of the rotarod are available.
The other three tests described in this article (horizontal bar, static rods and parallel bars) all measure coordination on static apparatus. The horizontal bar also requires strength for adequate performance, particularly of the forelimbs as the mouse initially grips the bar just with the front paws. 
Adult rats do not perform well on tests such as the static rods and parallel bars (personal observations); they appear less well coordinated than mice. I have only tested male rats, however, and male mice seem generally less well coordinated than females. Mice appear to have a higher strength:weight ratio than rats; the Latin name, Mus musculus, seems entirely appropriate. The rotarod, the variations of the foot fault test12 or the Catwalk (Noldus)15 apparatus are generally used to assess motor coordination in rats.

Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

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

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

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

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

Evaluation of Hemisphere Lateralization with Bilateral Local Field Potential Recording in Secondary Motor Cortex of Mice

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical areas of mice, which are useful for evaluating possible laterality deficits, as well as for assessing brain connectivity and coupling of neural network activities in rodents. The pathological mechanisms underlying Alzheimer&#39;s disease (AD), a common neurodegenerative disease, remain largely unknown. Altered brain laterality has been demonstrated in aging people, but whether or not abnormal lateralization is one of the early signs of AD has not been determined. To investigate this, we recorded bilateral LFPs in 3-5-month-old AD model mice, APP/PS1, together with littermate wild type (WT) controls. The LFPs of the left and right secondary motor cortex (M2), specifically in the gamma band, were more synchronized in APP/PS1 mice than in WT controls, suggesting a declined hemispheric asymmetry of bilateral M2 in this AD mouse model. Notably, the recording and data analysis processes are flexible and easy to carry out, and can also be applied to other brain pathways when conducting experiments that focus on neuronal circuits.

We present in vivo electrophysiological recording of the local field potential (LFP) in bilateral secondary motor cortex (M2) of mice, which can be applied to evaluate hemisphere lateralization. The study revealed altered levels of synchronization between the left and right M2 in APP/PS1 mice compared to WT controls.

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical ...

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.

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 ...

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

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

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

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

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...