This study was supported by the Merck Investigator Studies Program (#54843), a KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas, &#34;WillDynamics&#34; (16H06401) (T.S.), and a KAKENHI Grant-in-Aid for Exploratory Research on Innovative Areas (T.S.) (18H02595).

All experiments here were approved by the Animal Experiment and Use Committee of the University of Tsukuba, complying with NIH guidelines.
1. Animal Surgery, Virus Injection, Electrode for EEG/EMG, and Optical Fiber Implantation
CAUTION: Appropriate protection and handling techniques should be selected based on the biosafety level of the virus to be used. AAV should be used in an isolated P1A graded room for injection, and the tube carrying AA...

<ol>
	<li>Spoormaker, V. I., Montgomery, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disturbed+sleep+in+post-traumatic+stress+disorder:+Secondary+symptom+or+core+feature?.">Disturbed sleep in post-traumatic stress disorder: Secondary symptom or core feature?.</a> Sleep Medicine Reviews. 12 (3), 169-184 (2008).</li><li>Dworak, M., Wiater, A., Alfer, D., Stephan, E., Hollmann, W., Struder, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+slow+wave+sleep+and+reduced+stage+2+sleep+in+children+depending+on+exercise+intensity.">Increased slow wave sleep and reduced stage 2 sleep in children depending on exercise intensity.</a> Sleep Medicine. 9 (3), 266-272 (2008).</li><li>Mellman, T. A. <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+anxiety+disorders.">Sleep and anxiety disorders.</a> Psychiatric Clinics of North America. 29 (4), 1047-1058 (2006).</li><li>Scammell, T. E., Arrigoni, E., Lipton, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+circuitry+of+wakefulness+and+sleep.">Neural circuitry of wakefulness and sleep.</a> Neuron. 93 (4), 747-765 (2017).</li><li>Chemelli, R. 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=Narcolepsy+in+orexin+knockout+mice:+Molecular+genetics+of+sleep+regulation.">Narcolepsy in orexin knockout mice: Molecular genetics of sleep regulation.</a> Cell. 98 (4), 437-451 (1999).</li><li>Borb&#233;ly, A. A., Daan, S., Wirz-Justice, A., Deboer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+two-process+model+of+sleep+regulation:+A+reappraisal.">The two-process model of sleep regulation: A reappraisal.</a> Journal of Sleep Research. 25 (2), 131-143 (2016).</li><li>Daan, S., Beersma, D. G., Borbely, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+of+human+sleep:+recovery+process+gated+by+a+circadian+pacemaker.">Timing of human sleep: recovery process gated by a circadian pacemaker.</a> American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 246 (2), R161-R183 (1984).</li><li>Saper, C. B., Cano, G., 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=Homeostatic,+circadian,+and+emotional+regulation+of+sleep.">Homeostatic, circadian, and emotional regulation of sleep.</a> Journal of Comparative Neurology. 493 (1), 92-98 (2005).</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>LeDoux, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emotion+circuits+in+the+brain.">Emotion circuits in the brain.</a> Annual Review of Neuroscience. 23, 155-184 (2000).</li><li>Tovote, P., Fadok, J. P., L&#252;thi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+circuits+for+fear+and+anxiety.">Neuronal circuits for fear and anxiety.</a> Nature Reviews Neuroscience. 16 (6), 317-331 (2015).</li><li>Lebow, M. A., Chen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overshadowed+by+the+amygdala:+the+bed+nucleus+of+the+stria+terminalis+emerges+as+key+to+psychiatric+disorders.">Overshadowed by the amygdala: the bed nucleus of the stria terminalis emerges as key to psychiatric disorders.</a> Molecular Psychiatry. 21 (4), 450-463 (2016).</li><li>Wu, 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=Tangential+migration+and+proliferation+of+intermediate+progenitors+of+GABAergic+neurons+in+the+mouse+telencephalon.">Tangential migration and proliferation of intermediate progenitors of GABAergic neurons in the mouse telencephalon.</a> Development. 138 (12), 2499-2509 (2011).</li><li>Tye, K. M., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+investigation+of+neural+circuits+underlying+brain+disease+in+animal+models.">Optogenetic investigation of neural circuits underlying brain disease in animal models.</a> Nature Reviews Neuroscience. 13 (4), 251-266 (2012).</li><li>de Lecea, L., Carter, M. E., Adamantidis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shining+light+on+wakefulness+and+arousal.">Shining light on wakefulness and arousal.</a> Biological Psychiatry. 71 (12), 1046-1052 (2012).</li><li>Carter, M. E., Brill, J., Bonnavion, P., Huguenard, J. R., Huerta, R., de Lecea, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+for+hypocretin-mediated+sleep-to-wake+transitions.">Mechanism for hypocretin-mediated sleep-to-wake transitions.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (39), E2635-E2644 (2012).</li><li>Weber, F., Dan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circuit-based+interrogation+of+sleep+control.">Circuit-based interrogation of sleep control.</a> Nature Publishing Group. 538, 51-59 (2016).</li><li>Weber, F., Chung, S., Beier, K. T., Xu, M., Luo, L., Dan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+REM+sleep+by+ventral+medulla+GABAergic+neurons.">Control of REM sleep by ventral medulla GABAergic neurons.</a> Nature. 526, 435-438 (2015).</li><li>Oishi, 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=Slow-wave+sleep+is+controlled+by+a+subset+of+nucleus+accumbens+core+neurons+in+mice.">Slow-wave sleep is controlled by a subset of nucleus accumbens core neurons in mice.</a> Nature Communications. 8 (1), 1-12 (2017).</li><li>Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond-timescale,+genetically+targeted+optical+control+of+neural+activity.">Millisecond-timescale, genetically targeted optical control of neural activity.</a> Nature Neuroscience. 8 (9), 1263-1268 (2005).</li><li>Han, X., 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+high-light+sensitivity+optical+neural+silencer:+development+and+application+to+optogenetic+control+of+non-human+primate+cortex.">A high-light sensitivity optical neural silencer: development and application to optogenetic control of non-human primate cortex.</a> Frontiers in Systems Neuroscience. 5, 1-8 (2011).</li><li>Kim, C. K., Adhikari, A., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+optogenetics+with+complementary+methodologies+in+systems+neuroscience.">Integration of optogenetics with complementary methodologies in systems neuroscience.</a> Nature Reviews Neuroscience. 18 (4), 222-235 (2017).</li><li>Saito, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+neurons+in+the+preoptic+area+send+direct+inhibitory+projections+to+orexin+neurons.">GABAergic neurons in the preoptic area send direct inhibitory projections to orexin neurons.</a> Frontiers in Neural Circuits. 7 (December), 1-3 (2013).</li><li>Atasoy, D., Aponte, Y., Su, H. H., Sternson, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+FLEX+switch+targets+Channelrhodopsin-2+to+multiple+cell+types+for+imaging+and+long-range+circuit+mapping.">A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit mapping.</a> Journal of Neuroscience. 28 (28), 7025-7030 (2008).</li><li>Kodani, S., Soya, S., Sakurai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitation+of+GABAergic+neurons+in+the+bed+nucleus+of+the+stria+terminalis+triggers+immediate+transition+from+non-rapid+eye+movement+sleep+to+wakefulness+in+mice.">Excitation of GABAergic neurons in the bed nucleus of the stria terminalis triggers immediate transition from non-rapid eye movement sleep to wakefulness in mice.</a> Journal of Neuroscience. 37, 7174-7176 (2017).</li><li>Lin, F., Pichard, 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> Handbook of practical immunohistochemistry: frequently asked questions. , (2011).</li><li>Wiegert, J. S., Mahn, M., Prigge, M., Printz, Y., Yizhar, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+neurons:+tools,+applications,+and+experimental+constraints.">Silencing neurons: tools, applications, and experimental constraints.</a> Neuron. 95 (3), 504-529 (2017).</li><li>Yizhar, O., Fenno, L. E., Prigge, M., Schneider, F., Davidson, T. J., O&#8217;Shea, D. J., Sohal, V. S., Goshen, I., Finkelstein, J., Paz, J. T., Stehfest, K., Fudim, R., Ramakrishnan, C., Huguenard, J. R., Hegemann, P., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortical+excitation/inhibition+balance+in+information+processing+and+social+dysfunction.">Neocortical excitation/inhibition balance in information processing and social dysfunction.</a> Nature. 40 (6), 1301-1315 (2012).</li></ol>

We here presented a method to evaluate the effect of optogenetic stimulation of neurons with particular chemical identities on state transitions of sleep/wakefulness and gave an example of manipulation of GABABNST neurons. Our data showed that optogenetic excitation of GABABNST neurons results in immediate transition from NREM sleep to wakefulness.
Various experimental designs are available because of the development of numerous types of optogenetic tools. It is possible ...

Sleep is essential for optimal cognitive function.Recent findings also suggest that disturbances in sleep are associated with a wide range of diseases1,2,3. Although the functions of sleep are as yet largely unresolved, substantial progress has been made recently in understanding the neural circuits and mechanisms that control sleep/wakefulness states4. In mammals, there are three states of vigilance: wakefulness, non-rapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep. Wakefulness is characterized by fast EEG oscillations (5-12 Hz) of low amplitude with purposeful and sustained motor activity. NREM sleep is defined by slow oscillations (1-4 Hz) of high amplitude (delta waves), with lack of consciousness and purposeful motor activity. REM sleep is characterized by relatively fast oscillations (6-12 Hz) of low amplitude and almost complete bilateral muscle atonia5.
Borbely proposed a theory of sleep-wakefulness regulation known as the two process model6,7. A homeostatic process, also referred to as process S, represents sleep pressure that accumulates during wakefulness and dissipates during sleep. Another process, referred to as process C, is a circadian process, which explains why vigilance levels fluctuate in the 24 h cycle. In addition to these two processes, allostatic factors are also important for regulation of sleep/wakefulness8,9. Allostatic factors include nutritional states and emotion. Fear and anxiety are usually accompanied by an increase in arousal along with autonomic and neuroendocrine responses10,11,12. The limbic system is believed to play a role in regulation of fear and anxiety, and mechanisms underlying autonomic and neuroendocrine responses have been studied extensively, but the pathway by which the limbic system influences sleep/wakefulness states has not yet been revealed. A large number of recent studies using opto- and pharmacogenetics have suggested that neurons and neuronal circuits that regulate sleep/wakefulness states are distributed throughout the brain, including the cortices, basal forebrain, thalamus, hypothalamus, and brain stem. In particular, recent advances in optogenetics have allowed us to stimulate or inhibit specific neural circuits in vivo with high spatial and temporal resolutions. This technique will allow progress in our understanding of the neural substrates of sleep and wakefulness, and how sleep/wakefulness states are regulated by circadian processes, sleep pressure, and allostatic factors, including emotion. This paper aims to introduce how to use optogenetic manipulation combined with sleep/wake recording, which could have the potential to update our understanding of the connectomes and mechanisms in the brain that play a role in the regulation of NREM sleep, REM sleep, and wakefulness. Understanding of this mechanism by which the limbic system regulates sleep/wakefulness states is of paramount importance to health, because insomnia is usually associated with anxiety or fear of being unable to sleep (somniphobia).
The BNST is thought to play an essential role in anxiety and fear. GAD 67-expressing GABAergic neurons are a major population of the BNST12,13. We examined the effect of optogenetic manipulation of these neurons (GABABNST) on sleep/wakefulness states. One of the greatest advances in neuroscience in recent years has been methods that enable manipulation of neurons with particular chemical identities in vivo, with high spatial and temporal resolutions. Optogenetics is highly useful for demonstrating causal links between neural activity and specific behavioral responses14. We describe optogenetics as a method to examine the functional connectivity of defined neural circuits in the regulation of sleep/wakefulness states. By utilizing this technique, great progress has been achieved in understanding the neuronal circuits that regulate sleep/wakefulness states15,16,17,18,19. In many cases, opsins are specifically introduced in&#160;neurons with particular chemical identities in selective brain regions by a combination of Cre-driver mice and Cre-inducible AAV-mediated gene transfer.&#160;Further, focal expression of photo-sensitive opsins such as channelrhodopsin 2 (ChR2)20 or archaerhodopsin (ArchT)21 combined with a Cre-loxP or Flp-FRT system allows us to manipulate a selective neuronal population and specific neural pathway22.
We describe here experiments on GABAergic neurons in the BNST as an example. To express opsins in a designated neuronal population, appropriate Cre driver mice and Cre-dependent virus vectors are most frequently used. Transgenic or knock-in lines in which opsins are expressed in particular neuronal populations are also useful. In the following experiments, we used GAD67-Cre knock-in mice23 in which only GABAergic neurons express Cre recombinase with a C57BL/6J genetic background, and an AAV vector which contains ChR2 (hChR2 H134R) fused with EYFP or EYFP as a control with a &#34;FLEx (Flip-excision) switch&#34;24. The procedure specifically describes optogenetic excitation of GABAergic neurons in the BNST during monitoring of sleep/wakefulness states25.

<table border="0" cellpadding="0" cellspacing="0" style="width:1180px;" width="1180">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">1x1 Fiber-optic Rotary Joints</td>
			<td style="width:191px;">Doric</td>
			<td style="width:177px;">FRJ 1x1 FC-FC</td>
			<td style="width:437px;">for optogenetics</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">6-pin header</td>
			<td style="width:191px;">KEL corporation</td>
			<td style="width:177px;">DSP02-006-431G</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">6-pin socket</td>
			<td style="width:191px;">Hirose</td>
			<td style="width:177px;">21602X3GSE</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">A/D converter</td>
			<td style="width:191px;">Nippon koden</td>
			<td style="width:177px;">N/A</td>
			<td>Analog to digital converter</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:375px;">AAV10-EF1a-DIO-ChR2-EYFP</td>
			<td style="width:191px;"></td>
			<td style="width:177px;"></td>
			<td>3.70&#215;1013(genomic copies/ml)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:375px;">AAV10-EF1a-DIO-EYFP</td>
			<td style="width:191px;"></td>
			<td style="width:177px;"></td>
			<td>5.82&#215;1013(genomic copies/ml)</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Ampicillin</td>
			<td style="width:191px;">Fuji film</td>
			<td style="width:177px;">014-23302</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Amplifier</td>
			<td style="width:191px;">Nippon koden</td>
			<td style="width:177px;">N/A</td>
			<td>for EEG/EMG recording</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Anesthetic vaporizer</td>
			<td style="width:191px;">Muromachi</td>
			<td style="width:177px;">MK-AT-210D</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Automatic injecter</td>
			<td style="width:191px;">KD scientific</td>
			<td style="width:177px;">780311</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Carbide cutter</td>
			<td style="width:191px;">Minitor</td>
			<td style="width:177px;">B1055</td>
			<td>&#966;0.7 mm. Reffered as dental drill, used with high speed rotary micromotor&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Cyanoacrylate adhesion&#160; (Aron alpha A) and acceleration</td>
			<td style="width:191px;">Konishi</td>
			<td>#30533</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Dental curing light</td>
			<td style="width:191px;">3M</td>
			<td style="width:177px;">Elipar S10</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Epoxy adhesive</td>
			<td style="width:191px;">Konishi</td>
			<td>#04888</td>
			<td>insulation around the solder of 6-pin and shielded cable</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Fiber optic patch cord (branching)</td>
			<td style="width:191px;">Doric</td>
			<td>BFP(#)_50/125/900-0.22</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Gad67-Cre mice</td>
			<td style="width:191px;">provided by Dr. Kenji Sakimura</td>
			<td></td>
			<td>Cre recombinase gene is knocked-in in the Gad67 allele</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Hamilton syringe</td>
			<td style="width:191px;">Hamilton</td>
			<td>65461-01</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">High speed rotary micromotor kit</td>
			<td style="width:191px;">FOREDOM</td>
			<td style="width:177px;">K.1070</td>
			<td>Used with carbide cutter</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Interconnecting sleeve</td>
			<td style="width:191px;">Thorlab</td>
			<td>ADAF1</td>
			<td>&#966;2.5 mm Ceramic&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Isoflurane</td>
			<td style="width:191px;">Pfizer</td>
			<td>871119</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Laser&#160;&#160;</td>
			<td style="width:191px;">Rapp OptoElectronic</td>
			<td style="width:177px;">N/A</td>
			<td>473nm wave length</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Laser intesity checker</td>
			<td style="width:191px;">COHERENT</td>
			<td style="width:177px;">1098293</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Laser stimulator</td>
			<td style="width:191px;">Bio research center</td>
			<td style="width:177px;">STO2</td>
			<td>reffered as pulse generator in text</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Optic fiber with ferrule&#160;</td>
			<td style="width:191px;">Thorlab</td>
			<td>FP200URT-CANNULA-SP-JP</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">pAAV2-rh10</td>
			<td style="width:191px;">provided by PennVector Core</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">pAAV-EF1a-DIO-EYFP-WPRE-HGHpA</td>
			<td style="width:191px;">Addgene</td>
			<td>plasimid # 20296</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">pAAV-EF1a-DIO-hChR2(H134R)-EYFP-WPRE-HGHpA</td>
			<td style="width:191px;">provided by Dr. Karl Deisseroth</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Patch cord</td>
			<td style="width:191px;">Doric</td>
			<td>D202-9089-0.4</td>
			<td>0.4m length, laser conductor between laser and rotary joint</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">pHelper</td>
			<td style="width:191px;">Stratagene</td>
			<td style="width:177px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Photocurable dental cement</td>
			<td style="width:191px;">3M</td>
			<td style="width:177px;">56846</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Serafin clamp</td>
			<td style="width:191px;">Stoelting</td>
			<td style="width:177px;">52120-43P</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Shielded cable</td>
			<td style="width:191px;">mogami</td>
			<td style="width:177px;">W2780</td>
			<td>Soldering to 6-pin socket for EEG/EMG recording</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Sleep recording chamber</td>
			<td style="width:191px;">N/A</td>
			<td style="width:177px;">N/A</td>
			<td style="width:437px;">Custum-made (21cm&#215; 29cm &#215; 19cm) with water tank holder</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Sleep sign software</td>
			<td style="width:191px;">KISSEI COMTEC</td>
			<td style="width:177px;">N/A</td>
			<td>for EEG/EMG analysis</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Slip ring</td>
			<td style="width:191px;">neuroscience,inc</td>
			<td style="width:177px;">N/A</td>
			<td>for EEG/EMG analysis</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Stainless screw</td>
			<td style="width:191px;">Yamazaki</td>
			<td style="width:177px;">N/A</td>
			<td>&#966;1.0 x 2.0</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Stainless wire</td>
			<td style="width:191px;">Cooner wire</td>
			<td style="width:177px;">AS633</td>
			<td>&#160;0.0130 inch diameter</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Stereotaxic frame with digital console</td>
			<td style="width:191px;">Koph</td>
			<td style="width:177px;">N/A</td>
			<td>Model 940</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Syringe needle</td>
			<td style="width:191px;">Hamilton</td>
			<td>7803-05</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:375px;">Vital recorder software</td>
			<td style="width:191px;">KISSEI COMTEC</td>
			<td style="width:177px;">N/A</td>
			<td>for EEG/EMG recording</td>
		</tr>
	</tbody>
</table>

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.

The present study showed the effect of optogenetic excitation of GABABNST neurons on sleep state transition. ChR2-EYFP was focally expressed in GABA neurons in the BNST. An in situ hybridization histochemical study showed that ChR2-EYFP was colocalized in neurons expressing GAD 67 mRNA signals, indicating that these are GABAergic neurons. Immunohistochemical slice samples confirmed the position of the optic fiber, whose tip was just above the BNST25....

Watch this Scientific Journal Video about Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice at JoVE.com

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

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 ...

This procedure can help us identify the function of specific neuro circuit that are involved in the sleep wakefulness regulation with less invasive manipulation and high temporal resolution. This method allows us the monitoring surface EEG in real time in conjunction with manipulation of a specific neuro circuit to examine the causal relationship between circuits and sleep wakefulness states. Shota Kodani is going to demonstrate the procedure.After confirming a lack of response to toe pinch, apply ointment to the animal's eyes and use the ear bars and nose pin to the stereotactic apparatus to stabilize the head of the anesthetized mouse on an absorbent pad. When the head is fixed in position, make a mid-sagittal incision in the scalp to confirm that the intersection between the sutura sagittalis and sutura coronalis or sutura lambdoidea are in a horizontal line at the same level. To avoid a positioning gap, adjust the nose pinch and ear bars as necessary.Then using serafin clamps to the hold the skin open disinfect the exposed surface of the skull to enable the cranial sutures to be visualized more clearly. To inject the adeno-associated virus vector, load an ethanol sterilized 10 milliliter syringe with two microliters of mineral oil, followed by the experimental volume of virus to be injected. Use the microinjection pump to adjust the tip of the microinjection needle onto the bregma and note the coordinates as the point of origin.Then move the tip to the designated injection site and place the tip of the needle onto the position. Mark the skull at the injection site and use a dental drill equipped with a 0.7 millimeter carbide cutter to drill an approximately two millimeter diameter hole into the skull. After removing blood from around the hole with a cotton swab, slowly move the needle to the bed nucleus of the stria terminalis or BSNT and slowly inject the designated volume of virus solution.Then leave the needle in place for five minutes to allow the solution to sufficiently infiltrate the BNST tissue before carefully retracting the needle. For electroencephalogram, or EEG, and electromyogram, or EMG, electrode implantation, first solder two stainless steel wires from which one millimeter of insulation has been stripped from both ends to the EMG electrodes and place the center of the electrodes onto the bregma. Then mark the position for each EEG electrode.To determine the position of the optical fiber implant, attach an optic fiber ferrule to the manipulator and rotate the manipulator arm to a plus or minus 30 degree angle against a horizontal line. Put the fiber tip on the bregma and record the coordinates. Move the tip to the targeted insertion line and mark the position on the skull and the position for the anchor screw next to the insertion site.Use the dental drill to drill the skull at each site and use the manipulator to gently insert the optic fiber until it reaches above the BNST. The ferrule should rest on the remaining cranium. Secure the fiber to the skull with an anchor screw, taking care not to break the dura or damage any tissue.Then cover the fiber and screw with photo-curable dental cement. Next, drill holes for EEG/EMG electrodes and insert the tip of the first electrode into one hole. Holding the implant with one hand, apply cyanoacrylate adhesive to the space between the skull and the electrode and insert the electrode the rest of the way, taking care not to damage any tissue.When all of the electrodes have been placed, cover the circumference of the electrodes and the optic fibers with additional cyanoacrylate adhesive and cyanoacrylate accelerant to avoid causing any interruption at the ferrule to optic cable and electrode to lead wire connecting zones. Now expose the neck muscles, and insert the wires for the EMG electrode under the muscle. Adjust the length of the EMG electrode so that it fits just under the nuchal muscles and fill the implants with more cyanoacrylate adhesive and accelerant.Then place the mouse on a heat pad with monitoring until full recompensy. For EEG/EMG monitoring during the photo-excitation of targeted neurons, first use a scalar to adjust the laser intensity and use a ferrule to tether the tip of the laser cable to an unused optic fiber. Confirm that there is no space at the junction between the fiber and the cable.After 20 minutes, emit the warmed up laser to the intensity checker and adjust the intensity to 10 milliwatts per millimeter squared. Set the light pulse duration to 10 milliseconds, the rest period to 40 milliseconds, the cycle to 20, and the repeat to 20. Change the laser mode to transistor logic and confirm that light pulses are emitted from the fiber controlled by the pattern regulator.Connect the implanted electrode and cable adapter, then cover the junction with light impermeable material to prevent laser leakage. And when the laser is ready, move the mice to the experimental chamber for EEG/EMG recording. To assess the latency to wakefulness from non rapid eye movement or rapid eye movement sleep, limit the recording time and optimized site gain time and let the mice move freely in the experimental chamber for at least one hour.During the experimental period, monitor the EEG and EMG signals in the same recording screen. Evaluate the mouse's state as wakefulness, non rapid eye movement sleep, or rapid eye movement sleep using the gain control for each wave for ease of distinguishing each state. For measurement of the non rapid eye movement sleep to wakefulness latency, observe stable non rapid eye movement sleep for 40 seconds, then turn on the pattern generator for photo stimulation and confirm laser emission to the implanted optic fibers.Then record the EEG/EMG signals until the sleep state changes to wakefulness. Here represented EEG/EMG traces before and after photo stimulation during non rapid eye movement sleep are shown. A high voltage and slow frequency EEG with no EMG signals, represents non rapid eye movement sleep.Photo stimulation triggered an acute transition to wakefulness about two seconds after stimulation in channelrhodopsin-2 expressing mice while control mice did not demonstrate this transition, suggesting that the excitation of BNST GABA neurons during non rapid eye movement sleep triggers a rapid induction of wakefulness. Conversely, photo stimulation during rapid eye movement sleep had no effect so a transition effect only emerged in non rapid eye movement sleep. It is important to take care to precise coordinations for the virus injection and optic fiber implantation steps.The EEG/EMG traces captured during the sleep analysis can be used to assess the consequences of the photo manipulation on the different vigilant states in mice. This technique will also help us identify other components and circuits involved in regulating the sleep wakefulness states. Use protective goggles to avoid laser damage to the eye and be sure to always autoclave sterilize the adeno-associated virus vector container after use.

optogenetic-manipulation-neural-circuits-during-monitoring

Research

JoVE Journal

Neuroscience

9.5K vistas.  Kanazawa University. Este procedimiento puede ayudarnos a identificar la función de circuito neuro específico que intervienen en la regulación de la vigilia del sueño con menos manipulación invasiva y alta resolución temporal. Este método nos permite el monitoreo de la superficie EEG en tiempo real junto con la manipulación de un circuito neuro específico para examinar la relación causal entre los circuitos y los estados de vigilia del sueño. Shota Kodani va a demostrar el procedimiento.Después...