Research funded by Department of Defense (Defense Advanced Research Projects Agency, Young Faculty Award, Grant Number N66001-09-1-2117) and NINDS (R15NS070734).

1. Surgical Preparation of Animals
1. Experimental Subjects
Use mice of the B6.Cg-Tg(Thy1-COP4/eYFP)18Gfng/J transgenic line9; JAX strain #7612) or other mice expressing the blue light-sensitive cation channel, Channelrhodopsin-2, in cerebral cortical neurons. Application of blue light to the cerebral cortex of the B6.Cg-Tg(Thy1-COP4/eYFP)18Gfng/J transgenic line causes the pyramidal neurons expressing Channelrhodopsin-2 to depolarize and undergo action potent...

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+short-term+slow+wave+sleep+homeostasis+and+its+disruption+by+minocycline+in+the+laboratory+mouse.">Quantification of short-term slow wave sleep homeostasis and its disruption by minocycline in the laboratory mouse.</a> Neurosci. Lett. 490 (3), 165-169 (2011).</li><li>El Yacoubi, 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=Behavioral,+neurochemical,+and+electrophysiological+characterization+of+a+genetic+mouse+model+of+depression.">Behavioral, neurochemical, and electrophysiological characterization of a genetic mouse model of depression.</a> Proc. Natl. Acad. Sci. U.S.A. 100 (10), 6227-6232 (2003).</li><li>Tsunematsu, 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=Acute+optogenetic+silencing+of+orexin/hypocretin+neurons+induces+slow-wave+sleep+in+mice.">Acute optogenetic silencing of orexin/hypocretin neurons induces slow-wave sleep in mice.</a> J. Neurosci. 31 (29), 10529-10539 (2011).</li><li>Le, S., Gruner, J. A., Mathiasen, J. R., Marino, M. J., Schaffhauser, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+ex+vivo+receptor+occupancy+and+wake-promoting+activity+of+selective+H3+receptor+antagonists.">Correlation between ex vivo receptor occupancy and wake-promoting activity of selective H3 receptor antagonists.</a> J. Pharmacol. Exp. Ther. 325 (3), 902-909 (2008).</li><li>Burmeister, J. J., Palmer, M., Gerhardt, G. 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No conflicts of interest declared.

The methods presented here allow one to measure the relationship between sleep and changes in the brain concentration of the glycolytic intermediate lactate on a time scale not previously possible. Animals undergo spontaneous transitions between wake, NREMS and REMS. Furthermore, we are able to apply optogenetic stimuli while animals undergo these transitions. Data collected to date demonstrate that both spontaneous and induced waves impact on the readout of a lactate oxidase-based biosensor.
<p class="jove_content"...

<table border="1">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>BASi Mouse Guide Cannula</td>
			<td>Pinnacle Technology/BASi Inc</td>
			<td>7032</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lactate Biosensor</td>
			<td>Pinnacle Technology</td>
			<td>7004</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Head Mount</td>
			<td>Pinnacle Technology</td>
			<td>8402</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sleep/Biosensor Recording system</td>
			<td>Pinnacle Technology</td>
			<td>8400-K1-SL</td>
			<td>2 EEG channels, 1 EMG channel, &amp; 1 biosensor</td>
		</tr>
		<tr>
			<td>Tethered Mouse in-vitro Calibration kit</td>
			<td>Pinnacle Technology</td>
			<td>7000-K1-T</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fiber Optic Guide Cannula</td>
			<td>Plastics One</td>
			<td>C312G</td>
			<td>21 Gauge Guide Cannula</td>
		</tr>
		<tr>
			<td>Dummy Cannula</td>
			<td>Plastics One</td>
			<td>C312DC</td>
			<td>21 Gauge Dummy</td>
		</tr>
		<tr>
			<td>Diamond Fiber Scribe</td>
			<td>Thorlabs</td>
			<td>S90W</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fiber Connector Crimp Tool</td>
			<td>Thorlabs</td>
			<td>CT042</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Furcation Tubing</td>
			<td>Thorlabs</td>
			<td>FT030</td>
			<td>03.0 mm</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>Thorlabs</td>
			<td>T10S13</td>
			<td>Max Dia. 0.012</td>
		</tr>
		<tr>
			<td>Furcation Tube Stripper</td>
			<td>Thorlabs</td>
			<td>FTS3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bare Hard Cladding Multimode Fiber</td>
			<td>Thorlabs</td>
			<td>BFL37-200</td>
			<td>200 &mu;m Core, 0.37 NA</td>
		</tr>
		<tr>
			<td>Wire Snips/Kevlar Shears</td>
			<td>Thorlabs</td>
			<td>T865</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fiber Optic Epoxy</td>
			<td>Thorlabs</td>
			<td>F112</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fiber Stripper Tool</td>
			<td>Thorlabs</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass Polishing Plate</td>
			<td>Thorlabs</td>
			<td>CTG913</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rubber Polishing Pad</td>
			<td>Thorlabs</td>
			<td>NRS913</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Eye Loupe</td>
			<td>Thorlabs</td>
			<td>JEL10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Kim Wipes</td>
			<td>Thorlabs</td>
			<td>KW32</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Compressed Air</td>
			<td>Thorlabs</td>
			<td>CA3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polishing Puck</td>
			<td>Thorlabs</td>
			<td>D50-xx</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fiber Inspection scope</td>
			<td>Thorlabs</td>
			<td>CL-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polishing Films</td>
			<td>Thorlabs</td>
			<td>LFG5P, LFG3P, LFG1P, LFG03P</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FC/PC connector end</td>
			<td>Thorlabs</td>
			<td>30126G2-240</td>
			<td>240 &mu;m Bore, SS Ferrule</td>
		</tr>
		<tr>
			<td>MC Stimulus Unit</td>
			<td>Multi-Channel Systems</td>
			<td>STG-4002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MC Stimulus Software</td>
			<td>Multi-Channel Systems</td>
			<td>MC-Stimulus V 2.1.5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Blue Laser</td>
			<td>CrystaLaser</td>
			<td>CL473-050-0</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laser Power supply</td>
			<td>CrystaLaser</td>
			<td>CL2005</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fiber Optic Rotary Joint</td>
			<td>Doric Lenses</td>
			<td>FRJ-v4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Table 2. Supplies and equipment.</td>
		</tr>
	</tbody>
</table>

simultaneous electroencephalography, real-time measurement of lactate concentration and optogenetic manipulation of neuronal activity in the rodent cerebral cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

As shown in Figure 2, a mouse equipped for optogenetic stimulation and lactate/EEG/EMG data collection underwent spontaneous sleep/wake state transitions while EEG, EMG and cerebral lactate concentration were monitored continuously. Current at the lactate sensor increased during periods of low amplitude EEG and decreased during periods of high amplitude EEG. As shown in Figure 3, both channels of the EEG are responsive to optogenetic stimuli delivered in the frontal cort...

Watch this Scientific Journal Video about Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral C

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

simultaneous-electroencephalography-real-time-measurement-lactate

Research

JoVE Journal

Neuroscience

20.1K 조회수.  Washington State University. 절차는 뇌파, electromyogram, 그리고 뇌성 젖산 농도를 모니터링하는 동안 optogenetically 뇌성 대뇌 피질의 피라미드 뉴런의 활동을 조작하기위한 설명되어 있습니다. 사람들이 자연 수면 / 웨이크주기를 받아야 동안 실험 녹음 케이블 테 더링 마우스에 수행됩니다. Optogenetic 장비는 우리의 실험실에서 조립, 녹화 장비는 상업적으로 사용할 수 있습니다.

비디오: Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex