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 potentials9,10. As a consequence of pyramidal cell activation, local interneurons are activated, and the change in potential is propagated to neurons beyond the site of stimulation10. Perform surgery under sterile conditions in adherence to applicable regulatory guidelines: autoclave surgical tools and softpacks (surgical field, gauze pads); hot bead sterilize all heat-tolerant implanted metal devices (cannulas, screws and wires) for 30-sec; Disinfect heat-intolerant implantable devices (fiber optic cable, plastic connector) with a 10-sec dip in ethanol; blow ethanol-dipped items dry by application of canned air.
2. Anesthesia, Stereotaxic Placement and Clearance of Skull
Use the IMPAC6 Integrated Multi Patient (Vet Equip Inc) system to anesthetize the mouse. Use 5% Isoflurane/95% Oxygen for induction and 3% Isoflurane/97% oxygen for maintenance during surgery. Place the animal on a circulating-water blanket set to 37 C. It is not necessary to monitor body temperature if the blanket is kept at this temperature.
3. Preparing the Skull for Implantation
Prepare the incision site by shaving all fur from the top of the skull. The shaved area should extend laterally from ear to ear and anterior-posterior from the eyes to the posterior end of the skull. Disinfect the exposed skin with three consecutive swabs of betadine followed by ethanol. Make a medial incision on the top of the skull, from between the eyes to the back of the skull. Clean the skull with hydrogen peroxide and sterile saline (0.9% NaCl). Control bleeding with a cauterizing tool. Locate and mark bregma and lambda for determining stereotaxic coordinates. Stereotaxic coordinates for electrode/optogenetic stimulus configurations that we have used are presented in Table 1 and shown schematically in Figure 1A.
4. Preparation of the Implantation Sites on Skull
Use a high speed dental drill with a 0.5 mm ball burr bit to establish each implantation site in the skull. Chamfer the external margin of each hole using a 0.7 mm ball burr bit to facilitate screw insertion.
5. Insertion and Securing of Cannulas, EMG Leads and EEG Leads
Insert EEG screws (0.8 mm in length, with 2 cm of uncoated 30-gauge tinned-copper bus wire pre-soldered to screw head) into the holes and drive them in with a slotted hand screwdriver approximately 4-5 revolutions to obtain the desired depth. Hold guide cannulas in place with a stereotaxic cannula holder and then affix them to the skull and anchors with acrylic cement. Each guide cannula should contain a dummy cannula/stylet (Plastics One, part # C312 DC\1) from the time of surgery to the time of experimentation (10-14 days) to maintain patency.
6. Closing Surgical Site
Once EEGs and guide cannulas are positioned in the skull, bond them together with dental acrylic cement (Lang Dental - Ortho-Jet cement). After cement sets, position the plastic connector (Pinnacle Technology part # 8402) above the dried cement mound. Solder the ends of the wires emanating from EEG leads to the contacts on the plastic connector. Encase the wire leads in cement. Then draw the EMG wires through the nuchal muscles by sliding them into the barrel of a 21ga needle pierced through muscle. Tie a double surgeon's knot of 5-0 nylon suture around these wires just distal to where they exit the muscle. Suture back together the skin that was retracted to access the muscle tissue with single interrupted surgeon knots, using a reverse cutting P-3 needle and 5-0 nylon suture.
7. Post-operative Analgesia and Recovery
Administer buprenorphine as an analgesic (0.1 mg/kg, subcutaneous) and flunixin meglumine (0.1 mg/kg, subcutaneous) as an anti-inflammatory immediately after surgery, and daily on days 1-2 post-surgery. Allow animals to recover in standard colony housing conditions for at least seven days before experimentation. With no additional care beyond standard colony housing, cannulas can be expected to remain patent for at least two weeks with the indwelling stylets secured in place. EEG and EMG sensors can also be expected to retain functionality for this duration.
2. Generation of Timed Light Pulses
1. Programming of MC Stimulus Unit
Connect the MC-Stimulus unit (Multi-Channel Systems) via a USB connection to a desktop computer. Use the spreadsheet-like MC-Stimulus user interface to program the MC Stimulus unit. Enter the desired TTL (transistor-transistor logic) voltage for the on period, the durations of stimulus on and off periods, the number of cycles per second desired, and the total number of repeated cycles for the entire stimulus session. For further details see MC-Stimulus technical manual:
<a href="http://www.multichannelsystems.com/sites/multichannelsystems.com/files/documents/manuals/STG4002_Manual.pdf" target="_blank">http://www.multichannelsystems.com/sites/multichannelsystems.com/files/documents/manuals/STG4002_Manual.pdf</a>
3. Manufacture/Assembly of Fiber Optic Probes for Light Delivery to the Cerebral Cortex
<ol>
	<li>Supplies and equipment needed are found at: <a href="http://www.thorlabs.com/Thorcat/1100/1166-D02.pdf" target="_blank">http://www.thorlabs.com/Thorcat/1100/1166-D02.pdf</a></li>
	<li>Cutting and polishing of fiber optic cables. <a href="http://www.thorlabs.com/Thorcat/1100/1166-D02.pdf" target="_blank">http://www.thorlabs.com/Thorcat/1100/1166-D02.pdf</a></li>
</ol>
4. Assembly of Rodent Optogenetic Stimulation System
1. Interface of MC-Stimulus Unit and Laser
Once the MC-Stimulus unit is programmed, run it as a stand-alone signal generator of a 5-Volt binary ON/OFF signal. Connect the MC-Stimulus unit to the TTL enabled power unit of the laser with BNC connectors.
2. Producing Laser Output via Fiber Optic Cable
Use the digital display and variable power output dial of the laser power unit to manually adjust the power output of the laser. Connect the laser power unit to the laser via a ribbon cable. Connect the female FC (ferrule connector) fiber adapter of the laser to the male FC connector on the fiber optic patch cable (a 3 m length, 200 &mu;m diameter glass conduction fiber encased in light reflecting acrylate polymer cladding).
3. Conveying the Laser to the Animal: Rotary Commutator
Connect the originating fiber optic cable (3 m in length) and the delivery fiber optic cable (45 cm in length) to opposing ends of the optic rotary joint encasing two collimating lenses (Doric lenses). The fiber optic rotary joint serves as a commutator: as the animal moves about its cage, the rotary joint rotates to prevent breakage of the fiber optic cables. Use plastic cable ties to affix the commutator to a metal stand placed above the cylindrical cage in which the animal is housed.
4. Attaching Delivery Fiber Optic Cable to Head
Restrain the mouse by using one hand to pin the mouse under a cupped palm. Orient the head between the experimenter's middle and index finger. Remove the dummy cannula from the guide cannula placed for the fiber optic cannula. Clear the guide cannula of debris using a sterile 25ga needle. Insert the fiber optic cable by hand and fasten it to the fiber optic guide cannula with a threaded screw cap salvaged from a dummy cannula. Control the depth of insertion of the fiber optic cable in the brain by a suture knot tied on the fiber optic cable at a fixed distance from the flat-cleaved end. The entire experimental apparatus is shown in Figure 1B.
5. Measurement of EEG-defined Sleep and Lactate Concentration during Optogenetic Stimulation
1. Precalibration of the Lactate Sensor
Pre-calibration of the lactate sensor is performed with an in vitro kit (Pinnacle Technology part # 7000-K1-T). The sensor is equilibrated in phosphate buffered saline (pH 7.4), then exposed to three concentrations of L-lactate in stepwise fashion per manufacturer protocols.
2. Insertion of the Lactate Sensor and EEG Cable
Insert the pre-calibrated sensor into the skull-mounted lactate guide cannula in a manner identical to the fiber optic cable insertion procedure (section 4.4). Connect the lactate sensor to the preamplifier of the Pinnacle 8,400 biosensor with bipolar plug connectors. Attach this preamplifier to the 8-pin connector on the surgically-implanted headmount.
3. Establishing Optogenetic Stimulus Intensity
Before collecting data, use the laser intensity control knob to adjust the intensity of the optogenetic stimulus. The amplitude of the EEG response will vary across animals, due to factors that have not been studied systematically, and must be verified by visual inspection at the onset of stimulation. In practice, an intensity of 80-120 &mu;Watts will typically produce an increase in EEG amplitude of approximately 50% at the frequency of stimulation. Post-hoc fast Fourier transform analysis (see section 5.5) is necessary to quantify the absolute magnitude of the response.
4. Data Collection
Collect data using the Pinnacle 8400 system: <a href="http://pinnaclet.com/pal-8400.html" target="_blank">http://pinnaclet.com/pal-8400.html</a>.
5. Data Processing with Neuroscore
Classify sleep states by visual inspection of the EEG and EMG data with the Neuroscore interface (DataSciences, International: <a href="http://www.datasci.com/products/software/NeuroScore_CNS_Software.asp" target="_blank">http://www.datasci.com/products/software/NeuroScore_CNS_Software.asp</a>) or the Sirenia interface (Pinnacle Technology: <a href="http://www.pinnaclet.com/sirenia.html" target="_blank">http://www.pinnaclet.com/sirenia.html</a>). Process data in ten-second epochs as wake, non-rapid eye movement sleep, or rapid eye movement sleep based on EEG and EMG. Save data as Microsoft Excel spreadsheets for additional analysis and statistical testing.

<ol>
	<li>Magistretti, P., Zigmond, M. J., Bloom, F. E., Landis, S. C., Roberts, J. L., Squire, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+Energy+Metabolism.">Brain Energy Metabolism.</a> Fundamental Neuroscience. , 389-413 (1999).</li><li>Maquet, P., 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=Cerebral+glucose+utilization+during+sleep-wake+cycle+in+man+determined+by+positron+emission+tomography+and+[18F]2-fluoro-2-deoxy-D-glucose+method.">Cerebral glucose utilization during sleep-wake cycle in man determined by positron emission tomography and [18F]2-fluoro-2-deoxy-D-glucose method.</a> Brain Res. 513 (1), 136-143 (1990).</li><li>Buchsbaum, M. 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=Regional+cerebral+glucose+metabolic+rate+in+human+sleep+assessed+by+positron+emission+tomography.">Regional cerebral glucose metabolic rate in human sleep assessed by positron emission tomography.</a> Life Sci. 45 (15), 1349-1356 (1989).</li><li>Kennedy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+cerebral+glucose+utilization+in+non-rapid+eye+movement+sleep.">Local cerebral glucose utilization in non-rapid eye movement sleep.</a> Nature. 297 (5864), 325-327 (1982).</li><li>Pappenheimer, J. R., Koski, G., Fencl, V., Karnovsky, M. 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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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Protoc. 5 (2), 247-254 (2010).</li><li>Destexhe, A., Contreras, D., Steriade, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortically-induced+coherence+of+a+thalamic-generated+oscillation.">Cortically-induced coherence of a thalamic-generated oscillation.</a> Neuroscience. 92 (2), 427-443 (1999).</li><li>Liu, Z. W., Faraguna, U., Cirelli, C., Tononi, G., Gao, X. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+for+wake-related+increases+and+sleep-related+decreases+in+synaptic+strength+in+rodent+cortex.">Direct evidence for wake-related increases and sleep-related decreases in synaptic strength in rodent cortex.</a> J. 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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 ...

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Research

JoVE Journal

Neuroscience

20.1K Views.  Washington State University. 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.

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

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Combined Transcranial Magnetic Stimulation and Electroencephalography of the Dorsolateral Prefrontal Cortex

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic field pulses. The initiation of cortical activation or its modulation depends on the background activation of the neurons of the cortical region activated, the characteristics of the coil, its position and its orientation with respect to the head. TMS combined with simultaneous electrocephalography (EEG) and neuronavigation (nTMS-EEG) allows for the assessment of cortico-cortical excitability and connectivity in almost all cortical areas in a reproducible manner. This advance makes nTMS-EEG a powerful tool that can accurately assess brain dynamics and neurophysiology in test-retest paradigms that are required for clinical trials. Limitations of this method include artifacts that cover the initial brain reactivity to stimulation. Thus, the process of removing artifacts may also extract valuable information. Moreover, the optimal parameters for dorsolateral prefrontal (DLPFC) stimulation are not fully known and current protocols utilize variations from the motor cortex (M1) stimulation paradigms. However, evolving nTMS-EEG designs hope to address these issues. The protocol presented here introduces some standard practices for assessing neurophysiological functioning from stimulation to the DLPFC that can be applied in patients with treatment resistant psychiatric disorders that receive treatment such as transcranial direct current stimulation (tDCS), repetitive transcranial magnetic stimulation (rTMS), magnetic seizure therapy (MST) or electroconvulsive therapy (ECT).

The protocol presented here is for TMS-EEG studies utilizing intracortical excitability test-retest design paradigms. The intent of the protocol is to produce reliable and reproducible cortical excitability measures for assessing neurophysiological functioning related to therapeutic interventions in the treatment of neuropsychiatric diseases such as major depression.

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...