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 AAV must be sterilized with an autoclave after all the volume is used up. The surgical site and all implanted material should be clean and sterile during use. 
NOTE: See Figure 1.
<ol>
	<li>Disinfect the surgical equipment with the autoclave.</li>
	<li>Anesthetize mice with isoflurane using an anesthetic vaporizer. Observe until the mouse has reached the desired depth of anesthesia, determined by loss of response to pinching the tail with forceps. Apply ophthalmic ointment to the eyes to prevent them drying.</li>
	<li>Disinfect the surgical field with iodine solution or 70% EtOH (3x) and dry sufficiently. Allow the virus to thaw on ice as the surgery is being performed. Cover the surgical area with absorbent lab bench paper.</li>
	<li>Fix the mouse&#39;s head in the stereotactic apparatus with ear bars and a nose pinch. After confirming the head is held stably, make a midsagittal incision in the scalp to ensure the positions of the bregma and lambda are located at the same level on a horizontal line.</li>
	<li>To avoid a positioning gap, appropriately adjust the levels of the nose pinch and ear bars up and down. The bregma and lambda refer to the intersection between the sutura saggitalis and sutura coronalis or sutura lambdoidal, respectively (Figure 2).</li>
	<li>Use Serafin clamps to hold the skin to maintain access to the cranium. After exposure of the skull, disinfect the surface of the skull with iodine or 5% H2O2, to enable the cranial sutures including the bregma and lambda to be visualized more clearly.</li>
	<li>Prepare AAV vector injection:
	<ol>
		<li>Wash the inside of a 10 mL syringe (see Table of Materials) sequentially with 70% EtOH, 100% EtOH, and sterilized water, 5 times each. Secure the syringe in the clamp of a microinjection pump arm and make sure all solution in the syringe is discharged.</li>
		<li>Carefully aspirate 2 &#181;L of mineral oil without air bubbles, then aspirate the designated volume of the virus solution. After aspiration, manipulate the plunger button and confirm that virus solution emerges at the tip of the needle. 
		NOTE: The injection volume of the virus solution was determined in pilot experiments using the same mouse strain and same virus product. The relation between the volume of virus solution and extent of the infection area should be estimated in advance.</li>
	</ol>
	</li>
	<li>Inject AAV vector:
	<ol>
		<li>Adjust the tip of the microinjection needle on the bregma and note the coordinates as the original point. Move the tip to the designated injection site (for the BNST: anteroposterior + 0.2 mm, mediolateral &#177; 1.0 mm, dorsoventral - 4.2 mm) and place the tip of the needle onto the position. Put a mark on the skull and drill holes of approximately 2 mm in diameter using a dental drill with a 0.7 mm carbide cutter. Be careful not to damage the dura or brain tissue.</li>
		<li>After removing blood from around the holes with a cotton swab, slowly move the needle into the position of the BNST. Slowly inject the designated amount of virus solution (0.07 &#181;l/min) with a mechanical microinjector. After completing the injection, leave the needle for 5 min to allow the solution to sufficiently infiltrate the BNST tissue. Carefully take out the needle.</li>
		<li>For bilateral injection, repeat steps 1.8.1-1.8.2 on the other side. Throughout the procedure, keep the skull moist with applications of sterile saline. 
		NOTE: We used custom EEG/EMG implants (W: 5 mm, D: 7 mm, H: 1 mm) with four EEG electrodes (4 mm), two EMG electrodes (2 mm; cut the 4 mm electrode to 2 mm with nippers) and 6 electrodes (4.5 mm) (Figure 2A).</li>
	</ol>
	</li>
	<li>Solder two stainless steel wires (see Table of Materials) from which 1 mm of the insulation is stripped off both ends to the EMG electrodes. Adjust the center of the electrodes to the bregma and mark the position of each EEG electrode (anteroposterior &#177; 1.5 mm, mediolateral &#177; 1.0 mm), and determine the position of the implant (Figure 2B).</li>
	<li>Implant optical fibers:
	<ol>
		<li>Attach an optic fiber ferrule to the manipulator and rotate the manipulator arm so that it has an angle of &#177;30&#176; against a horizontal line (this process is only needed to avoid interference between electrodes and optic fibers, such as in the case of BNST stimulation). Put the fiber tip on the bregma and record the coordinates.</li>
		<li>Move the tip to the targeted insertion line and mark the position on the skull. Also put additional marks near the insertion site for the anchor screws. Drill the skull on each site with a dental drill to insert the optic fiber and fix the screw. Fix the screw on the skull. Be careful not to break the dura or damage any tissue by screw.</li>
		<li>Insert the optic fiber gently until reaching above the BNST with a manipulator. The ferrule should rest on the remaining cranium (Figure 1B).</li>
		<li>Apply photocurable dental cement (see Table of Materials) to cover the fiber and the screw. The reaction time to solidify the glue should be specified by the manufacturer&#39;s manual (Our material needs exposure to light for at least 10 s with specific wave length photo-generator. It is unnecessary to dry the glue after this).</li>
		<li>In this step, make sure that no materials (screw or glue) occupy the mounting space for the electrodes. In addition, avoid making any interruption in the cement for the ferrule connecting to the optic fiber and cable. Repeat steps 1.10.1-1.10.4 on the opposite side for bilateral stimulation.</li>
	</ol>
	</li>
	<li>Drill holes for EEG/EMG electrodes. Insert the tips of the electrodes into the holes. Hold the implant and apply cyanoacrylate adhesive to the space between the skull and the electrodes. Insert again with attention not to interfere with any materials.</li>
	<li>Cover the circumference of the electrodes and optic fibers with cyanoacrylate adhesive followed by application of cyanoacrylate accelerant on the adhesive. This step avoids causing any interruption at the ferrule-to-optic cable and electrode-to-lead wire connecting zone (Figure 1C). 
	NOTE: Cyanoacrylate adhesive and its accelerant are harmful to the mouse eye. Pay attention not to cause spillage of these chemical substances. Also, be careful not to strongly touch the electrodes and the fibers in order to avoid unexpected deviation immediately after adhesive solidification.</li>
	<li>Expose the mouse neck muscles and insert the wires for the EMG electrode under the muscle. Adjust the length of EMG electrode so that it locates just under the nuchal muscles. Light connection between tip of electrode and muscle fascia is enough to catch the EMG signal.</li>
	<li>Apply cyanoacrylate adhesive to fill the implants and solidify the adhesive with acceleration liquid. Then, put the mouse on a heat pad for recovery until the postural reflex appears. Adjust heat pad temperature to animal resting body temperature (36.0 &#176;C in ZT 0-12 in case of C57BL6 mice; do not exceed 38.0 &#176;C). 
	NOTE: An antibiotic is not required for sterile surgery.</li>
	<li>Follow your local institutional guidelines for post-operative analgesia. Keep the mice in a home cage for a recovery period of at least 7 days.</li>
</ol>
2. EEG/EMG Monitoring with Photo-excitation of Targeted Neurons in Specific Sleep States
CAUTION: This protocol includes use of class 3B laser equipment or LED devices. Experimenters should be aware of safety information. Protective eye goggles are required.
<ol>
	<li>Before connecting the laser cable to the optic fiber, adjust laser intensity with a scaler. Tether the tip of the laser cable to an unused optic fiber with a ferrule and confirm that there is no space at the junction between the fiber and the cable.</li>
	<li>Turn on the main switch of the laser and wait 20 min for it to warm up.</li>
	<li>Emit the laser to the intensity checker and adjust laser intensity to 10 mW/mm2. Change the laser mode to transistor logic and confirm that light pulses are emitted from the fiber controlled by the pattern regulator which is set at 10 ms for duration, 40 ms for rest, 20 times for cycle, and 20 times repeat (that is, 20 Hz of 10 ms light pulses for 20 s).</li>
	<li>After the recovery period, move the mice to the experimental chamber for recording EEG/EMG. House mice at a constant 23 &#176;C with a 12 h light/dark cycle with food and water available ad libitum.</li>
	<li>Connect the implanted electrode and cable adaptor which is tethered to a slip ring to avoid entanglement. &#65279;It is recommended to cover the junction with light-impermeable material such as aluminum foil to prevent laser leakage. If a bilateral experiment is required, use a slip ring with a bifurcate attachment for the cables.</li>
	<li>In this protocol, we assess latency to wakefulness from NREM sleep or REM sleep, so the recording time should be limited in optimized zeitgeber time (ZT0 is defined as the time when the light is on). This protocol was conducted between ZT4 - ZT10. Let the mice stay freely in the experimental chamber for at least 1 h as acclimatization.</li>
	<li>During the experimental period, monitor EEG and EMG signals in the same monitor and evaluate the mouse&#39;s state as wakefulness, NREM sleep or REM sleep. Use the gain control for each wave to make it easier to distinguish each state.</li>
	<li>For measurement of NREM sleep to wakefulness latency, observe stable NREM sleep for 40 s or stable REM sleep for 30 s, then turn on the switch of the pattern generator for photostimulation (this protocol generates 20 Hz of 10 ms light pulses for 20 s). Confirm laser emission to the implanted optic fibers.</li>
	<li>Record EEG/EMG signals until the sleeping state changes to wakefulness. If two or more experimental trials are needed, limit optogenetic manipulation to once a day because photostimulation is an artificial intervention which might affect sleep/wakefulness architecture.</li>
	<li>After the experiment, deeply anesthetize and perfuse with sterile saline and paraformaldehyde (PFA) for sampling the whole brain for immunohistochemical analysis26.</li>
</ol>
3. Analysis of Latency Time from NREM Sleep to Wakefulness
<ol>
	<li>After recording EEG/EMG signals, transfer the signal data to a computer for scoring sleep/wakefulness states. This protocol describes the method for EEG analysis with recording software (see Table of Materials).</li>
	<li>Start the sleep sign application and click the File tab and select Open to choose the recorded data (.kcd file). Click the Sleep tab to select Epoch time for adjusting the time window for each epoch (we use 1 epoch/4 sec).</li>
	<li>Manually score sleep/wakefulness states based on EEG/EMG signals, according to the following criteria: wakefulness, high EMG and low EEG voltage with high frequency; NREM, low EMG tone and high EEG voltage with high &#948; (0.5-4 Hz) frequency; and REM, EMG indicates muscle atonia and low EEG voltage with high &#952; (6-9 Hz) frequency. A state that does not consecutively continue for 16 s (i.e. 4 epochs) is not defined as a state change because it is not a stable state.</li>
	<li>Click and hold the left mouse button on the first epoch and drag the cursor until the specific trend of more than 16 s (4 epochs) is ended. Then, release the left mouse button and choose the appropriate state (wakefulness or NREM sleep or REM sleep) in the pop-up window. Repeat this procedure to score all EEG/EMG signals recorded in the file.</li>
	<li>Find the exact time of stimulation in the epoch where the EEG shows NREM or REM sleep, and the epoch showing state transition following the stimulation point. Count the number of epochs between the periods just after stimulation and just before state transition.</li>
	<li>Then multiply counted number of epochs by 4 s (A). In the epoch of stimulation, take a screen shot and measure the width between the stimulation point and the end of the epoch. Then, divide the measured length by the entire epoch length and multiply by 4 s (B). Similarly, calculate the duration of NREM sleep in the epoch of state change, which means the time between the start of the epoch and the state change point (C).</li>
	<li>Sum A, B and C to obtain the latency from NREM sleep to wakefulness. The same procedure is used for analysis of state transition from REM sleep to wakefulness.</li>
</ol>

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This project was partly supported financially by Merck &#38; Co.

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 to activate or inhibit neuronal activity of particular neurons using different kinds of opsins, such as ChR2, SSFO, halorhodopsin, ArchT, and iChloC27. ChR2 can activate neurons a few milliseconds after photo-stimulation and this can be used to evoke action potentials in a phase-lock manner by a pulse generator to examine the acute impact in specific sleep stages. A stably activating opsin such as stable step function opsin (SSFO), which induces depolarization of neurons for 15 to 30 min after stimulation, might also be useful for some kinds of experiments designed to observe a semi-chronic effect28. Depolarized cells with SSFO might become more sensitive to various physiological neuronal input and be deactivated by applying long wavelength light. Furthermore, we can activate axons by implantation of optic fibers at the site of an axonal projection. Fiber stimulation could provide information on the function of a particular axonal projection pathway.
EEG/EMG recording during optogenetic manipulation is a less invasive method to determine the direct consequences of selective excitation/inhibition of neural circuits on sleep/wakefulness states in mice. With this method, many neuronal populations and neural circuits have been shown to be involved in the regulation of sleep/wakefulness states. Towards further development of this technique, it is possible to implant multiple fibers to manipulate multiple pathways simultaneously, or this could be also used in combination with fiber photometory or miniscopes to monitor neuronal activities.
In conclusion, it is anticipated that optogenetics will accelerate progress in unlocking the mystery of sleep regulation by the brain and the development of innovative therapies for refractory insomnia and other sleep disorders.

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>
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		<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.
Figure 3A shows representative EEG/EMG traces before and after photostimulation during NREM sleep. High voltage and slow frequency EEG with no EMG signals represent NREM sleep. Photostimulation (10 ms pulses at 20 Hz for 20 sec) was applied following stable NREM sleep. Stimulation triggers acute transition to wakefulness (low voltage and high frequency EEG with active EMG signals) about 2 s after stimulation in ChR2-expressing mice. Control mice (EYFP) did not show transition after stimulation (latency of waking from NREM: EYFP, 295.39 &#177; 106.61 sec, n = 6; ChR2: 2.71 &#177; 0.59 sec, n = 6; t10 = 2.35, p &#60; 0.05; Figure 3B, upper). These data suggest that excitation of GABABNST neurons during NREM sleep triggers rapid induction of wakefulness. On the other hand, photostimulation during REM sleep had no effect (EYFP: 36.45 &#177; 13.08 sec, n = 6; ChR2: 37.29 &#177; 15.19 sec, n = 6; t10 = 0.04, p = 0.484; Figure 3B, bottom) so a transition effect only emerged in NREM sleep.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58613/58613fig1.jpg" /> 
Figure 1: Procedure to inject AAV, implant optic fibers and EEG/EMG implants. (A) Experimental procedure of virus injection. EYFP-fused ChR2 or EYFP (for control) gene incorporated in AAV vector whose transcription is activated by Cre recombinase was bilaterally injected into the BNST. (B) Optic fibers were inserted toward the BNST at a 30&#176; angle to the horizontal to prevent collision with the electrode. Two screws were inserted around it. (C) EEG/EMG recording device was implanted after secured placement of the optic fibers. (D) At the end of the operation, the entire surgical area should be covered with cyanoacrylate adhesive and strongly fixed. Make sure not to apply any agent to the region connecting the electrode and ferrules. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58613/58613fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58613/58613fig2.jpg" /> 
Figure 2: Custom EEG/EMG electrode and electrode pins insertion sites.&#160;(A) Top: Out of 6 electrode pins, the external two pins are cut down to 2 mm. Bottom: EEG/EMG electrodes. (B) These electrodes and EMG conduction wires are then soldered. The connecting zone should be isolated with any insulation like cyanoacrylate adhesive. Insertion sites of electrodes are relative to the bregma (anteroposterior &#177; 1.5mm, mediolateral &#177; 1.0 mm). EMG wires are inserted under the neck muscle with removal of insulation protecting the wire at the insertion site (1 mm). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58613/58613fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58613/58613fig3.jpg" /> 
Figure 3: Effect of GABABNST stimulation on state transition in NREM sleep and REM sleep. (A) Representative EEG and EMG wave and EEG power spectrum. Photostimulation (10 ms pulses at 20 Hz for 20 sec) was applied to ChR2-expressing GABABNST neurons following 40 s NREM sleep. Wakefulness was rapidly induced after a 2 s. The EEG showed low voltage and high frequency with EMG bursting. The EEG power spectrogram also showed transition from low to high frequency. (B) Optogenetic excitation of GABABNST neurons showed rapid transition from NREM sleep to wakefulness (upper), but this effect was not seen in the case of applying the same manipulation in REM sleep (bottom). *p &#60; 0.05, Welch&#39;s t-test.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58613/58613fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

optogenetic-manipulation-neural-circuits-during-monitoring

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.6K Views.  Kanazawa University. 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.

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

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents the return of fear. Pavlovian fear-conditioning paradigms are commonly employed to create a controlled, novel fear association in the laboratory. After pairing an innocuous stimulus (conditioned stimulus, CS) with an aversive outcome (unconditioned stimulus, US) we can elicit a fear response (conditioned response, or CR) by presenting just the stimulus alone1,2 . Once fear is acquired, it can be diminished using extinction training, whereby the conditioned stimulus is repeatedly presented without the aversive outcome until fear is no longer expressed3. This inhibitory learning creates a new, safe representation for the CS, which competes for expression with the original fear memory4. Although extinction is effective at inhibiting fear, it is not permanent. Fear can spontaneously recover with the passage of time. Exposure to stress or returning to the context of initial learning can also cause fear to resurface3,4.
Our protocol addresses the transient nature of extinction by targeting the reconsolidation window to modify emotional memory in a more permanent manner. Ample evidence suggests that reactivating a consolidated memory returns it to a labile state, during which the memory is again susceptible to interference5-9. This window of opportunity appears to open shortly after reactivation and close approximately 6hrs later5,11,16, although this may vary depending on the strength and age of the memory15. By allowing new information to incorporate into the original memory trace, this memory may be updated as it reconsolidates10,11. Studies involving non-human animals have successfully blocked the expression of fear memory by introducing pharmacological manipulations within the reconsolidation window, however, most agents used are either toxic to humans or show equivocal effects when used in human studies12-14. Our protocol addresses these challenges by offering an effective, yet non-invasive, behavioral manipulation that is safe for humans.
By prompting fear memory retrieval prior to extinction, we essentially trigger the reconsolidation process, allowing new safety information (i.e., extinction) to be incorporated while the fear memory is still susceptible to interference. A recent study employing this behavioral manipulation in rats has successfully blocked fear memory using these temporal parameters11. Additional studies in humans have demonstrated that introducing new information after the retrieval of previously consolidated motor16, episodic17, or declarative18 memories leads to interference with the original memory trace14. We outline below a novel protocol used to block fear recovery in humans.

Conditioned fear can be diminished through an inhibitory process called extinction, but can resurface under conditions such as the passage of time or exposure to stress. Our protocol presents a novel way of preventing fear recovery by introducing extinction during the reconsolidation window (the re-storage phase of a reactivated memory).

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents ...

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.

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 ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

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 ...

Disruption of Frontal Lobe Neural Synchrony During Cognitive Control by Alcohol Intoxication

Decision making relies on dynamic interactions of distributed, primarily frontal brain regions. Extensive evidence from functional magnetic resonance imaging (fMRI) studies indicates that the anterior cingulate (ACC) and the lateral prefrontal cortices (latPFC) are essential nodes subserving cognitive control. However, because of its limited temporal resolution, fMRI cannot accurately reflect the timing and nature of their presumed interplay. The present study combines distributed source modeling of the temporally precise magnetoencephalography (MEG) signal with structural MRI in the form of &#34;brain movies&#34; to: (1) estimate the cortical areas involved in cognitive control (&#34;where&#34;), (2) characterize their temporal sequence (&#34;when&#34;), and (3) quantify the oscillatory dynamics of their neural interactions in real time. Stroop interference was associated with greater event-related theta (4 - 7 Hz) power in the ACC during conflict detection followed by sustained sensitivity to cognitive demands in the ACC and latPFC during integration and response preparation. A phase-locking analysis revealed co-oscillatory interactions between these areas indicating their increased neural synchrony in theta band during conflict-inducing incongruous trials. These results confirm that theta oscillations are fundamental to long-range synchronization needed for integrating top-down influences during cognitive control. MEG reflects neural activity directly, which makes it suitable for pharmacological manipulations in contrast to fMRI that is sensitive to vasoactive confounds. In the present study, healthy social drinkers were given a moderate alcohol dose and placebo in a within-subject design. Acute intoxication attenuated theta power to Stroop conflict and dysregulated co-oscillations between the ACC and latPFC, confirming that alcohol is detrimental to neural synchrony subserving cognitive control. It interferes with goal-directed behavior that may result in deficient self-control, contributing to compulsive drinking. In sum, this method can provide insight into real-time interactions during cognitive processing and can characterize the selective sensitivity to pharmacological challenge across relevant neural networks.

This experiment uses an anatomically-constrained magnetoencephalography (aMEG) method to examine brain oscillatory dynamics and long-range functional synchrony during engagement of cognitive control as a function of acute alcohol intoxication.

Decision making relies on dynamic interactions of distributed, primarily frontal brain regions. Extensive evidence from functional magnetic resonance ...

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and smaller functional subgroups, it becomes paramount to record from projections and/or genetically-defined subpopulations of neurons. Fiber photometry is an accessible and powerful approach that can overcome these challenges. By combining optical and genetic methodologies, neural activity can be measured in deep brain structures by expressing genetically-encoded calcium indicators, which translate neural activity into an optical signal that can be easily measured. The current protocol details the components of a multi-fiber photometry system, how to access deep brain structures to deliver and collect light, a method to account for motion artifacts, and how to process and analyze fluorescent signals. The protocol details experimental considerations when performing single and dual color imaging, from either single or multiple implanted optic fibers.

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...