Name: Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice (Video) | JoVE Journal
Uploaded: 2018-06-28T14:30:00.000+00:00
Duration: 7 min 51 s
Description: Learn optogenetic single-unit recording in awake mice using a custom glass optrode. This method enables reliable identification of GABAergic and glutamatergic neurons in vivo with cost-effective materials.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The authors were supported by the Japan Society for the Promotion of Science KAKENHI Grant JP16K11200 and 17H02223, and the Grant for Research from Kanazawa Medical University S2016-8 and C2017-3. We thank Yuhichi Kuda for his support in taking the photos.

All procedures are done in accordance with the guiding principles of the Physiological Society of Japan and with the approval of the Animal Care Committee of Kanazawa Medical University.
1. Construction of the Glass Optrode Holder
NOTE: To build a glass optrode holder, a commercial electrode holder is used (Figure 1A).
<ol>
	<li>Gently pull out the steel tube for pressure control from the barrel of the holder.</li>
	<li>By drilling, broaden the hole of the pin seat side in the barrel of the holder. Make the hole 3 mm in diameter and 12 mm in depth.</li>
	<li>Put a stainless-steel pipe (3 mm in diameter, 0.5 mm in thickness, and 8 mm in length; Figure 1A2) in the hole.</li>
	<li>Insert a ceramic split mating sleeve for &#8960;2.5 mm ferrules (Figure 1A1) into the hole.</li>
	<li>Fix the barrel of the electrode holder to an L-shaped rabbet with epoxy adhesive.</li>
	<li>Make the holes (3 mm in diameter) in the rabbet and attach it to a manipulator with screws.</li>
</ol>
2. Head Post Installation
<ol>
	<li>Prepare a custom-built head post made of a circuit board spacer. Make the columnar circuit board spacer into a cuboidal shape with a milling cutter and cut it into 2 head posts with a saw (Figure 1B). Flatten the bottom of the head post with a milling cutter.</li>
	<li>Prepare the transgenic animal in which a light-sensitive opsin is expressed in a specific neuron type for the study. In this work, the transgenic mouse (VGAT-ChR2 mice) in which the inhibitory neurons express channelrhodopsin-2 (ChR2)11 is used.</li>
	<li>Anesthetize the animal with a mixture of 0.3 mg/kg of medetomidine, 4.0 mg/kg of midazolam, and 5.0 mg/kg of butorphanol by intraperitoneal administration. To confirm that the animal is fully anesthetized, test its reactions to tail pinching.</li>
	<li>Place the mouse in a stereotaxic frame on a heating pad. Clean the stereotaxic frame and the heating pad with 70% alcohol in advance. Place the teeth in the hole of the bite bar, and lightly tighten the nose clamp. Fix both sides of the head using ear bars. When the head is correctly fixed by the ear bars, tighten the nose clamp. Apply an ophthalmic ointment to the eyes.</li>
	<li>Shave the head skin with small scissors and clean the scalp with a cotton swab of chlorhexidine gluconate. Before the surgery, sterilize all the surgical instruments and a head post with an autoclave (121 &#176;C, 15 min).</li>
	<li>Cut the scalp along the midline with scissors and push it aside. Make the incision from the back of the head to the eyes (approximately 20 mm). Remove the periosteum with a cotton swab dipped in 70% alcohol and dry the skull.</li>
	<li>Attach the head post to the manipulator. Position the head post flat on the skull around the bregma using the manipulator.</li>
	<li>Mix the monomer (4 drops), catalyst (1 drop), and polymer (1 small spoon) of dental cement on a small dish that is chilled in the refrigerator in advance. Put the dental cement (below 1 g) on the frontal and parietal bone of the skull to fix the head post to the skull.</li>
	<li>After the dental cement becomes hard, remove the mouse from the stereotaxic frame. Put an antibiotic ointment on the exposed skin. Inject the medetomidine antagonist (0.3 mg/kg) and the antibiotics (oxytetracycline; 20 mg/kg). Place and monitor the mouse on the heat pad in a cage until it awakes, and return it to a housing cage.</li>
	<li>House the animal in an individual housing cage with some enrichment device. Change the animal paper bedding daily to keep the cage clean and prevent infection. Carefully monitor whether the animal shows any sign of discomfort. Apply lidocaine to the wound if any pain is suspected.&#160;Administer meloxicam or carprofen once every 24 h for 48 h after both the head post installation and craniotomy surgeries. The local lidocaine can be given in addition if the NSAID is not adequate.</li>
</ol>
3. Acclimation
<ol>
	<li>Two days (minimum) after the recovery from the anesthesia, acclimate the animals to the head-fixed state in the recording chamber. Perform the acclimation sessions for at least 5 days.</li>
	<li>During the acclimation sessions, place the animal in the recording chamber with the head post fixed to a custom-built clamp. During the session, in order to limit movement, cover the body with a custom-made resin half tube (Figure 1C), made using a 3D printer. 
	NOTE: On day 1, the acclimation session should be 5 min long. It is then increased daily to 10, 20, 40, and 60 min for days 2, 3, 4, and 5, respectively.</li>
	<li>At the end of each session, give the animal a food reward. If the animal struggles continuously during the acclimation, stop the session.</li>
</ol>
4. Craniotomy
NOTE: After the acclimation, a craniotomy is made over the brain region for recording. The craniotomy is performed in the stereotaxic frame under anesthesia, as with the head post installation. The post-operation procedure is the same as the head post installation.&#160;
<ol>
	<li>Before the craniotomy, clean the skull with a cotton swab with chlorhexidine gluconate and 70% alcohol.</li>
	<li>Carefully scrape the skull over the recording site with a dental drill. When the bone becomes thin enough, cut it with the tip of the scalpel and remove it.</li>
	<li>Put dental cement around the exposed region in order to strengthen the skull.</li>
	<li>Cover the exposed brain area with silicone adhesive after the dental cement becomes hard. The post-surgery procedure is as described in step 2.8.</li>
</ol>
5. Electrophysiological Recording
<ol>
	<li>Allow the animal to recover for at least 1 day after the craniotomy before performing the electrophysiological recording. Place the animal in the recording chamber in the same way it was done during the acclimation sessions when the recording starts.</li>
	<li>Make glass pipettes from borosilicate glass capillaries (OD = 1.5 mm, ID = 0.9 mm, 90.0 mm long) pulled on an electrode puller. Their tip diameter is 2&#8211;3 &#181;m, and their resistance is 4&#8211;6 M&#937; when they are filled with 10 mM PBS. The length of the electrode is 40&#8211;50 mm. In some recordings, 2% neurobiotin is added to the 10 mM PBS.</li>
	<li>Attach the filled glass pipette to the custom-made optrode holder.</li>
	<li>Put the Ag-Cl coated silver wire (0.2 mm in diameter) into the glass pipette. Connect the silver wire to a pin via a thin coated electric wire. Connect the pin to the headstage of the recording amplifier.</li>
	<li>Connect the fiber-optic patch cord (core = 960 &#181;m, cladding = 1,000 &#181;m, NA = 0.63) with a zirconia ferrule (OD = 2.5 mm) to the split mating sleeve on the optrode holder. Push the patch cord into the sleeve until the tip of the ferrule contacts the back of the glass pipette. The patch cord delivers the light (wavelength = 465 nm) from LED. The LED light is controlled by a single-channel LED driver.</li>
	<li>Remove the silicone adhesive over the craniotomy. Put warmed saline (38 &#176;C) on the brain surface periodically to prevent the brain surface from drying during the recording session. If necessary, remove the dura with sharp tweezers.</li>
	<li>Insert the glass electrode into the brain tissue with a manipulator. Monitor the electrode resistance when it is inserted. Replace the electrode when the resistance is lower than expected because the tip of the electrode may be damaged. At the depth of interest, search and isolate the single unit spikes by adjusting the depth in 2 to 5 &#181;m steps.</li>
	<li>After the single unit is isolated, deliver the LED light and observe the response to the light stimuli.</li>
	<li>Record the neural activities after the identification of the cell types. Perform the recordings for 2&#8211;3 h. If the recording is made on consecutive days (maximum of 2 days), cover the craniotomy with silicone adhesive. When the recording is finished, and the animal is sacrificed, retrieve the head post and put it in acetone to wash and reuse it.</li>
</ol>

<ol><li>Rajasethupathy, P., Ferenczi, E., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+neural+circuits%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Targeting neural circuits.</a> Cell. 165 (3), 524-534 (2016).</li>
<li>Gore, F., Schwartz, E. C., Salzman, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Manipulating+neural+activity+in+physiologically+classified+neurons%3A+triumphs+and+challenges%5BTitle%5D)+AND+%22Philosophical+Transactions+of+the+Royal+Society+of+London.+Series+B%2C+Biological+Sciences%22%5BJournal%5D)">Manipulating neural activity in physiologically classified neurons: triumphs and challenges.</a> Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 370 (1677), 20140216(2015).</li>
<li>Ono, M., Bishop, D. C., Oliver, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identified+GABAergic+and+glutamatergic+neurons+in+the+mouse+inferior+colliculus+share+similar+response+properties%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Identified GABAergic and glutamatergic neurons in the mouse inferior colliculus share similar response properties.</a> Journal of Neuroscience. 37 (37), 8952-8964 (2017).</li>
<li>Ono, M., Bishop, D. C., Oliver, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-lasting+sound-evoked+afterdischarge+in+the+auditory+midbrain%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Long-lasting sound-evoked afterdischarge in the auditory midbrain.</a> Scientific Reports. 6, 20757(2016).</li>
<li>Munoz, W., Tremblay, R., Rudy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Channelrhodopsin-assisted+patching%3A+in+vivo+recording+of+genetically+and+morphologically+identified+neurons+throughout+the+brain%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Channelrhodopsin-assisted patching: in vivo recording of genetically and morphologically identified neurons throughout the brain.</a> Cell Reports. 9 (6), 2304-2316 (2014).</li>
<li>Lima, S. Q., Hromadka, T., Znamenskiy, P., Zador, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PINP%3A+a+new+method+of+tagging+neuronal+populations+for+identification+during+in+vivo+electrophysiological+recording%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">PINP: a new method of tagging neuronal populations for identification during in vivo electrophysiological recording.</a> PLoS One. 4 (7), e6099(2009).</li>
<li>Kuwada, S., Batra, R., Stanford, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monaural+and+binaural+response+properties+of+neurons+in+the+inferior+colliculus+of+the+rabbit%3A+effects+of+sodium+pentobarbital%5BTitle%5D)+AND+%22Journal+of+Neurophysiology%22%5BJournal%5D)">Monaural and binaural response properties of neurons in the inferior colliculus of the rabbit: effects of sodium pentobarbital.</a> Journal of Neurophysiology. 61 (2), 269-282 (1989).</li>
<li>Populin, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anesthetics+change+the+excitation%2Finhibition+balance+that+governs+sensory+processing+in+the+cat+superior+colliculus%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Anesthetics change the excitation/inhibition balance that governs sensory processing in the cat superior colliculus.</a> Journal of Neuroscience. 25 (25), 5903-5914 (2005).</li>
<li>Duque, D., Malmierca, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stimulus-specific+adaptation+in+the+inferior+colliculus+of+the+mouse%3A+anesthesia+and+spontaneous+activity+effects%5BTitle%5D)+AND+%22Brain+Structure+and+Function%22%5BJournal%5D)">Stimulus-specific adaptation in the inferior colliculus of the mouse: anesthesia and spontaneous activity effects.</a> Brain Structure and Function. 220 (6), 3385-3398 (2015).</li>
<li>Cai, R., Richardson, B. D., Caspary, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Responses+to+predictable+versus+random+temporally+complex+stimuli+from+single+units+in+auditory+thalamus%3A+impact+of+aging+and+anesthesia%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Responses to predictable versus random temporally complex stimuli from single units in auditory thalamus: impact of aging and anesthesia.</a> Journal of Neuroscience. 36 (41), 10696-10706 (2016).</li>
<li>Zhao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+type-specific+channelrhodopsin-2+transgenic+mice+for+optogenetic+dissection+of+neural+circuitry+function%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function.</a> Nature Methods. 8 (9), 745-752 (2011).</li>
<li>Ito, T., Bishop, D. C., Oliver, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two+classes+of+GABAergic+neurons+in+the+inferior+colliculus%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Two classes of GABAergic neurons in the inferior colliculus.</a> Journal of Neuroscience. 29 (44), 13860-13869 (2009).</li>
<li>Ono, M., Yanagawa, Y., Koyano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GABAergic+neurons+in+inferior+colliculus+of+the+GAD67-GFP+knock-in+mouse%3A+electrophysiological+and+morphological+properties%5BTitle%5D)+AND+%22Neuroscience+Research%22%5BJournal%5D)">GABAergic neurons in inferior colliculus of the GAD67-GFP knock-in mouse: electrophysiological and morphological properties.</a> Neuroscience Research. 51 (4), 475-492 (2005).</li>
<li>Chen, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+neural+activity+using+Thy1-GCaMP+transgenic+mice%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Imaging neural activity using Thy1-GCaMP transgenic mice.</a> Neuron. 76 (2), 297-308 (2012).</li>
<li>Hirai, Y., Nishino, E., Ohmori, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+recording+of+fluorescence+and+electrical+signals+by+photometric+patch+electrode+in+deep+brain+regions+in+vivo%5BTitle%5D)+AND+%22Journal+of+Neurophysiology%22%5BJournal%5D)">Simultaneous recording of fluorescence and electrical signals by photometric patch electrode in deep brain regions in vivo.</a> Journal of Neurophysiology. 113 (10), 3930-3942 (2015).</li>
<li>LeChasseur, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+microprobe+for+parallel+optical+and+electrical+recordings+from+single+neurons+in+vivo%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">A microprobe for parallel optical and electrical recordings from single neurons in vivo.</a> Nature Methods. 8 (4), 319-325 (2011).</li>
<li>Abaya, T. V., Blair, S., Tathireddy, P., Rieth, L., Solzbacher, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+3D+glass+optrode+array+for+optical+neural+stimulation%5BTitle%5D)+AND+%22Biomedical+Optics+Express%22%5BJournal%5D)">A 3D glass optrode array for optical neural stimulation.</a> Biomedical Optics Express. 3 (12), 3087-3104 (2012).</li>
<li>Bittner, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conjunctive+input+processing+drives+feature+selectivity+in+hippocampal+CA1+neurons%5BTitle%5D)+AND+%22Nature+Neuroscience%22%5BJournal%5D)">Conjunctive input processing drives feature selectivity in hippocampal CA1 neurons.</a> Nature Neuroscience. 18 (8), 1133-1142 (2015).</li>
</ol>

The authors have nothing to disclose.

Optogenetics has become a powerful tool in neuroscience. It has been utilized for identifying specific neuron types in vivo as well as manipulating the activities of specific neuronal pathways. The clarification of the neural activities of different neuronal types promotes the understanding of the mechanism of the neural circuits. Here, we demonstrated a method to deliver the light to the recording site through a glass electrode in the IC of awake VGAT-ChR2 mice.
There are several cri...

The central nervous system consists of various types of neurons, which have different functions. How these different types of neurons work within the neural circuit is one of the major concerns in neuroscience. However, in many brain regions, it has been impossible to distinguish the neuronal types in in vivo recordings of electrical activities because there is no clear difference in the electrical spike signal itself, with some exceptions. Recent advances in optogenetics have made a breakthrough1,2. Using transgenic animals in which light-sensitive opsin (e.g., channelrhodopsin-2) is expressed in specific neuronal types, it became possible to distinguish the neuronal types efficiently in in vivo recordings3,4,5,6. In these animals, the neurons with light-sensitive opsin are excited by giving light stimuli during the electrical recordings, but other neurons are not. The opsin-positive neurons, therefore, are easily distinguished from other neuron types by their responses to light.
In optogenetics experiments, it is critical to deliver the light to the recording site. As a non-invasive method, the light is often directed from the brain&#39;s surface. However, because the light&#39;s strength reduces as it goes through brain tissue, it is hard to stimulate the deep brain regions from the brain&#39;s surface. Especially, it is difficult for the stimulation light to reach the deep brain regions when the optical transparency of the brain surface is low, as is often the case with recordings from awake animals. Electrophysiological experiments have often been performed on anesthetized animals because the body movement causes noise in the recordings. As is well documented, however, anesthesia is known to change the neural responses7,8,9,10. Thus, it is necessary to use awake animals in order to study neural responses without the artificial effects of anesthesia. Unlike the experiments with anesthetized animals, the electrophysiological recordings are performed after the recovery from surgery in the experiments with awake animals. During the interval between the surgery and the recordings, the tissue exudate often accumulates on the brain surface and makes the optical transparency of the brain surface low.
Here, we describe a method to record single-unit recordings from an awake mouse using a custom-made glass optrode. In this method, the light is delivered through the recording glass electrode so that it is possible to reliably stimulate the recorded neuron with light in deep brain regions. This custom-made optrode system consists of accessible and inexpensive materials and is easy to assemble.

<table><tbody><tr><td>Electrode holder</td><td>Molecular Device</td><td>1-HL-U</td><td>pipette holder for microelectrode amplifier</td></tr><tr><td>Ceramic split mating sleeve</td><td>Thorlabs</td><td>ADAF1</td><td>f2.5 mm ferrule</td></tr><tr><td>Circuit board spacer</td><td>Teishin Denki</td><td>SPA-320</td><td>f8.0 mm, 20.0 mm long</td></tr><tr><td>Stereotaxic frame for mice</td><td>Narishige</td><td>SR-6M-HT</td><td>Stereotaxic instruments for mice</td></tr><tr><td>Manipulator</td><td>Narishige</td><td>NA</td><td>Manual manipulator</td></tr><tr><td>Superbond</td><td>Sun Medical</td><td>M: 204610557</td><td>Dental adhesive resin cement</td></tr><tr><td>Form2</td><td>Formlabs</td><td>NA</td><td>3D printer</td></tr><tr><td>Kwik-Sil</td><td>WPI</td><td>KWIK-SIL</td><td>Low toxicity silicone adhesive</td></tr><tr><td>Borosilicate glass capillaries</td><td>Narishige</td><td>GD-1.5</td><td>OD 1.5 mm, ID 0.9 mm, 90.0 mm long</td></tr><tr><td>Fiber-optic patch cord</td><td>Doric Lenses</td><td>MFP_960/1000/2200-0.63_1m_FCM-ZF2.5</td><td>Monofiberoptic patchcord, OD, 2.5 mm, core = 960 mm, cladding = 1000 mm, NA = 0.63</td></tr><tr><td>Connectrized LED</td><td>Doric Lenses</td><td>LEDC-1B_FC</td><td>Central wave length = 465 nm, output power = 45 mW (Core 960 mm 0.63 NA )</td></tr><tr><td>LED driver</td><td>Doric Lenses</td><td>LEDRV_1CH_1000</td><td>1 ch LED driver, maximum output = 1000 mA</td></tr><tr><td>Electrode puller</td><td>Narishige</td><td>PB-7</td><td>Dual-stage glass micropipette puller</td></tr><tr><td>Borosilicate glass capillary</td><td>Narishige</td><td>GD-1.5</td><td>Bolosilicate glass capillary, OD, 1.5mm, ID, 0.9 mm, 90.0 mm long</td></tr><tr><td>GENTACIN</td><td>MSD CO., Ltd</td><td>185711173</td><td>Antibiotic ointment</td></tr><tr><td>Terramycin&amp;reg;-LA</td><td>Zoetis</td><td>G 333</td><td>Oxytetracycline</td></tr><tr><td>Tg(Slc32a1- COP4*H134R/EYFP)8Gfng/J</td><td>Jackson Labs</td><td>#14548</td><td>VGAT-ChR2 mice</td></tr><tr><td>Multiclamp 700B</td><td>Molecular Devices</td><td>2500-0157</td><td>Microelectrode amplifier</td></tr></tbody></table>

optogenetics identification of a neuronal type with a glass optrode in awake mice

Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the identification of the neuronal type in in vivo electrophysiological experiments in broad brain regions. In optogenetics experiments, it is critical to deliver the light to the recording site. However, it is often hard to deliver the stimulation light to the deep brain regions from the brain&#39;s surface. Especially, it is difficult for the stimulation light to reach the deep brain regions when the optical transparency of the brain surface is low, as is often the case with recordings from awake animals. Here, we describe a method to record spike responses to the light from an awake mouse using a custom-made glass optrode. In this method, the light is delivered through the recording glass electrode so that it is possible to reliably stimulate the recorded neuron with light in the deep brain regions. This custom-made optrode system consists of accessible and inexpensive materials and is easy to assemble.

In Figure 2, we examined the effects of the tip size and the length of the glass pipettes on the light power at the tip of the pipettes (Figure 2A-B). The light power was measured by an optical power meter placed 1 mm away from the tip. The full length was set to 50 ± 2 mm when the tip size varied, while the tip size was set to 2.5 µm when the full length varied. The shank of the glass pipette was set to 8 mm. Th...

This work introduces a method to perform an optogenetic single-unit recording reliably from an awake mouse using a custom-made glass optrode.

It is a major concern in neuroscience how different types of neurons work in neural circuits. Recent advances in optogenetics have enabled the ...

optogenetics-identification-neuronal-type-with-glass-optrode-awake

Research

JoVE Journal

Neuroscience

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Source: Kodani, S., et.al. Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice. J. Vis. Exp. (2019)This video demonstrates the optogenetic manipulation of neural circuits involved in the sleep-to-wakefulness transition in mice. The procedure involves surgically implanting an optic fiber and electrodes into the brain and muscles to enable light stimulation and electrophysiological recording. By delivering light to activate light-sensitive cation...

Optogenetic Manipulation of Neural Circuits for the Sleep-to-Wakefulness Transition in Mice

This video demonstrates the brain's neural activity recording through an optical window in transgenic mice. The mice express a fluorescent protein that enables the detection of neural activities. A small portion of the skull is carefully removed to install an optical window, and the skull is then covered with black cement to block light before imaging.

Installation of Optical Windows in the Brain of Transgenic Mice to Image Neural Activities

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations in vitro.

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

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.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

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.

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

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Here we present a protocol describing viral transduction of discrete brain regions with optogenetic constructs to permit synapse-specific electrophysiological characterization in acute rodent brain slices.

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Here, we present the fabrication method of an optrode system with optical fibers for light delivery and an electrode array for neural recording. In vivo experiments with transgenic mice expressing channelrhodopsin-2 show the feasibility of the system for simultaneous optogenetic stimulation and electrophysiological recording.

Optrode Array for Simultaneous Optogenetic Modulation and Electrical Neural Recording

Learn optogenetic single-unit recording in awake mice using a custom glass optrode. This method enables reliable identification of GABAergic and glutamatergic neurons in vivo with cost-effective materials.

Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice

Explore More Videos