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, b...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" style="width:971px;" width="971">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Electrode holder</td>
			<td style="width:140px;">Molecular Device</td>
			<td style="width:393px;">1-HL-U</td>
			<td style="width:191px;">pipette holder for microelectrode amplifier</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ceramic split mating sleeve</td>
			<td>Thorlabs</td>
			<td>ADAF1</td>
			<td>f2.5 mm ferrule</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Circuit board spacer</td>
			<td>Teishin Denki</td>
			<td>SPA-320</td>
			<td>f8.0 mm, 20.0 mm long</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Stereotaxic frame for mice</td>
			<td>Narishige</td>
			<td>SR-6M-HT</td>
			<td>Stereotaxic instruments for mice</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Manipulator</td>
			<td>Narishige</td>
			<td>NA</td>
			<td>Manual manipulator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Superbond</td>
			<td>Sun Medical</td>
			<td>M: 204610557</td>
			<td>Dental adhesive resin cement</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Form2</td>
			<td>Formlabs</td>
			<td>NA</td>
			<td>3D printer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Kwik-Sil</td>
			<td>WPI</td>
			<td>KWIK-SIL</td>
			<td>Low toxicity silicone adhesive</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">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 height="40">
			<td height="40" style="height:40px;">LED driver</td>
			<td>Doric Lenses</td>
			<td>LEDRV_1CH_1000</td>
			<td>1 ch LED driver, maximum output = 1000 mA</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electrode puller</td>
			<td>Narishige</td>
			<td>PB-7</td>
			<td>Dual-stage glass micropipette puller</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Borosilicate glass capillary</td>
			<td style="width:140px;">Narishige</td>
			<td style="width:393px;">GD-1.5</td>
			<td style="width:191px;">Bolosilicate glass capillary, OD, 1.5mm, ID, 0.9 mm, 90.0 mm long</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">GENTACIN</td>
			<td>MSD CO., Ltd</td>
			<td>185711173</td>
			<td>Antibiotic ointment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Terramycin&#174;-LA</td>
			<td>Zoetis</td>
			<td>G 333</td>
			<td>Oxytetracycline</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tg(Slc32a1- COP4*H134R/EYFP)8Gfng/J</td>
			<td>Jackson Labs</td>
			<td>#14548</td>
			<td>VGAT-ChR2 mice</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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

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 &#177; 2 mm when the tip size varied, while the tip size was set to 2.5 &#181;m when the full length varied. The shank of the glass pipette was set t...

Watch this Scientific Journal Video about Optogenetics Identification of a Neuronal Type with a Glass Optrode in Awake Mice at JoVE.com

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

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 ...

This method can help answer key questions in the neuroscience field, such as the functions of different types of neurons. The main advantage of this technique is that it makes it possible to reliably identify the different types of neurons in vivo with inexpensive and accessible materials. I will be demonstrating the procedure together with Shinji Muramoto, our technician, and Lanlan Ma, our assistant professor from our laboratory.To begin this procedure, gently pull out the steel tube for pressure control from the barrel of the holder. Broaden the hole of the pin seat side in the barrel of the holder by drilling. Then, place a stainless steel pipe in the hole.Next, insert a ceramic split mating sleeve for 2.5 millimeter diameter ferrule into the hole. Make a hole in an L-shaped rabbit and attach the barrel of the electrode holder to the rabbit with epoxy adhesive. Subsequently, attach it to a manipulator with screws.Prepare a custom-built head post by making the columnar circuit board spacer into a cuboidal shape with a milling cutter, and cut it into two head posts with a saw. Then, flatten the bottom of the head post with a milling cutter. Before the surgery, sterilize all the surgical instruments and a head post with an autoclave.Clean the stereotaxic frame and the heating pad with 70 percent alcohol. Next, anesthetize the mouse and place it in the stereotaxic frame on a heating pad. In this experiment, a transgenic mouse in which the inhibitory neurons expressing channelrhodopsin-II is used.Place the teeth in the hole of the bite bar and lightly tighten the nose clamp. Subsequently, 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. Trim the hair over the head with small scissors and clean the scalp with a cotton swab of chlorhexidine gluconate. Then, cut the scalp along the midline and push it aside.Remove the periosteum with a cotton swab dipped in 70 percent alcohol and let the skull dry. Then, attach the head post to the manipulator. Position the head post flat on the skull around the bregma using the manipulator.Mix four drops of the monomer, one drop of catalyst, and one small small spoon of polymer on a small dish that is chilled in the refrigerator. Apply dental cement on the frontal and parietal bone and fix the head post to the skull. After the dental cement has hardened, remove the mouse from the stereotaxic frame.Apply an antibiotic ointment on the exposed skin and inject a cocktail of medetomidine antagonist and antibiotic. Then, place the mouse on the heat pad in a cage until it awakes and return it to a housing cage with enrichments. To perform craniotomy over the target brain region for recording, clean the skull with a cotton swab soaked with chlorhexidine gluconate and 70 percent alcohol.Carefully scrape the skull over the recording site with a dental drill. When the bone is thin enough, cut it with the tip of the scalpel and remove it. Then apply dental cement around the exposed region in order to strengthen the skull.Cover the exposed brain area with silicone adhesive after the dental cement has hardened. Then, place the animal on the heat pad in a cage until it awakes, and return it to a housing cage. Allow the animal to recover for at least one day after craniotomy, before performing the electrophysiological recording.Before performing the electrophysiological recording, make some glass pipettes from borosilicate glass capillaries using an electrode puller. Fill the pipettes with 10 millimolar PBS. In some recordings, two percent neurobiotin is added to the 10 millimolar PBS.Next, place the animal in the recording chamber. Attach a filled glass pipette to the custom-made optrode holder. Insert a silver chloride coated wire into the glass pipette.Afterward, connect the pin to the head stage of the recording amplifier. Subsequently, connect the fiber-optic patch cord with a zirconia ferrule 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 from LED, which is controlled by a single-channel LED driver. Afterward, remove the silicone adhesive over the craniotomy. Apply warm saline on the brain surface periodically to prevent the brain surface from drying during the recording session.If necessary, remove the dura with sharp tweezers. Here, the spike responses were recorded in the inferior colliculus of the awake VGAT channelrhodopsin-II mice. In the inferior colliculus of the VGAT channelrhodopsin-II mice, the GABAergic neurons specifically express channelrhodopsin-II.In the awake mice, both the GABAergic and glutamatergic neurons were excited or suppressed respectively when they were given light stimuli. When the light stimulus is delivered through the glass electrode, it evoked spike responses in some neurons. In contrast, in other neurons, the light stimuli suppressed the spike responses evoked by sound stimuli.These results show that a glass optrode can record the well-isolated single unit activity and reliably stimulate the recorded neurons by light. After the development, this technique paved the way for researchers in the field of neuroscience to explore how different types of neurons work in the neural socket of various brain regions.

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Neuroscience

6.7K vues.  Kanazawa Medical University. This method can help answer key questions in the neuroscience field, such as the functions of different types of neurons. The main advantage of this technique is that it makes it possible to reliably identify the different types of neurons in vivo with inexpensive and accessible materials. I will be demonstrating the procedure together with Shinji Muramoto, our technician, and Lanlan Ma, our assistant professor from our laboratory.To begin this procedure, gently pull out the steel tube...