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

Summary

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

Abstract

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'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.

Introduction

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 breakthrough^1,2. Using transgenic animals in which light-sensitive opsin (e.g., channelrhodopsin-2) is express....

Protocol

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).

1. Gently pull out the steel tube for pressure control from the barrel of the holder.
2. 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.
3. Put a stainless-steel p....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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