Application of Optogenetics Combined with Fiber Photometry Recording in the Regulation of Neural Circuits-visceral Functions by Electroacupuncture

Summary

The purpose of this experiment is to establish a novel approach for the simultaneous integration of optogenetics, intracerebral calcium signal recording, and gastric motility monitoring, offering a novel paradigm for real-time monitoring of the relationship between somatic stimulation and neural circuit-visceral function interactions, which is applied to acupuncture research.

Abstract

This protocol describes a technique for optogenetic manipulation of specific types of neurons and real-time recording of the effects of electroacupuncture at Zusanli (ST36) on nuclear calcium signals and gastric motility in mice under anesthesia in vivo. The exploration of the central mechanisms underlying the efficacy of acupuncture has been limited by technological constraints. However, the advancement of optogenetic and optical imaging technology has propelled the development of neuroscience. Fiber photometry recording, initially leveraging genetically encoded calcium indicators to visualize changes in calcium dynamics indicative of neuronal activity, stands out as a key technology for characterizing brain-behavior correlations in vivo. The application of optogenetics enables the genetic encoding of neurons, enabling their activation or inhibition in response to light stimulation. This capability facilitates the establishment of causal links between neural circuitry function and behavioral outcomes. Simultaneous fiber photometry, calcium recording, and optogenetic stimulation provide a method for real-time recording and manipulation of neuronal activity, serving as an effective approach to investigate the central mechanisms of acupuncture. Nonetheless, there remains a scarcity of studies documenting the concurrent utilization of fiber photometry calcium recording, optogenetics, and visceral function monitoring in acupuncture research. The results of this experiment show that EA-ST36 might regulate gastric motility related to PBN^Glu-NTS^Glu-DMV^ChAT circuit. Optogenetic stimulation of this circuit produces the same effect. After the superposition of the two, the changes in calcium signal and gastric motility reach their maximum. This study established a novel approach for the simultaneous integration of optogenetic, intracerebral calcium signal recording, and gastric motility monitoring, offering a novel paradigm for real-time monitoring of the relationship between somatic stimulation and neural circuit-visceral function interactions.

Introduction

In recent years, the proliferation of biomolecular fluorescent sensors has propelled the utilization of fiber photometry in molecular biology, facilitating in vivo recording¹. This advancement enables the real-time monitoring of neuronal activity alterations in the brain. The integration of optogenetics allows for neuronal manipulation, offering the advantage of immediately conducting optogenetics and optical fiber recording by simply inserting the optical fiber. Combined with visceral function recording, this method offers good temporal resolution and nuclei-level targeting to observe how acupuncture modulates visceral functions via n....

Protocol

All procedures followed the guidelines for laboratory animal care and use established by the National Institutes of Health and received approval from the Animal Care and Use Ethics Committee of the Institute of Acupuncture & Moxibustion, Chinese Academy of Medical Sciences. Adult male C57 mice weighing 25 g were employed for viral injection and optical fiber implantation. The animal experiment protocol strictly adhered to the requirements of the national standard "Guidelines for Ethical Review of Welfare of Laboratory Animals" (GB/T 35892-2018) and was approved by the Ethics Committee of the institution.

NOTE: Keep the environme....

Results

Viruses were injected into the parabrachial nucleus (PBN), nucleus tractus solitarius (NTS), and dorsal motor nucleus of the vagus (DMV) of mice, with a fiber implanted in DMV (Figure 1A-G). During the peak viral expression period, variations in intragastric pressure and calcium signals within the DMV were recorded following electroacupuncture at ST36 and optogenetic stimulation. The aim of this study is to observe the regulatory effects of electroacupuncture at ST36 and opt.......

Discussion

The central scientific inquiry of acupuncture pertains to the regulation of target organs through acupoint stimulation of autonomic nerves. The findings suggested by Q. Ma suggested that acupuncture's influence on remote organ function modulation arises from somatic autonomic reflexes that activate sympathetic and/or parasympathetic pathways, especially those related to gastrointestinal diseases and systemic inflammation¹⁸. Nonetheless, extant research has predominantly centered on peripheral .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Virus
rAAV-CaMKIIα-CRE-WPRE-hGH polyA (AAV2/9)	BrainVTA	Cat#PT-0020
rAAV-CaMKIIα-DIO-ChrimsonR-WPREs (AAV2/9)	BrainVTA	Cat#PT-12637
rAAV-ChAT-GCamp6s-WPRE-hGH-polyA (AAV2/9)	BrainVTA	Cat#PT-10973
Other
1 μL microinjector	Hamilton	80135
3M EPSP Single Bond Universal Adhesive	Dental World Official	41269
Acupuncture Needle	ZhongYanTaiHe	0.25/13s
CED Micro1401-4	CED(England)	1401-4
Dental cement/Room temperature hardened resin	YAMAHACHI DENTAL	N/A
Fine Forceps	RWD	F11028-13
Glass tube	World Precision Instruments	504949
HANS-200A	HANS Institute	HANS-200
Inper Signal (software)	Inper	N/A
Inper Studio(Venus)	Inper	B1502
Latex balloon	Harward Apparatus	BS4 73-2787
Multichannel optical fiber recording system(Vista Pro)	Inper	R5268
Neurolog digitimer Model	Digitimer	NL900D
Optical fiber ferrule	Inper	V8475
Spike 2(software)	CED(England)	N/A
Stereotaxic instruments (with coordinates calculator)	David Kopf Instruments	KOPF 942
Tribromoethanol	Sigma	T48402
tribromoethanol	Sigma	T48402