Visualizing Spatiotemporal Dynamics of Somatosensory Cortex Neurons During Electroacupuncture Using Two-Photon In Vivo Imaging

Summary

This protocol integrates cranial windows in Thy1-GCaMP6f transgenic mice, standardized electroacupuncture, and two-photon imaging to visualize millisecond-scale cortical ensemble dynamics during stimulation, revealing layer-specific plasticity.

Abstract

This protocol establishes a comprehensive experimental framework for investigating cortical circuit dynamics during EA (electroacupuncture) stimulation. The methodology integrates three key components: chronic cranial window implantation in Thy1-GCaMP6f transgenic mice, standardized EA stimulation at the ST36 acupoint using 2/100 Hz biphasic pulses, and high-resolution two-photon calcium imaging. This integrated approach enables real-time visualization of neural ensemble activity in the primary somatosensory (S1) cortex with millisecond temporal precision, capturing previously inaccessible details of network-level responses to neuromodulation. The technique successfully reveals distinct layer-specific activation patterns and longitudinal plasticity changes, providing critical insights into the cortical mechanisms underlying the effects of EA. By overcoming the fundamental spatiotemporal limitations inherent in conventional fMRI and electrophysiological techniques, this high-resolution platform offers unprecedented analytical capabilities for mapping dynamic neural circuits. The protocol's robust design and reproducible outcomes make it particularly valuable for optimizing targeted neuromodulation therapies and advancing our understanding of circuit-level plasticity in response to peripheral stimulation.

Introduction

Electroacupuncture, a modern adaptation of traditional acupuncture incorporating controlled electrical currents applied via needles at specific acupoints, is recognized for modulating neural circuits involved in pain processing, motor control, and autonomic function. Its effects are believed to involve complex signaling cascades initiated by peripheral sensory nerve activation (primarily Aδ and C fibers), leading to neurotransmitter release (e.g., opioids, serotonin, GABA) and subsequent modulation at spinal, brainstem, and cortical levels^1,2. Key structures implicated include somatosensory cortices. With....

Protocol

All experiments utilized 8-12-week-old transgenic Thy1-GCaMP6f mice¹⁸. All experimental procedures and animal care protocols were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and were approved by the Huazhong University of Science and Technology Animal Care and Use Committee (IACUC; Protocol Approval Number: 3603). Every effort was made to minimize the number of animals used and their suffering. A schematic of the experimental setup is shown in Figure 1.

1. Chronic cranial window implantation

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Results

Structural characterization of S1 cortex and baseline neural activity
We began by establishing a high-fidelity structural and functional baseline for our imaging experiments. Tissue morphology analysis revealed discernible structural differences between non-capplied (Figure 2A) and coverslip-applied (Figure 2B) cranial window preparations. While the application of a coverslip introduced minor compression artifacts (arrowheads,

Discussion

To bridge the critical gap in observing electroacupuncture's (EA) cortical effects in real-time, we present a comprehensive integrated protocol combining chronic cranial window implantation for optical access, standardized EA stimulation, and longitudinal two-photon calcium imaging targeted at the primary somatosensory (S1) cortex.

This integrated protocol offers several distinct advantages over traditional methods for studying EA's cortical effects. First, it provides unprecedented spatiotemp.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Carprofen	MCE	N/A	5 mg/kg for analgesia
Digital stereotaxic frame	RWD	Kopf Model 1900
Electroacupuncture stimulator	HANS LH202 (2/100 Hz biphasic pulses)	N/A	HANS LH202 (2/100 Hz biphasic pulses)
Isoflurane	RWD	R510-22-10	Medical grade, 100% oxygen carrier
Micro-drill system	RWD		0.5-mm carbide burr, saline-cooled
Povidone-iodine	MCE	HY-B2234	10% solution
ScanImage	ScanImage	https://scanimage.org/
Sterile saline	Beyuntian	ST341-500ml	0.9% NaCl
Surgical microscope	Zeiss	https://www.zeiss.com/meditec/en/products/surgical-microscopes.html	10-40× magnification
Thy1-GCaMP6f transgenic mice	Cygen	5523978	8-12 weeks old, male/female
Two-photon microscope	Nikon A1MP	Nikon A1MP	Ti:Sapphire laser (920 nm excitation)