A Dual-Color Fiber Photometry Method for Recording Astrocyte-Neuron Activity Across Multiple Brain Regions During Learning and Memory Behaviors

Summary

This protocol provides a comprehensive description of a method for the simultaneous recording of calcium signals from neurons and astrocytes across multiple memory-related brain regions and demonstrates its application during a learning and memory behavior paradigm.

Abstract

Learning and memory depend critically on the coordination between neurons and astrocytes across distributed brain regions. Astrocytes modulate synaptic transmission and neuronal excitability, thereby contributing to the encoding and consolidation of information within memory-related circuits. The intra- and inter-regional correlations between astrocytes and neurons are crucial for understanding memory and cognitive disorders. However, simultaneous monitoring of astrocytic and neuronal activity across multiple regions during memory behavior remains technically challenging. Here, a method is presented that facilitates the concurrent recording of these calcium signals from astrocytes and neurons in several memory-related regions. This strategy combines cell-type-specific dual-color genetically encoded calcium indicators (GECIs), a multi-channel fiber photometry system, and synchronized behavioral tracking to achieve real-time monitoring of neuronal and astrocytic activity in the medial prefrontal cortex (mPFC), hippocampal CA1 region, and medial entorhinal cortex (MEC). Representative results demonstrate coordinated calcium dynamics across regions and cell types during the object recognition task. This method provides a reproducible and efficient experimental framework for investigating astrocyte–neuron interactions underlying learning and memory processes.

Introduction

Learning and memory are fundamental cognitive processes that involve acquiring, storing, and retrieving information, which are crucial for the formation of adaptive behaviors. These processes underpin a variety of complex functions, such as spatial navigation, object recognition, contextual association, and decision-making. In addition, cognitive dysfunctional diseases, including Alzheimer's disease, age-related cognitive disease, and traumatic brain injury, exhibit abnormalities in memory formation^1,2,3. Therefore, a comprehensive understanding of the neural basis of lea....

Protocol

All animal experiments were approved by the Animal Care and Use Committee of the Third Military Medical University (Animal Ethics Approval No.: AMUWEC20230126) and were conducted in accordance with the Third Military Medical University Guide for the Care and Use of Laboratory Animals to ensure the ethical treatment of animals. The reagents and the equipment used are listed in the Table of Materials.

To ensure the safety of the experimenter, all experimental procedures involving live animals and viral vectors were performed while wearing protective clothing, sterile gloves, and a mask. All instruments were thoroughly sterili....

Results

Based on the protocol described above, dual-color multi-site fiber recording was performed targeting the mPFC, CA1, and MEC of 4-month-old C57 mice. In the present study, we implemented a two-step viral injection strategy to sequentially introduce the two indicators. Under our experimental conditions, this strategy can be reliably and stably implemented, and the resulting expression pattern is consistent with the expected cell-type-specific promoter profile.

On Day 0, AAV5-GfaABC1D-GCaMP6f was.......

Discussion

In this study, a method was developed for the simultaneous recording of astrocytic and neuronal calcium activity in multiple memory-relevant brain areas of mice during learning and memory-related behaviors. Different from single-region imaging²⁸ or electrophysiological techniques, multi-region synchronous recording captures coordinated dynamics across distributed memory networks, thus proving a powerful tool for investigating learning and memory^9,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
75 RN 5uL SYR W/O NEEDLE	Hamilton	7634-01	Connects to micropipette for viral injection
AAV5-GfaABC1D-GCaMP6f	Addgene	52925	Astrocyte-specific GCaMP6f expression
AAV9-hSyn-NES-jRGECO1a	Addgene	100854	Neuron-specific red-shifted calcium indicator
Bepanthen Eye and Nasal Ointment	Bepanthen	MA0008	Prevents corneal drying
Bundled optical fibers with ceramic sleeves	Inper	BFO-1x3-W1.25	Connects implanted fibers to the photometry system
Camera	Mind Vision	MV-SUA502C-T	Behavioral videos synchronized with calcium signals
Dental cement	NISSIN	Super-Bond C&B	Used to secure ferrules to the skull
Ferrule holder	Inper	SCH-1.25	Holds the ceramic ferrule securely during implantation
Isoflurane vaporizer & Anesthesia system	RWD Life Science	R510-29	For anesthesia induction and maintenance
Multi-channel fiber photometry system	RWD Life Science	R821	For dual-color recording
OFRS software	RWD Life Science		Analyze recorded calcium signals
Optical fibers	RWD Life Science	R-FOC-BL200C-39N	Implanted in the target brain region
Stereotaxic instrument	RWD Life Science	68801	Used for precise viral injection and fiber implantation
Syringe pump	KD Scientific	788130	Delivers virus at controlled rates
Tissue glue	3M Animal Care Products	1469SB Vetbond	For incision closure