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 learning and memory requires elucidating the dynamic interplay among different cell types. This interplay occurs within and across these regions during the encoding and retrieval of information.

Traditionally, memory formation was thought to be predominantly determined by neuronal activity. However, extensive research has demonstrated that astrocytes significantly regulate information encoding, consolidation, and retrieval. They achieve this by modulating synaptic transmission, regulating neuronal excitability, releasing gliotransmitters, and providing metabolic support^4,5,6,7,8. Astrocytes dynamically interact with neurons, and the bidirectional communication is collectively referred to as the "astrocyte-neuron network"^9,10,11. Therefore, understanding the coordinated interactions between astrocytes and neurons is crucial for understanding the cellular and network mechanisms of learning and memory.

Learning and memory depend on the dynamic integration of information across multiple brain regions. The interactions between astrocytes and neurons are not confined to local microcircuits but extend across distributed brain regions implicated in memory, including the medial prefrontal cortex (mPFC), hippocampal CA1 region, and medial entorhinal cortex (MEC). The mPFC has been implicated in memory consolidation and long-term memory regulation; CA1 is central to episodic memory encoding and reactivation; and MEC serves as a hub for spatial and multimodal information integration, providing essential input to the hippocampal–cortical circuit^{12,13,14,15,16}. Accumulating evidences suggest that astrocytes in these regions participate in coordinated cross-regional dynamics with neurons, playing key roles in information flow and state transitions during learning and memory^17,18.

Although recognition of the synergistic function of astrocytes and neurons in memory processing has increased, a significant technical challenge remains in simultaneously monitoring calcium activity in both astrocytes and neurons across multiple memory-associated brain regions during the behavior of freely moving animals. Two-photon imaging offers high spatial resolution imaging but is restricted by depth and head fixation^19,20; multi-channel electrophysiology enables high-temporal-resolution recordings but lacks cell-type specificity and cannot distinguish astrocytic calcium signals. Thus, a method was developed that enables simultaneous recording of astrocytic and neuronal calcium signals across multiple memory-related brain regions in freely moving mice. Genetically encoded calcium indicators (GECIs) report intracellular Ca²⁺ dynamics through Ca²⁺-dependent conformational changes in CaM/M13 that modulate the fluorescence of genetically encoded fluorophores. Dual-color GECI approaches leverage spectrally separable indicators, such as green and red calcium sensors, together with cell-type-specific promoters to enable simultaneous recording of astrocytic and neuronal Ca²⁺ activity within the same brain region. By combining cell-type-specific dual-color GECIs (GCaMP6f and jRGECO1a) and a multi-channel fiber photometry system, real-time monitoring of calcium activity in neurons and astrocytes in mPFC, CA1, and MEC has been achieved. Furthermore, taking the novel object recognition (NOR) task as an example, coordinated calcium dynamics across brain regions and cell types in learning tasks have been demonstrated. This method is a reproducible, reliable, and scalable protocol for investigating astrocyte-neuron ensemble activity across brain regions during learning and memory.

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 sterilized before use, and experimental surfaces were cleaned with 70% ethanol before and after each experiment. Remaining materials were disposed of in accordance with institutional waste disposal regulations. All reagents and equipment were handled to avoid direct contact with skin or eyes.

1. Animal preparation and viral handling

1. House 4-month-old C57BL/6 mice (both sexes) under a 12 h light/dark cycle with ad libitum access to food and water.
2. Thaw aliquots of AAV5-GfaABC1D-GCaMP6f or AAV9-hSyn-jRGECO1a on ice at 0–4 °C immediately prior to use.
3. Connect a pulled glass micropipette (tip diameter 15–25 μm) to a syringe and mount it on a microinjection pump.
4. Place the mouse on a sterile surgical platform and disinfect all instruments with 70% ethanol.

2. Stereotaxic viral injection

NOTE: A two-step virus injection strategy was used in this study. Under the current 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.

1. First surgery: AAV5-GfaABC1D-GCaMP6f (Titer: 7 × 10¹² vg/mL) injection for astrocytes
   1. Induce anesthesia with 3% isoflurane in a chamber, then maintain anesthesia with 0.8%–1.2% isoflurane via a nose cone (following institutionally approved protocols).
   2. Maintain body temperature at 37–38 °C using a heating pad and apply ophthalmic ointment to prevent corneal desiccation.
   3. Remove the scalp hair and disinfect the skin thoroughly. Make a midline incision to reveal the skull surface.
   4. Place the head in the stereotaxic instrument, adjust the bregma and lambda sutures to the same horizontal plane, and further adjust the frame to ensure that points 2 mm lateral to the bregma on both sides are level.
   5. Identify stereotaxic coordinates using a mouse brain atlas, with bregma as the reference point.
      mPFC: AP +1.8 mm, ML +0.3 mm, DV −3.0 mm
      CA1: AP −2.1 mm, ML +1.6 mm, DV −1.2 mm
      MEC: AP −4.5 mm, ML −2.9 mm, DV −2.0 mm
   6. Drill a 0.6-mm (diameter) circle craniotomy above each site, keep the dura mater moist and intact after opening the skull.
   7. Fill the GCaMP6f virus into the syringe at 5 nL/s and install the syringe into the stereotaxic arm.
   8. Lower the pipette slowly, inject 150 nL at 0.6 nL/s, hold for 8 min, then withdraw at 0.1 mm/s. Repeat this procedure for all regions.
   9. Seal the incision with tissue adhesive and allow 2 weeks for viral expression.
2. Secondary surgery: AAV9-hSyn-jRGECO1a (Titer: 1 × 10¹³ vg/mL) injection for neurons.
   ​NOTE: The surgery was performed 14 days after the first virus injection.
   1. Induce and maintain anesthesia as described above (Step 2.1.1).
   2. Maintain body temperature and protect the eyes.
   3. Reopen the scalp along the midline and level the skull.
      NOTE: The skull was leveled by aligning the bregma and lambda in the same horizontal plane.
   4. Clear tissue debris at the same locations as the craniotomy windows from the first surgery, exposing the dura mater intact.
   5. Adjust coordinates using bregma as the reference point, perform injections strictly at the same coordinates and depths as described previously.
   6. Fill jRGECO1a virus at 5 nL/s and install the syringe.
   7. Inject 150 nL at 0.6 nL/s, hold for 8 min, and withdraw slowly. Repeat this procedure for all regions and allow 3 weeks for viral expression.

3. Multi-site fiber photometry probe implantation

NOTE: Perform immediately after jRGECO1a injection.

1. Install the optical fiber ferrule in a stereotaxic holder.
2. Position the fiber (1.25-mm ferrule, 200-μm core, NA 0.39) 100–150 μm above the viral injection depth:
   mPFC: AP +1.8 mm, ML +0.3 mm, DV −2.9 mm
   CA1: AP −2.1 mm, ML +1.6 mm, DV −1.1 mm
   MEC: AP −4.5 mm, ML −2.9 mm, DV −1.9 mm
3. Seal the craniotomy with tissue adhesive and secure the ferrule with dental cement.

4. Multi-channel fiber photometry recording

NOTE: To ensure stable and full expression of the two calcium indicators, multi-channel fiber optic photometry was performed three weeks after the second virus injection for recording.

1. Habituate the mice to the fiber system and arena once per day for three days.
   1. After 3 weeks of viral expression, briefly anesthetize the mouse with 3% isoflurane delivered via an induction chamber.
   2. Connect one end of the bundled optical fiber to the fiber photometry acquisition system.
   3. Set the excitation light to two wavelengths: 470 nm and 560 nm.
   4. Set the acquisition frequency to 30 Hz.
   5. Connect the three branches at the other end of the bundled fiber to three brain regions: Channel 1 to mPFC, Channel 2 to CA1, Channel 3 to MEC.
   6. Place the mice in an open area with a side length of 50 cm and allow free exploration.
   7. After 10 min, disconnect the fiber and bring the mice back to their home cages. 
2. Fiber photometry recording during the Novel Object Recognition Task
   NOTE: Clean the platform and objects with 75% ethanol before each trial.
   1. Place two identical objects in diagonally opposite quadrants.
   2. Connect the fiber bundle in a light anesthetized state.
   3. Allow the mice to explore objects for 10 min, then return them to home cages without removing the fiber.
   4. Perform fiber photometry recording during the novel object recognition test in mice.
      ​NOTE: Exclude the trial if motion artifacts are excessive for stable recording data.
      1. Adjust stimulation lasers:
         470 nm for GCaMP6f (astrocytes)
         560 nm for jRGECO1a (neurons)
      2. Record calcium signals at 30 Hz, synchronized with video tracking at 30 Hz. Position a laser diode within the video frame (outside the mouse’s visual field) upon initiating acquisition to serve as a temporal marker.
      3. Ten minutes after the learning phase, replace one object with a novel object and place the mouse back for 10 min of exploration while recording.
      4. Disconnect the fiber with ceramic ferrule and bring the mice back to home cages.

5. Data processing and analysis

NOTE: Classify a Ca^2⁺ transient in fiber photometry recordings as a genuine signal only if its amplitude exceeds three times the standard deviation of the noise band^21,22,23. Novel Object Recognition Test (NORT) Protocol²⁴:Define object interactions as events where mice make physical contact with the object, either by poking it or sniffing it with their noses. For signal quantification, define the baseline as the mean signal during the -2 to -1 s window (relative to interaction onset at t=0). Set the peri-event analysis window to span from 2 s before to 4 s after the initiation of each object interaction, following established protocols.

1. Import fluorescence traces into analysis software.
   NOTE: Fluorescence signal processing was performed using proprietary analysis software following standard fiber photometry workflows. The software itself is not open source and cannot be shared. For general methodological considerations regarding fiber photometry signal processing, see recent methodology reports²⁵.
2. Perform baseline correction and align calcium signals to the onset of the NOR task using synchronized video.
3. Convert ΔF/F to Z-scores for inter-region and dual-color comparisons⁹:
   
   
   NOTE:
   (ΔF/F)_mean: mean of ΔF/F.
   (ΔF/F)_std: standard deviation of ΔF/F.
   F_raw: raw fluorescence intensity.
   F_baseline: baseline fluorescence intensity (mean over predefined baseline period).​
4. Quantify metrics such as area under the curve (AUC) for astrocyte and neuronal calcium responses during novel vs. familiar object exploration.

6. Histological verification after recording

1. Anesthetize the mouse with an overdose of pentobarbital administered intraperitoneally until complete loss of reflexes is confirmed.
2. Perform transcardial perfusion with normal saline, followed by 4% paraformaldehyde (PFA).
3. Dissect the brain carefully and post-fix it in 4% PFA at 4 °C for 12–24 h.
4. Transfer the brain to 30% sucrose in PBS at 4 °C until the tissue sinks for cryoprotection.
5. Embed the brain in optimal cutting temperature compound, freeze it, and cut coronal sections (40 μm thickness) using a cryostat.
6. Perform immunofluorescence staining to verify viral expression and fiber placement.

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