Name: A Dual-Color Fiber Photometry Method for Recording Astrocyte-Neuron Activity Across Multiple Brain Regions During Learning and Memory Behaviors (Video) | JoVE Journal
Uploaded: 2026-04-28T12:30:00.000+00:00
Duration: 9 min 24 s
Description: Learn dual-color fiber photometry to record astrocyte-neuron calcium signals across mPFC, CA1, and MEC during learning and memory behaviors. This method enables real-time monitoring of cell-type-specific activity.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

Ms. Jia Lou is acknowledged for technical assistance, and Sai Te Xin Si (AI tool) is acknowledged for language editing assistance. This work received support from the National Natural Science Foundation of China (grant no. 82371485, 82588301, 32400932) and the Natural Science Foundation of Chongqing, China (grant no. CSTB2024NSCQ-JQX0024, 2024NSCQ-MSX1104).

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
<ol>
	<li>House 4-month-old C57BL/6 mice (both sexes) under a 12 h light/dark cycle with ad libitum access to food and water.</li>
	<li>Thaw aliquots of AAV5-GfaABC1D-GCaMP6f or AAV9-hSyn-jRGECO1a on ice at 0–4 °C immediately prior to use.</li>
	<li>Connect a pulled glass micropipette (tip diameter 15–25 μm) to a syringe and mount it on a microinjection pump.</li>
	<li>Place the mouse on a sterile surgical platform and disinfect all instruments with 70% ethanol.</li>
</ol>
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.
<ol>
	<li>First surgery: AAV5-GfaABC1D-GCaMP6f (Titer: 7 × 1012 vg/mL) injection for astrocytes
	<ol>
		<li>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).</li>
		<li>Maintain body temperature at 37–38 °C using a heating pad and apply ophthalmic ointment to prevent corneal desiccation.</li>
		<li>Remove the scalp hair and disinfect the skin thoroughly. Make a midline incision to reveal the skull surface.</li>
		<li>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.</li>
		<li>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</li>
		<li>Drill a 0.6-mm (diameter) circle craniotomy above each site, keep the dura mater moist and intact after opening the skull.</li>
		<li>Fill the GCaMP6f virus into the syringe at 5 nL/s and install the syringe into the stereotaxic arm.</li>
		<li>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.</li>
		<li>Seal the incision with tissue adhesive and allow 2 weeks for viral expression.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Induce and maintain anesthesia as described above (Step 2.1.1).</li>
		<li>Maintain body temperature and protect the eyes.</li>
		<li>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.</li>
		<li>Clear tissue debris at the same locations as the craniotomy windows from the first surgery, exposing the dura mater intact.</li>
		<li>Adjust coordinates using bregma as the reference point, perform injections strictly at the same coordinates and depths as described previously.</li>
		<li>Fill jRGECO1a virus at 5 nL/s and install the syringe.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Multi-site fiber photometry probe implantation
NOTE: Perform immediately after jRGECO1a injection.
<ol>
	<li>Install the optical fiber ferrule in a stereotaxic holder.</li>
	<li>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</li>
	<li>Seal the craniotomy with tissue adhesive and secure the ferrule with dental cement.</li>
</ol>
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.
<ol>
	<li>Habituate the mice to the fiber system and arena once per day for three days.
	<ol>
		<li>After 3 weeks of viral expression, briefly anesthetize the mouse with 3% isoflurane delivered via an induction chamber.</li>
		<li>Connect one end of the bundled optical fiber to the fiber photometry acquisition system.</li>
		<li>Set the excitation light to two wavelengths: 470 nm and 560 nm.</li>
		<li>Set the acquisition frequency to 30 Hz.</li>
		<li>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.</li>
		<li>Place the mice in an open area with a side length of 50 cm and allow free exploration.</li>
		<li>After 10 min, disconnect the fiber and bring the mice back to their home cages. </li>
	</ol>
	</li>
	<li>Fiber photometry recording during the Novel Object Recognition Task 
	NOTE: Clean the platform and objects with 75% ethanol before each trial.
	<ol>
		<li>Place two identical objects in diagonally opposite quadrants.</li>
		<li>Connect the fiber bundle in a light anesthetized state.</li>
		<li>Allow the mice to explore objects for 10 min, then return them to home cages without removing the fiber.</li>
		<li>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.
		<ol>
			<li>Adjust stimulation lasers: 
			470 nm for GCaMP6f (astrocytes) 
			560 nm for jRGECO1a (neurons)</li>
			<li>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.</li>
			<li>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.</li>
			<li>Disconnect the fiber with ceramic ferrule and bring the mice back to home cages.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Data processing and analysis
NOTE: Classify a Ca2⁺ transient in fiber photometry recordings as a genuine signal only if its amplitude exceeds three times the standard deviation of the noise band21,22,23. 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.
<ol>
	<li>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.</li>
	<li>Perform baseline correction and align calcium signals to the onset of the NOR task using synchronized video.</li>
	<li>Convert ΔF/F to Z-scores for inter-region and dual-color comparisons9: 
	<img alt="Z-score calculation formula, showing standard deviation and mean adjustments in statistical data analysis." src="/files/ftp_upload/70582/70582eq1.jpg" style="margin: auto;" data-altupdatedat="1784001604000" data-alt="Equation 1"> 
	<img alt="ΔF/F calculation formula for fluorescence change; relevant in data analysis for experiments." src="/files/ftp_upload/70582/70582eq2.jpg" style="margin: auto;" data-altupdatedat="1784001604000" data-alt="Equation 2"> 
	NOTE: 
	(ΔF/F)mean: mean of ΔF/F. 
	(ΔF/F)std: standard deviation of ΔF/F. 
	Fraw: raw fluorescence intensity. 
	Fbaseline: baseline fluorescence intensity (mean over predefined baseline period).​</li>
	<li>Quantify metrics such as area under the curve (AUC) for astrocyte and neuronal calcium responses during novel vs. familiar object exploration.</li>
</ol>
6. Histological verification after recording
<ol>
	<li>Anesthetize the mouse with an overdose of pentobarbital administered intraperitoneally until complete loss of reflexes is confirmed.</li>
	<li>Perform transcardial perfusion with normal saline, followed by 4% paraformaldehyde (PFA).</li>
	<li>Dissect the brain carefully and post-fix it in 4% PFA at 4 °C for 12–24 h.</li>
	<li>Transfer the brain to 30% sucrose in PBS at 4 °C until the tissue sinks for cryoprotection.</li>
	<li>Embed the brain in optimal cutting temperature compound, freeze it, and cut coronal sections (40 μm thickness) using a cryostat.</li>
	<li>Perform immunofluorescence staining to verify viral expression and fiber placement.</li>
</ol>

<ol><li>Eichenbaum, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Memory%3A+Organization+and+Control%5BTitle%5D)+AND+%22Annu+Rev+Psychol%22%5BJournal%5D)">Memory: Organization and Control.</a> Annu Rev Psychol. 68, 19-45 (2017).</li>
<li>Kamatham, P. T., Shukla, R., Khatri, D. K., Vora, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathogenesis%2C+diagnostics%2C+and+therapeutics+for+Alzheimer%E2%80%99s+disease%3A+Breaking+the+memory+barrier%5BTitle%5D)+AND+%22Ageing+Res+Rev%22%5BJournal%5D)">Pathogenesis, diagnostics, and therapeutics for Alzheimer’s disease: Breaking the memory barrier.</a> Ageing Res Rev. 101, 102481(2024).</li>
<li>Graff-Radford, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+insights+into+atypical+Alzheimer%E2%80%99s+disease+in+the+era+of+biomarkers%5BTitle%5D)+AND+%22Lancet+Neurol%22%5BJournal%5D)">New insights into atypical Alzheimer’s disease in the era of biomarkers.</a> Lancet Neurol. 20 (3), 222-234 (2021).</li>
<li>Araque, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gliotransmitters+travel+in+time+and+space%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Gliotransmitters travel in time and space.</a> Neuron. 81 (4), 728-739 (2014).</li>
<li>Khakh, B. S., Sofroniew, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diversity+of+astrocyte+functions+and+phenotypes+in+neural+circuits%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Diversity of astrocyte functions and phenotypes in neural circuits.</a> Nat Neurosci. 18 (7), 942-952 (2015).</li>
<li>Di Castro, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Local+Ca2%2B+detection+and+modulation+of+synaptic+release+by+astrocytes%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Local Ca2+ detection and modulation of synaptic release by astrocytes.</a> Nat Neurosci. 14 (10), 1276-1284 (2011).</li>
<li>Savtchouk, I., Volterra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gliotransmission%3A+Beyond+Black-and-White%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Gliotransmission: Beyond Black-and-White.</a> J Neurosci. 38 (1), 14-25 (2018).</li>
<li>Perea, G., Navarrete, M., Araque, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tripartite+synapses%3A+Astrocytes+process+and+control+synaptic+information%5BTitle%5D)+AND+%22Trends+Neurosci%22%5BJournal%5D)">Tripartite synapses: Astrocytes process and control synaptic information.</a> Trends Neurosci. 32 (8), 421-431 (2009).</li>
<li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+multi-regional+functional+connectivity+of+astrocyte-neuronal+networks+during+behaviors%5BTitle%5D)+AND+%22Neurophotonics%22%5BJournal%5D)">Mapping multi-regional functional connectivity of astrocyte-neuronal networks during behaviors.</a> Neurophotonics. 11 (4), 045010(2024).</li>
<li>Lines, J., Martin, E. D., Kofuji, P., Aguilar, J., Araque, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Astrocytes+modulate+sensory-evoked+neuronal+network+activity%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Astrocytes modulate sensory-evoked neuronal network activity.</a> Nat Commun. 11 (1), 3689(2020).</li>
<li>Lines, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Astrocyte-neuronal+network+interplay+is+disrupted+in+Alzheimer%E2%80%99s+disease+mice%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">Astrocyte-neuronal network interplay is disrupted in Alzheimer’s disease mice.</a> Glia. 70 (2), 368-378 (2022).</li>
<li>Brincat, S. L., Miller, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Frequency-specific+hippocampal-prefrontal+interactions+during+associative+learning%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Frequency-specific hippocampal-prefrontal interactions during associative learning.</a> Nat Neurosci. 18 (4), 576-581 (2015).</li>
<li>DeNardo, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Temporal+evolution+of+cortical+ensembles+promoting+remote+memory+retrieval%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Temporal evolution of cortical ensembles promoting remote memory retrieval.</a> Nat Neurosci. 22 (3), 460-469 (2019).</li>
<li>Kitamura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engrams+and+circuits+crucial+for+systems+consolidation+of+a+memory%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Engrams and circuits crucial for systems consolidation of a memory.</a> Science. 356 (6333), 73-78 (2017).</li>
<li>Gonzalo Cogno, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Minute-scale+oscillatory+sequences+in+medial+entorhinal+cortex%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Minute-scale oscillatory sequences in medial entorhinal cortex.</a> Nature. 625 (7994), 338-344 (2024).</li>
<li>Roudi, Y., Moser, E. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Grid+cells+in+an+inhibitory+network%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Grid cells in an inhibitory network.</a> Nat Neurosci. 17 (5), 639-641 (2014).</li>
<li>Williamson, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Learning-associated+astrocyte+ensembles+regulate+memory+recall%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Learning-associated astrocyte ensembles regulate memory recall.</a> Nature. 637 (8045), 478-486 (2025).</li>
<li>Curreli, S., Bonato, J., Romanzi, S., Panzeri, S., Fellin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complementary+encoding+of+spatial+information+in+hippocampal+astrocytes%5BTitle%5D)+AND+%22PLoS+Biol%22%5BJournal%5D)">Complementary encoding of spatial information in hippocampal astrocytes.</a> PLoS Biol. 20 (3), e3001530(2022).</li>
<li>Oheim, M., Michael, D. J., Geisbauer, M., Madsen, D., Chow, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Principles+of+two-photon+excitation+fluorescence+microscopy+and+other+nonlinear+imaging+approaches%5BTitle%5D)+AND+%22Adv+Drug+Deliv+Rev%22%5BJournal%5D)">Principles of two-photon excitation fluorescence microscopy and other nonlinear imaging approaches.</a> Adv Drug Deliv Rev. 58 (7), 788-808 (2006).</li>
<li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deep+tissue+two-photon+microscopy%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Deep tissue two-photon microscopy.</a> Nat Methods. 2 (12), 932-940 (2005).</li>
<li>Zhang, K., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensory+response+in+host+and+engrafted+astrocytes+of+adult+brain+in+vivo%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">Sensory response in host and engrafted astrocytes of adult brain in vivo.</a> Glia. 65 (12), 1867-1884 (2017).</li>
<li>Qin, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+visual-cue-dependent+memory+circuit+for+place+navigation%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">A visual-cue-dependent memory circuit for place navigation.</a> Neuron. 99 (1), 47-55.e44 (2018).</li>
<li>Yao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+corticopontine+circuit+for+initiation+of+urination%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">A corticopontine circuit for initiation of urination.</a> Nat Neurosci. 21 (11), 1541-1550 (2018).</li>
<li>Mirzayi, P., Shobeiri, P., Kalantari, A., Perry, G., Rezaei, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optogenetics%3A+Implications+for+Alzheimer%E2%80%99s+disease+research+and+therapy%5BTitle%5D)+AND+%22Mol+Brain%22%5BJournal%5D)">Optogenetics: Implications for Alzheimer’s disease research and therapy.</a> Mol Brain. 15 (1), 20(2022).</li>
<li>Simpson, E. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lights%2C+fiber%2C+action%21+A+primer+on+in+vivo+fiber+photometry%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Lights, fiber, action! A primer on in vivo fiber photometry.</a> Neuron. 112 (5), 718-739 (2024).</li>
<li>Chen, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrasensitive+fluorescent+proteins+for+imaging+neuronal+activity%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Ultrasensitive fluorescent proteins for imaging neuronal activity.</a> Nature. 499 (7458), 295-300 (2013).</li>
<li>Dana, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensitive+red+protein+calcium+indicators+for+imaging+neural+activity%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Sensitive red protein calcium indicators for imaging neural activity.</a> Elife. 5, 12727(2016).</li>
<li>Serikov, A., Martsishevska, I., Shin, W., Kim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protocol+for+in+vivo+dual-color+fiber+photometry+in+the+mouse+thalamus%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">Protocol for in vivo dual-color fiber photometry in the mouse thalamus.</a> STAR Protoc. 5 (2), 102931(2024).</li>
<li>Gilbert, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Horizontal+integration+and+cortical+dynamics%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Horizontal integration and cortical dynamics.</a> Neuron. 9 (1), 1-13 (1992).</li>
<li>de Pasquale, F., Corbetta, M., Betti, V., Della Penna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cortical+cores+in+network+dynamics%5BTitle%5D)+AND+%22Neuroimage%22%5BJournal%5D)">Cortical cores in network dynamics.</a> Neuroimage. 180 (Pt B), 370-382 (2018).</li>
<li>Suthard, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engram+reactivation+mimics+cellular+signatures+of+fear%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Engram reactivation mimics cellular signatures of fear.</a> Cell Rep. 44 (10), 116409(2025).</li>
<li>Sandoval, K. C., Rychlik, J., Choe, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Calcium+dynamics+in+hypothalamic+paraventricular+oxytocin+neurons+and+astrocytes+associated+with+social+and+stress+stimuli%5BTitle%5D)+AND+%22eNeuro%22%5BJournal%5D)">Calcium dynamics in hypothalamic paraventricular oxytocin neurons and astrocytes associated with social and stress stimuli.</a> eNeuro. 12 (5), (2025).</li>
<li>Srinivasan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ca%282%2B%29+signaling+in+astrocytes+from+Ip3r2%28-%2F-%29+mice+in+brain+slices+and+during+startle+responses+in+vivo%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Ca(2+) signaling in astrocytes from Ip3r2(-/-) mice in brain slices and during startle responses in vivo.</a> Nat Neurosci. 18 (5), 708-717 (2015).</li>
<li>Spellman, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hippocampal-prefrontal+input+supports+spatial+encoding+in+working+memory%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Hippocampal-prefrontal input supports spatial encoding in working memory.</a> Nature. 522 (7556), 309-314 (2015).</li>
<li>Hu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optogenetics+sheds+new+light+on+tissue+engineering+and+regenerative+medicine%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Optogenetics sheds new light on tissue engineering and regenerative medicine.</a> Biomaterials. 227, 119546(2020).</li>
<li>Liddelow, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurotoxic+reactive+astrocytes+are+induced+by+activated+microglia%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Neurotoxic reactive astrocytes are induced by activated microglia.</a> Nature. 541 (7638), 481-487 (2017).</li>
<li>Shah, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Astrocyte+calcium+dysfunction+causes+early+network+hyperactivity+in+Alzheimer%E2%80%99s+disease%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Astrocyte calcium dysfunction causes early network hyperactivity in Alzheimer’s disease.</a> Cell Rep. 40 (8), 111280(2022).</li>
<li>Jiménez-Balado, J., Eich, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GABAergic+dysfunction%2C+neural+network+hyperactivity+and+memory+impairments+in+human+aging+and+Alzheimer%E2%80%99s+disease%5BTitle%5D)+AND+%22Semin+Cell+Dev+Biol%22%5BJournal%5D)">GABAergic dysfunction, neural network hyperactivity and memory impairments in human aging and Alzheimer’s disease.</a> Semin Cell Dev Biol. 116, 146-159 (2021).</li>
<li>Lin, R., Guo, Y., Jiang, W., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advanced+technologies+for+the+study+of+neuronal+cross-organ+regulation%3A+a+narrative+review%5BTitle%5D)+AND+%22Adv+Technol+Neurosci%22%5BJournal%5D)">Advanced technologies for the study of neuronal cross-organ regulation: a narrative review.</a> Adv Technol Neurosci. 1 (2), 166-176 (2024).</li>
<li>Reitman, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Norepinephrine+links+astrocytic+activity+to+regulation+of+cortical+state%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Norepinephrine links astrocytic activity to regulation of cortical state.</a> Nat Neurosci. 26 (4), 579-593 (2023).</li>
</ol>

No conflicts of interest, financial or otherwise, are declared by the authors.

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 memory9,<sup class="xref...

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&#39;s disease, age-related cognitive disease, and traumatic brain injury, exhibit abnormalities in memory formation1,2,3. 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 support4,5,6,7,8. Astrocytes dynamically interact with neurons, and the bidirectional communication is collectively referred to as the &#34;astrocyte-neuron network&#34;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&#8211;cortical circuit12,13,14,15,16. 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 memory17,18.
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 fixation19,20; 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.

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

a dual-color fiber photometry method for recording astrocyte-neuron activity across multiple brain regions during learning and memory behaviors

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

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&#8211;neuron interactions underlying learning and memory processes.

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...

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.

Learning and memory depend critically on the coordination between neurons and astrocytes across distributed brain regions. Astrocytes modulate synaptic ...

This protocol enables recording of calcium signals from neurons and astrocytes across multiple brain regions during learning and memory behaviors. This protocol can be applied to study astrocytes-neuron network dynamics during learning, memory, and other behaviors. To perform the first surgery for astrocyte injection, take an anesthetized mouse and maintain body temperature at 37 to 38 degrees Celsius with a heating pad.Apply ophthalmic ointment to prevent corneal desiccation. Then, remove the scalp hair and disinfect the skin thoroughly. Make a midline incision to reveal the skull surface.Place the head in the stereotaxic instrument. Adjust the bregma and lambda sutures to the same horizontal plane and adjust the frame further to ensure that points two millimeters lateral to the bregma on both sides are level. Identify the stereotaxic coordinates with a mouse brain atlas using the bregma as the reference point.Drill a 0.6-millimeter-diameter circular craniotomy above each site. Keep the dura mater moist and intact after opening the skull. Load the GCaMP6f virus into the syringe at five nanoliters per second and install the syringe in the stereotaxic arm.Lower the syringe slowly into the target region and inject 150 nanoliters at 0.6 nanoliters per second. Hold for eight minutes. And withdraw at 0.1 millimeters per second.After repeating this procedure for all the regions, seal the incision with tissue adhesive and allow two weeks for viral expression. To perform the secondary surgery for neuronal injection with AAV-hSyn-jRGECO1a, prepare the mouse as demonstrated earlier. Reopen the scalp along the midline and level the skull.Clear tissue debris at the same location as the craniotomy windows from the first surgery to expose the dura mater intact. Adjust coordinates using the bregma as the reference point. Then, perform injections at the same coordinates and depths as described previously.Next, load the jRGECO1a virus into the syringe at five nanoliters per second and install the syringe in the stereotaxic arm. Inject 150 nanoliters at 0.6 nanoliters per second. Hold for eight minutes and withdraw slowly.Repeat this procedure for all regions and allow three weeks for viral expression. Next, install the optical fiber ferrule in the stereotaxic holder using the presented coordinates. Position the fiber at 100 to 150 micrometers above the viral injection depth.Lower the fiber slowly to minimize tissue damage. Seal the craniotomy with tissue adhesive and secure the ferrule with dental cement. Perform multichannel fiber photometry, recording three weeks after the second virus injection to ensure stable and full expression of both calcium indicators.To habituate the mice to the fiber system and arena, first connect one end of the bundled optical fiber to the fiber photometry acquisition system. Connect the three branches at the other end of the bundled fiber to the three brain regions. Connect channel one to the medial prefrontal cortex, channel two to CA1, and channel three to the medial entorhinal cortex.After briefly anesthetizing the mouse, place it in an open area with a side length of 50 centimeters and allow free exploration. After 10 minutes, disconnect the fiber and return the mouse to the home cage. Repeat once per day for three days.For fiber photometry recording during the novel object recognition task, place two identical objects in diagonally opposite quadrants. Connect the fiber bundle in a lightly anesthetized state. Allow the mouse to explore the objects for 10 minutes.After that, return the mouse to the home cage without removing the fiber. Adjust the stimulation parameters of the lasers. Set 470 nanometers for GCaMP6f and 560 nanometers for jRGECO1a.Record calcium signals at 30 hertz, synchronized with video tracking at 30 hertz. Then, position a laser diode within the video frame outside the mouse's visual field at the start of fiber photometry acquisition to serve as a temporal marker. After 10 minutes from the learning phase, replace one object with a novel object.Place the mouse back into the arena for 10 minutes of exploration while recording. Disconnect the fiber from the ceramic ferrule and return the mouse to the home cage. After imaging, quantify the calcium responses during novel versus familiar object exploration using the appropriate analysis software.Confocal imaging revealed strong overlap between GCaMP6f fluorescence and the astrocyte marker S100 beta and robust co-localization of jRGECO1a with the neuronal marker NeuN in the mPFC, CA1, and MEC. These results confirmed stable and specific expression of GCaMP6f in astrocytes and jRGECO1a in neurons within the targeted memory-related brain regions. Statistical analysis of event frequency, duration, and cross-regional distribution demonstrated that this method reliably and stably measured dual cell type specific calcium signals across multiple brain regions.The cross-correlation coefficients between astrocytes and neurons across multiple brain regions during the stationary state were significantly lower than during the freely-moving state. Astrocytic calcium signals exhibited lower peak amplitudes during exploration of the novel object compared to the familiar object. Neuronal calcium signals also showed a similar decreasing trend during exploration of the novel object compared to the familiar object.Quantitative analysis of the area under the curve further confirmed that calcium responses in astrocytes and neurons were significantly attenuated during novel object exploration. This protocol enables simultaneous moment of astrocytes-neuron activity and the functional coupling across multiple brain regions. The key challenge is maintaining precise alignment across safe surgeries, while minimizing tissue damage during injection and the fiber implantation.Future studies can apply this method to delays models to examine how astrocytes-neuron coordination is altered.

a-dual-color-fiber-photometry-method-for-recording-astrocyte-neuron

Research

JoVE Journal

Neuroscience

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

This video demonstrates a procedure for observing changes in neuronal activity in a brain slice stained with a voltage-sensitive dye. The dye reflects changes in the neuron's membrane potential by altering its fluorescence intensity. Through the application of electrical stimuli and advanced imaging techniques, this process enables visual tracking of real-time changes in neuronal membrane potential based on VSD fluorescence.

Optical Recording of Neuronal Activity in Brain Slices Stained with a Voltage-Sensitive Dye

This video demonstrates a method for recording neural activity in a freely moving mouse using fiber photometry. In this method, neurons in a specific brain region express a genetically encoded calcium indicator. A stimulus is applied to this brain region, and the stimulus-dependent change in fluorescence is measured to identify neural activity.

Recording Neural Activity in Freely Moving Mice Using Fiber Photometry

Source: Maruoka, H., et. al. Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice. J. Vis. Exp. (2022)     This video shows a two-photon microscopy procedure for imaging neuronal activity and microglial dynamics in an awake mouse during visual stimulation, highlighting calcium transients and cellular interactions.

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in an Awake Mouse

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Using an all-optical approach, ischemic stroke is induced while simultaneously recording neuronal Ca2+ activity using a fluorescent biosensor in the emerging stroke core of an awake, behaving mouse.

Simultaneous Photothrombosis and Fiber Photometry to Induce and Monitor Ischemic Stroke in Behaving Mice

This protocol provides an integrated method that includes viral delivery, optrode implantation, photothrombotic induction, simultaneous recording of astrocyte and neuronal activity, and optogenetic stimulation of astrocytes. This integrated method aims to provide concurrent investigation of the astrocyte-neuron relationship across the acute-to-chronic stages of ischemic stroke.

An Integrated Method for Photothrombotic Stroke Modeling and In Vivo Optrode Recording of Neuronal and Astrocytic Activity in Behaving Mice

Learn dual-color fiber photometry to record astrocyte-neuron calcium signals across mPFC, CA1, and MEC during learning and memory behaviors. This method enables real-time monitoring of cell-type-specific activity.

A Dual-Color Fiber Photometry Method for Recording Astrocyte-Neuron Activity Across Multiple Brain Regions During Learning and Memory Behaviors (Video) | JoVE Journal

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

Explore More Videos