Mar 23, 2026

Public workspaceStereotaxic viral injection surgeries for multifiber photometry recordings and optical stimulation of dopamine neurons

DOI
https://dx.doi.org/10.17504/protocols.io.bp2l6e2nrgqe/v1
Stereotaxic viral injection surgeries for multifiber photometry recordings and optical stimulation of dopamine neurons
  • Safa Bouabid1,2,
  • Mark Howe1,2
  • 1Department of Psychological & Brain Sciences, Boston University, Boston, MA, USA;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA
Safa Bouabid
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.bp2l6e2nrgqe/v1
Protocol Citation: Safa Bouabid, Mark Howe 2026. Stereotaxic viral injection surgeries for multifiber photometry recordings and optical stimulation of dopamine neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6e2nrgqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 21, 2026
Last Modified: March 23, 2026
Protocol Integer ID: 239100
Keywords: optical stimulation of dopamine neuron, midbrain dopamine neuron optogenetic stimulation, chronic optical fiber implantation in the mouse striatum, optogenetic stimulation, optical stimulation, dopamine neuron, midbrain dopamine neuron, stereotaxic viral injection, stereotaxic viral injection surgeries for multifiber photometry recording, stereotaxic viral injection surgery, surgical procedures for stereotaxic viral injection, chronic optical fiber implantation, mouse striatum, pavlovian learning
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-020370; ASAP-025192
Abstract
This protocol outlines the surgical procedures for stereotaxic viral injections of genetically encoded sensors and opsins and chronic optical fiber implantation in the mouse striatum for monitoring acetylcholine and dopamine during spontaneous behavior, unpredicted reward, Pavlovian learning and extinction, and midbrain dopamine neuron optogenetic stimulation.
Guidelines
Post-surgical care and recovery procedures ensure animal well-being before experimental data collection.
Materials
Equipment:
  • Pulled glass pipette (tip diameter 30-50 μm)

Viral Vectors:

Troubleshooting
Anaesthetic Induction & Surgical Preparation
Anesthetise mice with isoflurane (3-4%) and place it in a stereotaxic frame (Kopf instruments) on an electric heating pad (Physitemp instruments).
Administer buprenorphine extended release for pre-operative analgesia (3.25 mg kg-1 subcutaneous, Ethiqa XR).
Following induction, maintain isoflurane at 1-2% (in 0.8-1 L min -1 pure oxygen) and body temperature at 37°C throughout the surgical procedure.
Injection of Viral Vectors
Monitoring simultaneous Dopamine and Acetylcholine release:
Using a pulled glass pipette, pressure-inject the genetically-encoded fluorescent dopamine (DA) GRAB-rDA3m (pAAV-hSyn-rDA3m) and Acetylcholine (ACh) GRAB-ACh3.0 (AAV9-hSyn-ACh3.0) mixed together, into the striatum (right hemisphere) of wild-type mice at ~ 30 separate striatum locations.
Note
The striatum locations were chosen to maximize expression around fiber tips (350 nL at each location at a rate of 100 nL/min).

For control experiments, inject mutant version of the genetically-encoded fluorescent acetycholine (ACh) sensor (AAV9-hSyn-ACh3.0-mut(Ach4.3-mut)) mixed with pAAV-CAG-tdTomato into the striatum of wild-type mice using the same strategy.
Midbrain optogenetic stimulation experiments with simultaneous Dopamine and Acetylcholine release measurements
Using a pulled glass pipette, pressure-inject the genetically-encoded fluorescent dopamine (DA) GRAB-rDA3m (pAAV-hSyn-rDA3m) and Acetylcholine (ACh) GRAB-ACh3.0 (AAV9-hSyn-ACh3.0) mixed together, into the striatum of DAT-IRES-cre mice (RRID:IMSR_JAX:006660) at ~ 30 separate striatum locations.
Three weeks post DA and ACh GRAB sensors viral injections and implants, drill craniotomies through metabond and skull above the midbrain at pre-marked coordinates.
Inject ChR2 opsin (pAAV-EF1a-doublefloxed-hChR2(H134R)-EYFP-WPRE-HGHpA) into the right SNc and VTA at 3 sites (200 nl/site at a rate of 100nl/min) at the following three coordinates in mm,
AP:-3.05, ML:± 0.6, DV: -4.25; AP:-3.15, ML: ± 1.3, DV: -4.1; AP:-3.5, ML: ± 1.15, DV: -4.1.
Implantation of multi-Fiber Arrays and opto-stimulation Fiber
Mount the multi-fiber array onto stereotaxic manipulator.
Remove dura gently, and slowly lower the multi-fiber array into striatum position.
Seal craniotomy with a thin layer of kwik-Sil (WPI), and secure the multi-fiber array to the skull surface using Metabond (Pakell).
For midbrain DA optogenetic stimulation experiments, slowly lower a 90 degree 100 µm core mono fiber-optic cannula (MFC_100/125 - 0.66 NA) attached to a zirconia ferrule (Doric) to a depth of -4 mm from the dura at the coordinates in mm, AP: -3.15 mm and ML: ±1.3 mm.
Seal craniotomy above the midbrain with a thin layer of Kwik-Sil (WPI), and secure the optical fiber to the skull surface using Metabond (Parkell).
Head-Fixation
Secure a metal head plate and ring (Atlas Tool and Die Works) to the skull with Metabond, and cover the implant surface with a mixture of Metabond and carbon powder (Sigma Aldrich) to reduce optical artefacts.
Protect the fiber bundle by a cylindrical plastic tube, extending ~ 1-2 mm above the fiber bundle, and secure around the bundle using a mixture of Metabond and carbon powder.
Post-Operative Recovery
Place each mouse in an individual cage with a heating pad and perform post-operative injections of meloxicam (5 mg kg -1 subcutaneous, Covertus) and 1 mL of saline per day subcutaneously for 4 days after surgery.
Allow them to recover in their cages for at least 2 weeks after surgery.
Protocol references
Jing, M., Li, Y., Zeng, J., Huang, P., Skirzewski, M., Kljakic, O., Peng, W., Qian, T., Tan, K., Zou, J., et al. (2020). An optimized acetylcholine sensor for monitoring in vivo cholinergic activity. Nat Methods 17, 1139–1146.
Patriarchi, T., Cho, J.R., Merten, K., Howe, M.W., Marley, A., Xiong, W.-H., Folk, R.W., Broussard, G.J., Liang, R., Jang, M.J., et al. (2018). Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors. Science 360 , eaat4422.
Zhang, Y., Zhao, S., Rodriguez, E., Takatoh, J., Han, B.-X., Zhou, X., and Wang, F. (2015). Identifying local and descending inputs for primary sensory neurons. J Clin Invest 125, 3782–3794.

Marvin, J.S., Scholl, B., Wilson, D.E., Podgorski, K., Kazemipour, A., Müller, J.A., Schoch, S., Quiroz, F.J.U., Rebola, N., Bao, H., et al. (2018). Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR. Nat Methods 15, 936–939.

Bouabid, S., Zhang, L., T. Vu, MA. et al. (2025) Distinct spatially organized striatum-wide acetylcholine dynamics for the learning and extinction of Pavlovian associations. Nat Commun 16, 5169.