Mar 23, 2026
Multi-fiber photometry in head-fixed mice during spontaneous behavior and dual-cue delay Pavlovian association
- 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
Protocol Citation: Safa Bouabid, Mark Howe 2026. Multi-fiber photometry in head-fixed mice during spontaneous behavior and dual-cue delay Pavlovian association. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl489jjvo5/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 22, 2026
Last Modified: March 23, 2026
Protocol Integer ID: 239165
Keywords: optical stimulation of dopamine neuron, cue delay pavlovian task, auditory cues with water reward, pavlovian task, optical stimulation, cue delay pavlovian association this protocol, pavlovian conditioning, dopamine release dynamic, cue delay pavlovian association, mice during spontaneous behavior, dopamine neuron, auditory cue, fluorescence signal, reward probability, water reward, random reward, encoded sensor, reward, unpredicted reward, delivered random reward, fixed mice, microscope with precise excitation, conditioning phase, behavioural response, stereotaxic viral injection surgeries for multifiber photometry recording, multifiber photometry recording, such as licking, mice, spontaneous behavior, licking
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-020370; ASAP-025192
Abstract
This protocol outlines multi-fiber photometry recordings in head-fixed mice during spontaneous behavior, unpredicted reward, and a dual-cue delay Pavlovian task. Mice are first placed on a controlled water schedule and habituated to head fixation on a spherical treadmill. In spontaneous behavior and unpredicted reward, mice are delivered random rewards. In the Pavlovian task, during the conditioning phase, they learn to associate visual and auditory cues with water rewards, followed by a partial extinction phase where reward probabilities are downshifted.
Behavioural responses, such as licking and movement, are tracked and synchronized with photometry data to assess acetylcholine and dopamine release dynamics using genetically encoded sensors (see Protocol: Stereotaxic viral injection surgeries for multifiber photometry recordings and optical stimulation of dopamine neurons) during spontaneous behavior, unpredicted reward, and during Pavlovian conditioning and extinction.
A custom-built microscope with precise excitation and detection systems is used to monitor fluorescence signals from genetically encoded sensors. Imaging is synchronized with behavioural data collection, and data processing is performed to ensure accurate analysis.
Materials
Equipment:
- Spherical Styrofoam Treadmill (Smoothfoam, 8in diameter)
- Optical Computer Mice (Logitech G203)
- Acquisition Board (NIDAQ, PCle 6343)
- Solenoid Valve (# 161T012, Neptune Research)
- Camera used to capture orofacial movements (Blackfly S USB3, BFS-U3-16S2M-CS, Teledyne Flir)
- LED lights (Thor labs, M470L3, 470 nm)
- Micromanipulator (Newport Model 96067-XYZ-R)
- Bandpass Filter (Chroma, No 525/50m)
- Tube lens (Thor labs, No TTL165-A)
- Hamamatsu Camera (Hamamatsu, Orca Fusion BT Gen III)
- Vibration isolation table (Newport)
- Programmable digital acquisition card (NIDAQ, National Instruments PCIe 6343)
Software:
- HCImage live (HCImage live, Hamamatsu)
Troubleshooting
Before start
Genetically encoded sensors to monitor acetylcholine and dopamine dynamics were injected in the striatum following the Protocol: Stereotaxic viral injection surgeries for multifiber photometry recordings and optical stimulation of dopamine neurons.
Habituation & Behaviour Setup
Three weeks post-surgery, place mice on a water schedule, receiving 1mL of water/day, adjusted as necessary to maintain them at 80-85% of their initial body weight for the duration of the experiments.
Three to four days before starting behavior experiments and photometry recordings, habituate the mice to head-fixation on a lightweight cylindrical running wheel or on a spehirical styrofoam treadmill until they freely run and transition spontaneously between resting and running.
Dispense water rewards (5μL/reward, (random inter-trial interval: 10-30 s) through a water spout operated by an electronically controlled solenoid valve, mounted on a post a few mm away from the mice’s mouths.
Monitor tongue spout contacts (a proxy for licking) by a capacitive touch circuit connected to the spout and confirmed with live video taken from a camera positioned to capture orofacial movements.
Measure mouse location
For the mice trained on lightweight cylindrical running wheel, sample the wheel's linear velocity at 2Khz using a rotary encoder (E2-5000, US Digital) attached to the wheel axle.
For the mice trained on spehirical styrofoam treadmill, measure the ball rotation in pitch, yaw, and roll directions using optical computer mice through an acquisition board.
Spontaneous Behaviour and Unpredicted Reward
Conduct two distinct daily sessions during photometry recordings:
Session 1: Perform photometry recordings while mice are spontaneously behaving on the wheel with no lick spout present and no reward available to capture purely spontaneous behavior.
Session 2: Provide access to the lick spout and deliver unpredicted water rewards (5μL) at random intervals (10-30s).
Midbrain Optogenetic Stimulation
For unilateral optogenetic stimulation of DA neurons with simultaneous DA and ACh release dynamics measurements during unpredicted reward sessions, connect implanted optical fibers to a 450 nm laser diode light source (Doric).
Use a 100 μm patch cord (Doric) with zirconia sleeves to connect fibers to the light source.
Calibrate laser power at the tip of optical fibers to 2.5 mW.
Deliver 4 ms blue light pulses at 30 Hz for 500 ms to activate dopamine neurons.
Tigger 5 stimulation trains each session at randomized intervals (outside unpredicted reward deliveries).
Dual-cue delay Pavlovian Conditioning Task and Partial Extinction
Pavlovian conditioning Phase
In each session (daily) present 60 cue presentations (30 light and 30 tone) in a pseudorandom order.
Deliver light cues via a 7 mW LED positioned 20 cm from the mouse and present tone cues (12 kHz, 80 dB) through a USB speaker placed 30 cm away.
Present each cue for a duration of 6 seconds with water reward delivery (5μL) 3 seconds after cue onset.
Randomly draw an inter-trial interval (ITI; time between separate trials) from a uniform distribution of 4-40s.
Include eight additional non-contingent rewards per session during the inter-trial interval (ITI; time between separate trials) periods.
Continue training for 26-38 days until mice learn to associate both cues with rewards.
Partial Extinction Phase
After conditioning is complete, begin the partial extinction phase.
Downshift the reward probability for one cue to 20% while maintaining the other cue at 80%.
Continue training until significant reductions in responses to the 20% cue are observed.
Reverse the cue probabilities (20% , 80%), counterbalancing the order across mice.
Microscope Setup for Multi-Fiber Photometry
As mice perform the behavioral tasks (spontaneous behavior, reward sessions, optogenetic stimulation, or Pavlovian conditioning), conduct fluorescence measurements from the multi-fiber arrays using a custom-built microscope setup mounted on a 4’ W x 8’ L x 12’ thick vibration isolation table.
Filter and focus emission light using a bandpass filter and a tube lens onto the CMOS sensor of the Hamamatsu camera, creating an image of the fiber bundle.
To enable precise manual focusing, connect microscope to a micromanipulator and mount on a rotatable arm extending over the head-fixation setup to facilitate positioning of the objective above the imaging surface over the mouse head.
Multi-Fiber Photometry Image Acquisition
Open the basic imaging application HCImage Live (Hamamatsu) that is included with the Hamamatsu camera.
Perform dual wavelength excitation in a quasi-simultaneous externally triggered imaging mode.
Alternate 470 nm excitation with 570 nm excitation at 36 Hz with 20 ms exposure time, to achieve a frame rate of 18 Hz for each excitation wavelength, respectively.
Control the timing and duration of TTL pulses through a custom MATLAB software and a programmable digital acquisition card (NIDAQ, National Instruments PCIe 6343).
Transmit voltage pulses to the NIDAQ from the camera following the exposure of each frame to confirm proper camera triggering and to synchronize imaging data with behavioral data.
Record videos of the fiber bundle surface throughout the behavioral session.
Image Preprocessing and Data Extraction
Apply motion correction to the acquired videos using a whole-frame cross-correlation algorithm (Dombeck et al., 2010; Miri et al., 2011).
Extract fluorescence changes from each fiber using previously described methods (Vu et al., 2024). See Github repositories: BouabidVu2026 and MultifiberProcessing
Extract mean fluorescence time series from each region of interest.
Fit a 2-term exponential function to the mean fluorescence time series to define the baseline.
Calculate ΔF/F signals by Normalize fluorescence signals to baselinn.
Remove low-frequency artifacts by high-pass filtering the ΔF/F signals using a finite impulse response filter.
For green sensors (ACh or other green fluorophores), set the passband frequency at 0.3 Hz, for red sensors (dopamine or other red fluorophores), set the passband frequency at 0.1 Hz.
Note
For experiments involving optogenetic stimulation of midbrain DA neurons, skip high-pass filtering to preserve longer timescale dynamics of stimulation effects on ACh signals.
Protocol references
Dombeck, D., Harvey, C., Tian, L. et al. Functional imaging of hippocampal place cells at cellular resolution during virtual navigation. Nat Neurosci 13, 1433–1440 (2010).
Miri, A., Daie, K. et al. Regression-Based Identification of Behavior-Encoding Neurons During Large-Scale Optical Imaging of Neural Activity at Cellular Resolution Journal of Neurophysiology, 105:2, 964-980 (2011).
Vu, M.-A.T., Brown, E.H., Wen, M.J., Noggle, C.A., Zhang, Z., Monk, K.J., Bouabid, S., Mroz, L., Graham, B.M., Zhuo, Y., et al. Targeted micro-fiber arrays for measuring and manipulating localized multi-scale neural dynamics over large, deep brain volumes during behavior. Neuron 112 , 909-923.e9 (2024).
Bouabid, S., Zhang, L., T. Vu, MA. et al. Distinct spatially organized striatum-wide acetylcholine dynamics for the learning and extinction of Pavlovian associations. Nat Commun 16, 5169 (2025).