Abstract
Optogenetics and calcium imaging can be combined to simultaneously stimulate and record neural activity in vivo. However, this usually requires two-photon microscopes, which are not portable nor affordable. Here we report the design and implementation of a miniaturized one-photon endoscope for performing simultaneous optogenetic stimulation and calcium imaging. By integrating digital micromirrors, the endoscope makes it possible to activate any neuron of choice within the field of view, and to apply arbitrary spatiotemporal patterns of photostimulation while imaging calcium activity. We used the endoscope to image striatal neurons from either the direct pathway or the indirect pathway in freely moving mice while activating any chosen neuron in the field of view. The endoscope also allows for the selection of neurons based on their relationship with specific animal behaviour, and to recreate the behaviour by mimicking the natural neural activity with photostimulation. The miniaturized endoscope may facilitate the study of how neural activity gives rise to behaviour in freely moving animals.
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Data availability
The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are too large to be publicly shared, yet they are available for research purposes from the corresponding author on reasonable request.
Code availability
The Matlab codes are available from the corresponding author on request.
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Acknowledgements
We thank N. Calakos, S. Je and M. Rossi for helpful comments on the manuscript. This paper is dedicated to the memory of our co-author Ryan N. Hughes (1987–2021).
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Authors and Affiliations
Contributions
H.H.Y. conceived the concept of the all-optical 1-photon system for freely moving mice. J.Z., N.K. and H.H.Y. designed the experiments. J.Z. designed, assembled and calibrated the circuit board and MAPSI. J.Z. and I.P.F performed testing in mice. J.Z., N.K. F.P. U.S. and K.B. analysed data. N.K., I.P.F., K.B. and R.N.H performed surgeries. R.N.H and J.K. performed histology and confocal imaging. R.N.H., J.Z. and H.H.Y. wrote the manuscript.
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H.H.Y. and J.Z. filed a provisional patent on the designs presented in this paper (63/289394, December 14, 2021, North Carolina, USA). The other authors declare no competing interests.
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Nature Biomedical Engineering thanks Dimitri Ryczko, Michael C. Wiest and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Free movement using MAPSI and stimulation pipeline.
To reduce the weight of MAPSI carried by the mouse, a custom commutator was built. a) The commutator is attached to a 4 g pulley, and allows the wires to rotate without being tangled. b) Top: Open field trajectory over 15 min of a representative mouse with 4 g UCLA miniscope compared to the trajectory of the same mouse carrying 7.8 g MAPSI. Bottom: Total movement distance, peak speed, and average speed (N = 7, 4 D1-cre mice and 3 A2A-cre mice, 15 minutes in the same open field platform). Unpaired t test analysis revealed no significant difference between mice carrying UCLA Miniscope and MAPSI in distance (p = 0.1274) or peak speed (p = 0.0826), but mice carrying MAPSI showed reduced average speed (p = 0.0238). * p < 0.05. c) Stimulation pipeline. Simultaneously record and analyze the calcium activity as well as the behavior. Isolate the behaviorally relevant neurons. Find the neurons that co-express both ChR2 and jRCaMP1b. Target neurons with co-expression and replay activity.
Extended Data Fig. 2 Contraversive turning behavior can be reliably elicited by stimulating as few as 3 dSPNs.
a) Calcium imaging of the selected direct pathway neurons for photostimulation. Anywhere from one to five neurons were selectively stimulated. b) Optogenetically stimulating 3 or more neurons reliably produced contraversive turning. c) Turning was significantly higher when exciting 3 or more neurons (One-way ANOVA, F(2,174) = 15.39, p = 0.0006). Tukey’s post hoc analysis revealed no significant difference between 2 neurons and 1 neuron (p = 0.9997), no significant difference between 3 neurons and 1 neuron(p = 0.3750), significant difference between 4 neurons and 1 neuron (p = 0.0454), significant difference between 5 neurons and 1 neuron (p < 0.0001), and significant difference between whole FOV stim and 1 neuron (p < 0.0001). (N = 2 D1-Cre mice, 15 trials for 1 neuron, 13 trials for 2 neurons, 13 trials for 3 neurons, 11 trials for 4 neurons, 19 trials for 5 neurons, and 14 trials for whole FOV). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Extended Data Fig. 3 Ipsiversive turning behavior can be reliably elicited by stimulating as few as 3 iSPNs.
a) Images of the selected indirect pathway neurons for photostimulation. b) Stimulating 3 or more neurons reliably produced contraversive turning. c) Higher number of stimulated neurons increased maximum ipsiversive turning angle (One-way ANOVA, F(4,21) = 6.104, p = 0.0037). Tukey’s post hoc analysis revealed no significant difference between 2 neurons and 1 neuron (p = 0.9999), no significant difference between 3 neurons and 1 neuron(p = 0.0953), significant difference between 4 neurons and 1 neuron (p = 0.0303), and significant difference between whole FOV stim and 1 neuron (p < 0.0001). 2 A2A-Cre mice, 10 trials for 1 neuron, 7 trials for 2 neurons, 9 trials for 3 neurons, 13 trials for 4 neurons, and 11 trials for whole FOV.
Extended Data Fig. 4 There is no significant difference in turning from direct and indirect pathway stimulation using different parameters of stimulation.
a) We optogenetically stimulated neurons in the calcium imaging FOV with 3 different parameters: continuous 500 ms, 20 Hz (10 pulses in 500 ms), and 10 Hz (5 pulses in 500 ms). b) Left) Optogenetic excitation of direct pathway neurons in the DLS using the MAPSI significantly increased contraversive turning. Right) There was no significant stimulation effect (One-way ANOVA, F(2,24)=1.315, p = 0.2872) (N = 2 D1-cre mice, 10 trials for 10 Hz, 8 trials for 20 Hz, and 12 trials for continuous). c) Left) Optogenetic excitation of indirect pathway neurons in the DLS using the MAPSI significantly increased ipsiversive turning behavior (One-way ANOVA, F(2,24)=4.099, p = 0.0294; continuous vs 20 Hz, p = 0.9367; continuous vs 10 Hz, p = 0.0407, 20 Hz vs 10 Hz, p = 0.1080) (2 A2A-cre mice, 12 trials for 10 Hz, 8 trials for 20 Hz, and 8 trials for continuous). * p < 0.05.
Extended Data Fig. 5 Behavioral effect of stimulation is stable across time.
a) Top, a representative A2A-cre mouse was placed in an open field arena where several neurons were selected for stimulation on day 1 of a simultaneous stimulation and recording experiment. Shown on the right are the neurons selected for stimulation. Bottom, the same mouse was then tested 10 days and 40 days later in the open field arena, and the same neurons from day 1 were selectively stimulated. b) There was no significant difference in turning from day 1 compared to day 40 (t(14) = 0.71, p = 0.49, 2 A2A-cre mice, 12 trials on day 1, 13 trials on day 40).
Supplementary information
Supplementary Information
Supplementary figures.
Supplementary Video 1
Photoactivation of a selected neuron in the dorsal striatum (direct pathway).
Supplementary Video 2
Stimulation of 4 indirect pathway neurons that are normally active during ipsiversive turning.
Supplementary Video 3
Sequence stimulation of 5 direct pathway neurons that are normally active during contraversive turning.
Supplementary Video 4
Sweep stimulation of indirect pathway neurons.
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Zhang, J., Hughes, R.N., Kim, N. et al. A one-photon endoscope for simultaneous patterned optogenetic stimulation and calcium imaging in freely behaving mice. Nat. Biomed. Eng 7, 499–510 (2023). https://doi.org/10.1038/s41551-022-00920-3
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DOI: https://doi.org/10.1038/s41551-022-00920-3
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