A complete pupillometry toolbox for real-time monitoring of locus coeruleus activity in rodents


The locus coeruleus (LC) is a region in the brainstem that produces noradrenaline and is involved in both normal and pathological brain function. Pupillometry, the measurement of pupil diameter, provides a powerful readout of LC activity in rodents, primates and humans. The protocol detailed here describes a miniaturized setup that can screen LC activity in rodents in real-time and can be established within 1–2 d. Using low-cost Raspberry Pi computers and cameras, the complete custom-built system costs only ~300 euros, is compatible with stereotaxic surgery frames and seamlessly integrates into complex experimental setups. Tools for pupil tracking and a user-friendly Pupillometry App allow quantification, analysis and visualization of pupil size. Pupillometry can discriminate between different, physiologically relevant firing patterns of the LC and can accurately report LC activation as measured by noradrenaline turnover. Pupillometry provides a rapid, non-invasive readout that can be used to verify accurate placement of electrodes/fibers in vivo, thus allowing decisions about the inclusion/exclusion of individual animals before experiments begin.

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Fig. 1: Pupillometry using a small and lightweight camera.
Fig. 2: Pupillometry as a real-time readout for LC activation.
Fig. 3: Pupillometry is a sensitive readout of physiologically relevant modes of LC activity.
Fig. 4: Repeated pupillometry recordings with various stimulation parameters in females and males.

Data availability

The datasets generated during the current study (‘Anticipated results’) are available from the corresponding authors upon request.

Code availability

All software and code described in this protocol are freely available online:

Raspberry Pi Code https://github.com/ein-lab/pupillometry-raspi

MATLAB Code https://github.com/ein-lab/pupillometry-matlab

Pupillometry App

https://bohaceklab.hest.ethz.ch/pupillometry/ (web version)

https://github.com/ETHZ-INS/pupillometry (source code)


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The authors acknowledge Rongrong Xiang and Matthew J. P. Barrett for their initial work on the MATLAB analysis, Marc Zuend for extensive testing of prototypes, Christa Schläppi for testing the pupillometry guidelines and Alexandra von Faber-Castell for testing the assembly guide.

Author information




Conceptualization, M.P., K.D.F., B.W. and J.B.; methodology, M.P., K.D.F. and O.S.; investigation, M.P., K.D.F, A.F.-S., S.N.D., Y.V. and M.T.W.; software, K.D.F., L.M.v.Z., P.-L.G. and O.S.; writing—original draft, M.P., K.D.F, L.M.v.Z., O.S., S.N.D. and J.B.; figures, M.P. and K.D.F.; writing—review and editing, M.P., K.D.F., L.M.v.Z., O.S., A.F.-S., P.-L.G., Y.V., S.N.D., M.T.W., P.P.D.D., B.W. and J.B.; funding acquisition, B.W., P.P.D.D. and J.B.; resources, B.W. and J.B.

Corresponding authors

Correspondence to Bruno Weber or Johannes Bohacek.

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The authors declare no competing interests.

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Key references using this protocol

Zerbi, V. et al. Neuron 103, 702–718.e5 (2019): https://doi.org/10.1016/j.neuron.2019.05.034

Zuend, M. et al. Nat. Metab. 2, 179–191 (2020): https://doi.org/10.1038/s42255-020-0170-4

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Supplementary Information

Supplementary Methods, Supplementary Figures 1 and 2 and Supplementary Manual.

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Privitera, M., Ferrari, K.D., von Ziegler, L.M. et al. A complete pupillometry toolbox for real-time monitoring of locus coeruleus activity in rodents. Nat Protoc 15, 2301–2320 (2020). https://doi.org/10.1038/s41596-020-0324-6

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