OptoPi: An open source flexible platform for the analysis of small animal behaviour

Behaviour is the ultimate output of neural circuit computations, and therefore its analysis is a cornerstone of neuroscience research. However, every animal and experimental para-digm requires different illumination conditions to capture and, in some cases, manipulate specific behavioural features. This means that researchers often develop, from scratch, their own solutions and experimental set-ups. Here, we present OptoPi, an open source, affordable (- & POUND;600), behavioural arena with accompanying multi-animal tracking soft-ware. The system features highly customisable and reproducible visible and infrared illu-mination and allows for optogenetic stimulation. OptoPi acquires images using a Raspberry Pi camera, features motorised LED-based illumination, Arduino control, as well as irradiance monitoring to fine-tune illumination conditions with real time feedback. Our open-source software (BioImageProcessing) can be used to simultaneously track multiple unmarked animals both in on-line and off-line modes. We demonstrate the functionality of OptoPi by recording and tracking under different illumination conditions the sponta-neous behaviour of larval zebrafish as well as adult Drosophila flies and their first instar lar-vae, an experimental animal that due to its small size and transparency has classically been hard to track. Further, we showcase OptoPi's optogenetic capabilities through a series of experiments using transgenic Drosophila larvae.& COPY; 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).