Two-photon microscopy is widely used to investigate brain function across multiple spatial scales. However, measurements of neural activity are compromised by brain movement in behaving animals. Brain motion-induced artefacts are typically corrected using post-hoc processing of 2D images, but this approach is slow and does not correct for axial movements. Moreover, the deleterious effects of brain movement on high speed imaging of small regions of interest and photostimulation cannot be corrected post-hoc . To address this problem, we combined random access 3D laser scanning using an acousto-optic lens and rapid closed-loop FPGA processing to track 3D brain movement and correct motion artifacts in real-time at up to 1 kHz. Our recordings from synapses, dendrites and large neuronal populations in behaving mice and zebrafish demonstrate real-time movement corrected 3D two-photon imaging with sub-micrometer precision.

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Golgi cells (GoCs) are the main inhibitory interneurons in the input layer of the cerebellar cortex and are electrically coupled together, forming syncytia. GoCs control the excitability of granule cells (GCs) through feedforward, feedback and spillovermediated inhibition. The GoC circuit therefore plays a central role in determining how sensory and motor information is transformed as it flows through the cerebellar input layer. Recent work has shown that GCs are activated when animals perform active behaviours, but the underlying mechanisms remain poorly understood. Norepinephrine (NE), also known as noradrenaline, is a powerful modulator of network function during active behavioral states and the axons of NE-releasing neurons in the locus coeruleus innervate the cerebellar cortex. Here we show that NE hyperpolarizes the GoC membrane potential, decreases spontaneous firing and reduces the gain of the spike frequency versus input-current relationship. The GoC membrane hyperpolarization can be mimicked with an α2-noradrenergic agonist, inhibited with a specific α2 antagonist and is abolished when G protein-coupled inwardly-rectifying potassium (GIRK) channels are blocked. Moreover, NE reduces the effective electrical coupling between GoCs through a persistent sodium current (INaP)-dependent mechanism. Our results suggest that NE controls the gain of the GoC inhibitory circuit by modulating membrane conductances that act to reduce membrane excitability and decrease electrical coupling. These mechanisms appear configured to reduce the level of GoC inhibition onto GCs during active behavioural states.