A method for exploring input-output relations of neurons in awake mice

Measuring input-output relations of single neurons in awake animals is essential for understanding how information is processed in the brain. So far, the most complete input-output relations were measured in brain slices. However, brain slices lack physiological input to dendrites and soma. Here we describe a method that combines two-photon microscopy and electrophysiology to simultaneously measure dendritic voltage and calcium signals (inputs) and somatic output from cerebellar Purkinje cells (PC) in anesthetized and awake mice. To record dendritic voltage changes we label single PCs with the synthetic, purely electrochromic voltage sensitive dye ANNINE-6plus (Fromherz et al. 2008), using a chronic cranial window with access port (Roome and Kuhn, 2014). To reduce phototoxicity and increase signal amplitude we excite at the red spectral excitation edge (Kuhn and Fromherz 2003; Kuhn, Fromherz, Denk 2004). The typical signal amplitude for dendritic voltage signals during a complex spike is 10 to 15%. We record dendritic activity with line scans at a temporal resolution of 2 kHz. For dendritic calcium recording, adeno-associated viruses delivering the gene of the genetically encoded calcium indicator GCaMP6f (Chen et al 2013), can be injected prior to ANNINE-6plus labelling. Extracellular electrophysiology can also be performed at the labelled PC soma to record their somatic activity. We combine these techniques with EEG and behavioural monitoring while the mouse is resting or moving on a cylindrical treadmill. By recording spontaneous input-output relations at a single cell level within the intact brain and simultaneous monitoring of behavior, we hope to address important questions concerning neuronal computations in vivo.