Extended Data Fig. 1: Awake two-photon imaging to study cortical lactate and calcium dynamics in neurons and astrocytes (Related to Fig. 1).

(a) Workflow scheme of preparing mice for awake two-photon imaging. Following recovery from surgery and intracortical AAV injections, mice were trained for head-restrained imaging using a suppression-response task and a water reward. On average, mice were ready for imaging at around 4 weeks after surgery. This protocol was repeated for all animals in this study with similar results. (b) Behavioral paradigm used during awake imaging. Each trial consisted of an 8 s imaging period (IM) which ended with a 1 ms auditory cue (AC) indicating the mouse to collect the water reward within a lick period of 2 s (LP). Each trial had an intertrial interval (ITI) of 2 s before the next trial started. (c) Mean behavioral performance (grey) and learning progress (black) during training. A trial was counted correct when no licking occurred during image acquisition (IM). N = number of animals, n = number of sessions. (d) Cortical expression of genetically-encoded lactate sensor Laconic (San Martin et al., ²⁵), calcium sensor RCaMP 1.07 (Ohkura et al., ²⁷) and GCaMP6s (Chen et al., ^26,32) imaged through a chronic cranial window. Visualization with single photon excitation. M = medial, L = lateral, C = cranial, R = rostral. (e) Representative images of Laconic expression in neurons and astrocytes (mTFP = blue; Venus = yellow), RCaMP 1.07 in neurons (mApple = red) and GCaMP6s in astrocytes (cpGFP = green) and a merge of calcium sensors expressed at the same site (neurons = red, astrocytes = green). Excitation wavelength for Laconic = 870 nm. Wavelength for simultaneous excitation of GCaMP6s and RCaMP 1.07 = 940 nm. Images were taken 121.4 ± 26.9 µm below cortical surface.