Extended Data Fig. 8 ∣. Persistent cAMP elevation in the MPOA.

a, Setup and protocol for optogenetic Chrimson stimulation of dopamine axons in the MPOA in awake, head-fixed males while measuring PKA activity using two-photon fluorescence lifetime imaging (FLIM) of the cAMP sensor, cADDis, via a GRIN lens. Stimulation protocol: 10-ms pulses at 10 Hz for 5 s, repeated every 30 seconds. FLIM frames were collected 10 min before stimulation as well as 30, 60, and 120 min after stimulation.

b, Left: mean cADDis fluorescence intensity image. Brighter regions: cell bodies. Scale bar: 200 μm. Right: dopamine stimulation (red dashed line) induced a persistent decrease in cADDis lifetime (increase in cAMP concentration) that gradually returned to baseline over tens of minutes. Scale bar: 200 μm.

c,d, Average lifetime changes across fields-of-view (c: n = 8, 6 fields of view) and across mice (d: n = 4 mice) demonstrated a persistent decrease in lifetime following stimulation (segmentation and neuropil ring subtraction were performed as in FLIM-AKAR imaging, cf. Extended Data Fig. 7c, d).

e, Left: experimental design. Right: head-fixed 2p-FLIM imaging sessions prior to priming (‘A’) and after priming (‘B’) show increased baseline cAMP concentration in the MPOA following priming (n = 6 fields of view from 3 mice, each value is the average of all cells in a field of view). Mean ± s.e.m. ***p<0.001. See Supplementary Table 1 for statistics.