Figure 4. Enhancing cAMP breakdown in BA glutamatergic neurons disrupts learning of motivational salience.

A) Schematic of phosphodiesterase 4D3 catalytic domain (PDE4D3-cat) enhancing the breakdown of cAMP production following biPAC photostimulation (left) and brain slice two-photon imaging (right).

B) Example images of amygdala brain slice (top left) and cADDis fluorescence in BA glutamatergic neurons (top right). Example image showing neurons expressing cADDis (bottom left) and mKate2-biPAC with mKate2-PDE4D3-cat (bottom right).

C) Left: stimulation of biPAC (2 s duration; 120 s ITI) in control neurons (black; n = 13 slices, 4 mice) or neurons expressing PDE4D3-cat (green; n = 14 slices, 5 mice). Right: same as left for 30 s ITI in control (black; n = 11 slices, 4 mice) or PDE4D3-cat (green; n = 7 slices, 3 mice) neurons.

D) Stimulation of biPAC (2 s) in neurons expressing PDE4D3-cat prior to (green; n = 5 slices, 4 mice) or following application of a phosphodiesterase inhibitor (IBMX, 100 μM; red; n = 5 slices, 4 mice).

E) Top: viral construct for PDE4D3-cat. Bottom: viral delivery of PDE4D3-cat in BA glutamatergic neurons.

F) Left: schematic of visual stimulus and Ensure delivery to an awake, food restricted, head-fixed mouse. Right: training paradigm: habituation following by consecutive days of pairing cue with Ensure (60 trials/day; 30 s ITI).

G) Mean baseline-subtracted lick rate per day throughout Pavlovian conditioning, for control (top; n = 7) or PDE4D3-cat expressing mice (bottom; n = 6).

H) Top: mean baseline-subtracted lick rate during the last second of the 2 s cue, normalized to maximum cue-evoked lick rate across days, for control (n = 7) or PDE4D3-cat expressing mice (n = 6). Bottom: maximum lick rate during each bin across control (black) or PDE4D3-cat expressing (green) mice.

I) Latency to half maximum cue-evoked lick rate. ***, p = 0.0007, two-tailed unpaired t-test. All panels: mean ± s.e.m.