NEUROMODULATION OF INHIBITORY BOUTON FORMATION VIA PKA

Neuromodulators, including endocannabinoids, norepinephrine, and dopamine, are well known to shape network activation patterns in response to environmental stimuli, providing a cellular integration of contextual information. We recently demonstrated that, after elevated excitatory activity, local endocannabinoid signaling promotes the formation of new boutons in inhibitory axons to maintain dendritic excitation/inhibition coordination. The underlying molecular mechanism involves increased cyclic adenosine monophosphate (cAMP) and activation of protein kinase A (PKA) in inhibitory axons. Because other neuromodulators also activate cAMP/PKA, this pathway might serve as a common mediator of inhibitory bouton formation. However, the spatiotemporal regulation of neuromodulator signaling in inhibitory neurons remains poorly understood. Here, we characterized bouton formation and PKA dynamics in response to norepinephrine and dopamine signaling in inhibitory axons of CA1 hippocampal organotypic slices using novel PKA biosensors for two-photon fluorescence lifetime imaging. We observed that 5 minutes dopaminergic receptor activation promoted inhibitory bouton formation, whereas adrenergic receptor activation was insufficient. Consistently, brief dopaminergic application robustly activated PKA, whereas norepinephrine elicited only a weak response. Sustained norepinephrine, however, robustly activated PKA to levels comparable to dopamine response. These results suggest a PKA activation threshold that determines inhibitory bouton formation. Together, our findings identify PKA as a key effector of neuromodulator signaling to trigger bouton formation in inhibitory axons. While primarily recognized for modulating brain network activation patterns, our results suggest that neuromodulators also control local neuronal network connectivity. Currently, we are characterizing the spatiotemporal dynamics of PKA activity following local receptor activation and its link to bouton formation.