PDE4 Control on cAMP/PKA Compartmentation Revealed by Biosensor Imaging in Neurons

The signaling pathway of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) is responsible for the modulation of various processes in the brain, including some specific forms of synaptic plasticity, control of excitability, regulation of nuclear factors and imprinting of long-term changes. Our interest focuses on how neuromodulatory signals such as monoamines or neuropeptides are integrated through the cAMP/PKA cascade within the complex tridimensional structure of the neuron, leading to appropriate adaptations of neuronal function.

We developed a new methodological approach to directly address the dynamics of the cAMP/PKA signal in morphologically intact neurons. FRET biosensors are expressed using viral vectors in brain slice preparations from neonatal rats or mice. Changes in the signaling cascade are imaged in real time with wide-field or two-photon microscopy. We measure free cAMP concentrations using Epac1-camps (Kd=2.4µM) or Epac2-camps300 biosensors (Kd=0.32µM). We monitor PKA activation with AKAR2.2 or AKAR3 in the cytosol and specifically in the nucleus with an added NLS tag. Besides, we use electrophysiology to monitor PKA activity at the membrane by its effect on a potassium current.

These combined approaches were applied to study the responses evoked by different neuromodulators like serotonin, noradrenaline, dopamine, VIP, CRF and PACAP, which activate the Gs-coupled receptors 5-HT7, beta1-adrenergic, D1-like, VPAC1, CRF1, PAC1 respectively. These experiments, performed in pyramidal cortical neurons and in thalamic neurons, revealed several important points: Without any added neuromodulator, there is a tonic cAMP production at the membrane which maintains a tonic PKA activity level. This tonic activity is detected either by electrophysiology or when imaging small dendrites.

In response to the stimulation of membrane receptors, the cAMP/PKA signal generated at the membrane is strong, then declines in efficacy and slows-down in kinetics as it propagates from the membrane towards the somatic cytosol and the nucleus.

In the somatic cytosol, the increases in free cAMP concentration remains surprisingly low, below micromolar level, in contrast to most other preparations like embryonic neurons, cardiomyocytes and cultured cell lines.

For different neuromodulators, the amplitude of the PKA response in the somatic cytosol ranged from 30% up to 75% of full probe activation, with correspondingly graded responses in the nucleus, indicating a well regulated mechanism specific for each neuromodulator.

PDE4 plays a critical role in degrading the tonically produced cAMP, maintaining the system in an equilibrium. PDE4 activity also controls the amplitude of the responses evoked by neuromodulators. These two effects may account for the beneficial effect of rolipram (which was tested as an antidepressant and procognitive drug), in compensating for pathologically low levels of neuromodulators and lack of neuronal activity.