4. Picrotoxin (10-6-10-5 M) was effective in blocking both the glutamate inhibitory responses. 10-4 m-picrotoxin, which was necessary to produce a 50% block of the GABA-K+-dependent response, had no effect on the GABA-Cl-response.5. fl-Guanidinopropionic acid (fl-GP) was found to be an agonist for the GABA-K+ response, but was ineffective in mimicking or blocking the GABA-C1-response.6. ACh applications produced large depolarizing responses with a pharmacological profile similar to that of the nicotinic ganglionic response in vertebrates.7. The muscarinic agonist, acetyl-,f-methyl choline (MeCh), produced depolarizations which decreased in amplitude as the membrane was hyperpolarized from -40 to -100 mV. Pilocarpine and oxotremorine produced changes in the endogenous activity of ganglionic neurones.8. Implications of these results for the identification of synaptic transmitters in the stomatogastric ganglion are discussed.

The cAMP-dependent protein kinase A (PKA) plays a ubiquitous role in the regulation of neuronal activity, but the dynamics of its activation have been difficult to investigate. We used the genetically encoded fluorescent probe AKAR2 to record PKA activation in the cytosol and the nucleus of neurons in mouse brain slice preparations, whereas the potassium current underlying the slow afterhyperpolarization potential (sAHP) in thalamic intralaminar neurons was used to monitor PKA activation at the membrane. Adenylyl cyclase was stimulated either directly using forskolin or via activation of 5-HT 7 receptors. Both stimulations produced a maximal effect on sAHP, whereas in the cytosol, the amplitude of the 5-HT 7 receptor-mediated response was half of that after direct adenylyl cyclase stimulation with forskolin. 5-HT 7 -mediated PKA responses were obtained in 30 s at the membrane, in 2.5 min in the cytosol, and in 13 min in the nucleus. Our results show in morphologically intact mammalian neurons the potential physiological relevance of PKA signal integration at the subcellular level: neuromodulators produce fast and powerful effects on membrane excitability, consistent with a highly efficient functional coupling between adenylyl cyclases, PKA, and target channels. Phosphorylation in the cytosol is slower and of graded amplitude, showing a differential integration of the PKA signal between the membrane and the cytosol. The nucleus integrates these cytosolic signals over periods of tens of minutes, consistent with passive diffusion of the free catalytic subunit of PKA into the nucleus, eventually resulting in a graded modulation of gene expression.

Key points• Dopamine D 1 receptors activate the cAMP/protein kinase A (PKA) pathway in both cortex and striatum, with different types of signalling enzymes being involved in cAMP/PKA signal integration. We investigated the functional implications of such differences.• Biosensor imaging in mouse brain slice preparations revealed that the cAMP/PKA signal increases faster, reaches higher levels and lasts longer in striatal neurones than in cortical neurones.• These differences result from faster cAMP production and lower degradation by type 4 phosphodiesterase activities in the striatum than in the cortex. In addition, DARPP-32 in the striatum prolongs the PKA response by inhibiting phosphatases.• These molecular features confers on striatal neurones a particular ability to temporally decode sub-second dopamine signals associated with reward and learning.Abstract The cAMP/protein kinase A (PKA) signalling cascade is ubiquitous, and each step in this cascade involves enzymes that are expressed in multiple isoforms. We investigated the effects of this diversity on the integration of the pathway in the target cell by comparing prefrontal cortical neurones with striatal neurones which express a very specific set of signalling proteins. The prefrontal cortex and striatum both receive dopaminergic inputs and we analysed the dynamics of the cAMP/PKA signal triggered by dopamine D 1 receptors in these two brain structures. Biosensor imaging in mouse brain slice preparations showed profound differences in the D 1 response between pyramidal cortical neurones and striatal medium spiny neurones: the cAMP/PKA response was much stronger, faster and longer lasting in striatal neurones than in pyramidal cortical neurones. We identified three molecular determinants underlying these differences: different activities of phosphodiesterases, particularly those of type 4, which strongly damp the cAMP signal in the cortex but not in the striatum; stronger adenylyl cyclase activity in the striatum, generating responses with a faster onset than in the cortex; and DARPP-32, a phosphatase inhibitor which prolongs PKA action in the striatum. Striatal neurones were also highly responsive in terms of gene expression since a single sub-second dopamine stimulation is sufficient to trigger c-Fos expression in the striatum, but not in the cortex. Our data show how L. R. V. Castro and M. Brito contributed equally to this work and are joint first authors.

We investigated the role of phosphodiesterases (PDEs) in the integration of cAMP signals and protein kinase A (PKA) activity following ␤-adrenergic stimulation, by carrying out real-time imaging of male mouse pyramidal cortical neurons expressing biosensors to monitor cAMP levels (Epac1-camps and Epac2-camps300) or PKA activity (AKAR2). In the soma, isoproterenol (ISO) increased the PKA signal to approximately half the maximal response obtained with forskolin, with a characteristic ␤ 1 pharmacology and an EC 50 of 4.5 nM. This response was related to free cAMP levels in the submicromolar range. The specific type 4 PDE (PDE4) inhibitor rolipram had a very small effect alone, but strongly potentiated the PKA response to ISO. Blockers of other PDEs had no effect. PDE4 thus acts as a brake in the propagation of the ␤ 1 -adrenergic signal from the membrane to the bulk somatic cytosol. The results for a submembrane domain were markedly different, whether recorded with a PKA-sensitive potassium current related to the slow AHP or by two-photon imaging of small distal dendrites. The responses to ISO were stronger than in the bulk cytosol. This is consistent with the cAMP/PKA signal being strong at the membrane, as shown by electrophysiology, and favored in cellular domains with a high surface area to volume ratio, in which this signal was detected by imaging. Rolipram alone also produced a strong cAMP/PKA signal, revealing tonic cAMP production. PDE4 thus appears as a crucial integrator with different physiological implications in different subcellular domains.

The NO-cGMP signaling plays an important role in the regulation of striatal function although the mechanisms of action of cGMP specifically in medium spiny neurons (MSNs) remain unclear. Using genetically encoded fluorescent biosensors, including a novel Epac-based sensor (EPAC-SH150) with increased sensitivity for cAMP, we analyze the cGMP response to NO and whether it affected cAMP/PKA signaling in MSNs. The Cygnet2 sensor for cGMP reported large responses to NO donors in both striatonigral and striatopallidal MSNs, this cGMP signal was controlled partially by PDE2. At the level of cAMP brief forskolin stimulations produced transient cAMP signals which differed between D1 and D2 MSNs. NO inhibited these cAMP transients through cGMP-dependent PDE2 activation, an effect that was translated and magnified downstream of cAMP, at the level of PKA. PDE2 thus appears as a critical effector of NO which modulates the post-synaptic response of MSNs to dopaminergic transmission.