Pierre Vincent

Simultaneous measurements of intracellular cAMP and L‐type Ca2+ current in single frog ventricular myocytes

1 The cAMP fluorescent probe FlCRhR was used to monitor changes in intracellular cAMP concentration ([cAMP]i) in isolated frog ventricular myocytes. The probe was introduced into the cell through a patch pipette which allowed simultaneous recording of the whole‐cell L‐type Ca2+ current (ICa). Ratiometric imaging was used to monitor [cAMP]i changes in response to the β‐adrenergic agonist isoprenaline (ISO) or to the direct adenylyl cyclase activator forskolin (FSK). 2 FlCRhR fluorescence was distributed in the cytosol in a striated pattern, with high fluorescence in the I‐bands and low fluorescence in the A‐bands. This pattern of distribution was mimicked by fluorescein dextran, another high molecular weight fluorescent molecule, and was therefore likely to be due to anisotropic diffusion of the probe in the cytosol due to the hindrance generated by sarcomeric proteins in the A‐bands. 3 Introduction of FlCRhR into the cell induced a small ≈70% stimulatory effect on basal ICa, attenuating about 2‐fold a subsequent response of ICa to 1‐10 μm ISO (from 400 to 200%). 4 Brief (10 s) application of a saturating concentration of ISO (1‐20 μm) to the cell induced a transient increase in both ICa and [cAMP]i. However, the [cAMP]i transient was ≈2‐fold shorter in duration than the ICa transient, i.e. ICa was still strongly enhanced when [cAMP]i had already returned to control level. This indicates that hydrolysis of cAMP by phosphodiesterases is not a rate limiting step in the recovery of ICa from ISO stimulation. 5 When the application of ISO was maintained, ICa and [cAMP]i responses followed a similar time course, with a half‐maximal response at ≈60 s. This suggests that activation of Ca2+ channels by cAMP‐dependent protein kinase occurs on a much faster time scale than the rise in [cAMP]i. 6 When the cells were exposed to FSK (13 μm), both responses of ICa and [cAMP]i were ≈2‐fold slower than with ISO. This demonstrates that the slower response of ICa to FSK is due to a slower rise in [cAMP]i rather than to some inhibitory effect of FSK on ICa or to a direct or priming effect of the stimulatory G protein Gs on Ca2+ channels. 7 Simultaneous measurements of [cAMP]i and ICa changes in intact cardiac myocytes opens the way to dissect the temporal sequence of events in the cAMP cascade mediating the response of the heart to a large number of hormones and inotropic agents.

Dynamics of protein kinase A signaling at the membrane, in the cytosol, and in the nucleus of neurons in mouse brain slices

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-HT7 receptors. Both stimulations produced a maximal effect on sAHP, whereas in the cytosol, the amplitude of the 5-HT7 receptor-mediated response was half of that after direct adenylyl cyclase stimulation with forskolin. 5-HT7-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.

Live imaging of neural structure and function by fibred fluorescence microscopy

Only a few methods permit researchers to study selected regions of the central and peripheral nervous systems with a spatial and time resolution sufficient to image the function of neural structures. Usually, these methods cannot analyse deep‐brain regions and a high‐resolution method, which could repeatedly probe dynamic processes in any region of the central and peripheral nervous systems, is much needed. Here, we show that fibred fluorescence microscopy—which uses a small‐diameter fibre‐optic probe to provide real‐time images—has the spatial resolution to image various neural structures in the living animal, the consistency needed for a sequential, quantitative evaluation of axonal degeneration/regeneration of a peripheral nerve, and the sensitivity to detect calcium transients on a sub‐second timescale. These unique features should prove useful in many physiological studies requiring the in situ functional imaging of tissues in a living animal.

Type 4 Phosphodiesterase Plays Different Integrating Roles in Different Cellular Domains in Pyramidal Cortical Neurons

We investigated the role of phosphodiesterases (PDEs) in the integration of cAMP signals and protein kinase A (PKA) activity following β-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 β1 pharmacology and an EC50 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 β1-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.

Serotonin suppresses the slow afterhyperpolarization in rat intralaminar and midline thalamic neurones by activating 5-HT7 receptors

While the highest expression level of 5‐HT7 receptors in the brain is observed in intralaminar and midline thalamic neurones, the physiological role of these receptors in this structure is unknown. In vivo recordings have shown that stimulation of the serotonergic raphe nuclei can alter the response of these neurones to a nociceptive stimulus, suggesting that serotonin modulates their firing properties. Using the patch‐clamp technique in rat thalamic brain slices, we demonstrate that activation of 5‐HT7 receptors can strongly modulate the excitability of intralaminar and midline thalamic neurones by inhibiting the calcium‐activated potassium conductance that is responsible for the slow afterhyperpolarization (sAHP) following a spike discharge. This sAHP was inhibited after activation of the cAMP pathway, either by bath application of forskolin or intracellular perfusion with 8‐bromo‐cAMP. The inhibitory effect of 5‐HT7 receptors on sAHPs was blocked by the protein kinase A antagonist RP‐cAMPS. Calcium‐imaging experiments showed no change in intracellular calcium levels during the 5‐HT7 response, indicating that in these neurones, a global calcium signal was not necessary to activate the cAMP cascade. Finally, bath application of serotonin produced a strong increase in cytosolic cAMP concentration, as measured using the fluorescent probe FlCRhR, and an inhibition of the sAHP. Taken together, these results suggest that 5‐HT7 receptors are implicated in the effect of 5‐HT on sAHP in intralaminar and midline thalamic neurones, an effect that is mediated by the cAMP second‐messenger cascade.

Striatal neurones have a specific ability to respond to phasic dopamine release

•  Dopamine D1 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.

Phosphodiesterase type 2 and the homeostasis of cyclic GMP in living thalamic neurons

The ubiquitous second messenger cyclic GMP (cGMP) is synthesized by soluble guanylate cyclases in response to nitric oxide (NO) and degraded by phosphodiesterases (PDE). We studied the homeostasis of cGMP in living thalamic neurons by using the genetically encoded fluorescence resonance energy transfer sensor Cygnet, expressed in brain slices through viral gene transfer. Natriuretic peptides had no effect on cGMP. Basal cGMP levels decreased upon inhibition of NO synthases or soluble guanylate cyclases and increased when PDEs were inhibited. Single cell RT‐PCR analysis showed that thalamic neurons express PDE1, PDE2, PDE9, and PDE10. Basal cGMP levels were increased by the PDE2 inhibitors erythro‐9‐(2‐hydroxy‐3‐nonyl) adenine (EHNA) and BAY60‐7550 but were unaffected by PDE1 or PDE10 inhibitors. We conclude that PDE2 regulates the basal cGMP concentration in thalamic neurons. In addition, in the presence of 3‐isobutyl‐1‐methylxanthine (IBMX), cGMP still decreased after application of a NO donor. Probenecid, a blocker of cGMP transporters, had no effect on this decrease, leaving PDE9 as a possible candidate for decreasing cGMP concentration. Basal cGMP level is poised at an intermediate level from which it can be up or down‐regulated according to the cyclase and PDE activities.

VIP, CRF, and PACAP Act at Distinct Receptors to Elicit Different cAMP/PKA Dynamics in the Neocortex

The functional significance of diverse neuropeptide coexpression and convergence onto common second messenger pathways remains unclear. To address this question, we characterized responses to corticotropin-releasing factor (CRF), pituitary adenylate cyclase-activating peptide (PACAP), and vasoactive intestinal peptide (VIP) in rat neocortical slices using optical recordings of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) sensors, patch-clamp, and single-cell reverse transcription-polymerase chain reaction. Responses of pyramidal neurons to the 3 neuropeptides markedly differed in time-course and amplitude. Effects of these neuropeptides on the PKA-sensitive slow afterhyperpolarization current were consistent with those observed with cAMP/PKA sensors. CRF-1 receptors, primarily expressed in pyramidal cells, reportedly mediate the neocortical effects of CRF. PACAP and VIP activated distinct PAC1 and VPAC1 receptors, respectively. Indeed, a selective VPAC1 antagonist prevented VIP responses but had a minor effect on PACAP responses, which were mimicked by a specific PAC1 agonist. While PAC1 and VPAC1 were coexpressed in pyramidal cells, PAC1 expression was also frequently detected in interneurons, suggesting that PACAP has widespread effects on the neuronal network. Our results suggest that VIP and CRF, originating from interneurons, and PACAP, expressed mainly by pyramidal cells, finely tune the excitability and gene expression in the neocortical network via distinct cAMP/PKA-mediated effects.

A TASK3 channel (KCNK9) mutation in a genetic model of absence epilepsy

Childhood absence epilepsy is an idiopathic, generalized, nonconvulsive epilepsy with a multifactorial genetic etiology. The KCNK9 gene coding for the TASK3 (Twik-like acid-sensitive K+) channel is present on chromosome 8 at position 8q24, a locus that has shown positive linkage to the human absence epilepsy phenotype. Sequencing of the KCNK9 gene in the genetic absence epilepsy rats from Strasbourg (GAERS), a well established genetic model of this disease, reveals an additional alanine residue in a polyalanine tract within the C-terminal intracellular domain. This additional alanine is absent in the inbred nonepileptic control (NEC) strain, Wistar, and Wistar albino Glaxo strain bred in Rijswijk, another inbred rat model of absence epilepsy. Expression of the mutant channel in CHO cells produces a K+ current that is blocked by acidic pH and millimolar concentrations of barium or ruthenium red and is not different from the wild-type channel. In brain slices, thalamic neurons display a prominent pH-sensitive tonic K+ current, but no difference was observed between GAERS and NEC or Wistar rats. Ruthenium red had no effect in cortical, reticular thalamic, or sensory thalamic neurons in either GAERS or NEC, indicating that the TASK3 homodimer is not present in these structures. Twik-like acid-sensitive K+ (TASK3) channels, therefore, are probably associated with TASK1 to form ruthenium red-insensitive heterodimers in these neurons. Finally, no difference was found between GAERS and NEC rats in the modulation of the leak K+ current following activation of muscarinic receptors. These studies describe the first mutation found in a genetic model of absence epilepsy. Although our experiments showed no difference in the leak K+ current between GAERS and NEC rats, further work is needed to ascertain whether this mutation contributes to the generation of absence seizures, possibly by mechanisms related to the expansion of the polyalanine run.

Cyclic AMP imaging in neurones in brain slice preparations.

The second messenger cascade of cyclic AMP (cAMP) plays an important physiological role in neurones, modulating neuronal excitability and synaptic transmission. The fluorescent probe FlCRhR allows real time ratiometric imaging of cAMP changes inside cells (Nature 349 (1991) 694). Until now, the only way to introduce FlCRhR into cells was microinjection, which restricted the use of FlCRhR to large invertebrate neurones. This report describes the use of the patch-clamp technique to deliver FlCRhR into the cytosol of several types of neurones in brain slice preparations. Direct activation of adenylate cyclase by forskolin produced marked increases in fluorescence ratio, confirming that the probe can report cAMP increases. However, some neurones failed to exhibit a cAMP response and this lack of response was related to the nucleus integrity. Stimulation of membrane receptors positively coupled to adenylate cyclase elicited cAMP increases in various neuronal cell types. This is the first report of a cAMP response to neuromodulators measured by an imaging technique in neurones in brain slices. The method described here could find many applications such as testing the ability of agonists to specifically activate the cAMP cascade in identified neurones, studying the kinetics of the cAMP response and determining the subcellular localisation of cAMP changes.