Selective Effects of PDE10A Inhibitors on Striatopallidal Neurons Require Phosphatase Inhibition by DARPP-32

Type 10A phosphodiesterase (PDE10A) is highly expressed in the striatum, in striatonigral and striatopallidal medium-sized spiny neurons (MSNs), which express D1 and D2 dopamine receptors, respectively. PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear. We analyzed the effects of PDE10A inhibition in vivo by immunohistochemistry, and imaged cAMP, cAMP-dependent protein kinase A (PKA), and cGMP signals with biosensors in mouse brain slices. PDE10A inhibition in mouse striatal slices produced a steady-state increase in intracellular cAMP concentration in D1 and D2 MSNs, demonstrating that PDE10A regulates basal cAMP levels. Surprisingly, the PKA-dependent AKAR3 phosphorylation signal was strong in D2 MSNs, whereas D1 MSNs remained unresponsive. This effect was also observed in adult mice in vivo since PDE10A inhibition increased phospho-histone H3 immunoreactivity selectively in D2 MSNs in the dorsomedial striatum. The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for protein phosphatase-1 inhibition. These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs. This study shows that PDE10A inhibitors share with antipsychotic medications the property of activating preferentially PKA-dependent signaling in D2 MSNs.

Decoding spatial and temporal features of neuronal cAMP/PKA signaling with FRET biosensors.

Cyclic adenosine monophosphate (cAMP) and the cyclic-AMP-dependent protein kinase (PKA) regulate a plethora of cellular functions in virtually all eukaryotic cells. In neurons, the cAMP/PKA signaling cascade controls a number of biological properties such as axonal growth, pathfinding, efficacy of synaptic transmission, regulation of excitability, or long term changes. Genetically encoded optical biosensors for cAMP or PKA are considerably improving our understanding of these processes by providing a real-time measurement in living neurons. In this review, we describe the recent progress made in the creation of biosensors for cAMP or PKA activity. These biosensors revealed profound differences in the amplitude of the cAMP signal evoked by neuromodulators between various neuronal preparations. These responses can be resolved at the level of individual neurons, also revealing differences related to the neuronal type. At the sub-cellular level, biosensors reported different signal dynamics in domains like dendrites, cell body, nucleus, and axon. Combining this imaging approach with pharmacology or genetic models points at phosphodiesterases and phosphatases as critical regulatory proteins. Biosensor imaging will certainly emerge as a forefront tool to decipher the subtle mechanics of intracellular signaling. This will certainly help us to understand the mechanism of action of current drugs and foster the development of novel molecules for neuropsychiatric diseases.

The NO/cGMP pathway inhibits transient cAMP signals through the activation of PDE2 in striatal neurons.

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-S(H150)) 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.

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

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 D1 receptors in these two brain structures. Biosensor imaging in mouse brain slice preparations showed profound differences in the D1 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 specific molecular elements of the cAMP/PKA signalling cascade selectively enable the principal striatal neurones to respond to brief dopamine stimuli, a critical process in incentive learning.

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.

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 beta-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 beta(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 beta(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.

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.

Pathway-specific action of gamma-hydroxybutyric acid in sensory thalamus and its relevance to absence seizures.

The systemic injection of gamma-hydroxybutyric acid (GHB) elicits spike and wave discharges (SWDs), the EEG hallmark of absence seizures, and represents a well established, widely used pharmacological model of this nonconvulsive epilepsy. Despite this experimental use of GHB, as well as its therapeutic use in narcolepsy and its increasing abuse, however, the precise cellular mechanisms underlying the different pharmacological actions of this drug are still unclear. Because sensory thalamic nuclei play a key role in the generation of SWDs and sleep rhythms, and because direct injection of GHB in the ventrobasal (VB) thalamus elicits SWDs, we investigated GHB effects on corticothalamic EPSCs and GABAergic IPSCs in VB thalamocortical (TC) neurons. GHB (250 microm-10 mm) reversibly decreased the amplitude of electrically evoked EPSCs and GABAA IPSCs via activation of GABAB receptors; however, approximately 60% of the IPSCs were insensitive to low (250 microm-1.0 mm) GHB concentrations. The putative GHB receptor antagonist NSC 382 applied alone had a number of unspecific effects, whereas it either had no action on, or further increased, the GHB-elicited effects on synaptic currents. Low GHB concentrations (250 microm) were also effective in increasing absence-like intrathalamic oscillations evoked by cortical afferent stimulation. These results indicate that low concentrations of GHB, similar to the brain concentrations that evoke SWDs in vivo, differentially affect excitatory and inhibitory synaptic currents in TC neurons and promote absence-like intrathalamic oscillations. Furthermore, the present data strengthen previous suggestions on the GHB mechanism of sleep promotion and will help focus future studies on the cellular mechanisms underlying its abuse.

Furosemide modulation of GABA(A) receptors in dopaminergic neurones of the rat substantia nigra.

Furosemide is a diuretic which has been shown to decrease recombinant GABA(A) receptor (GABA(A)R)-mediated currents and also to block epileptiform discharges. Here, we show that furosemide actions on GABA(A)Rs of rat substantia nigra dopaminergic neurones depend on both furosemide and GABA(A)R agonist concentrations. The whole-cell currents induced by low concentrations of GABA (5 microM) or by the selective GABA(A)R agonist isoguvacine (7-25 microM) were enhanced by 200 microM furosemide. However, furosemide did not affect GABA(A)R currents induced by 60 microM isoguvacine and even decreased those induced by 200 microM isoguvacine. At the single-channel level, furosemide had comparable effects. It increased the open time proportion with 7 microM isoguvacine but had no significant effect on the open time proportion with 60 microM isoguvacine. These effects resulted from a differential action on the multiple conductance levels activated by GABA(A)R agonists. The concentration-response relationship to isoguvacine in the whole-cell mode revealed the presence of a high and a low apparent affinity GABA(A)R population (EC(50) 4.8 vs 89 microM). These two populations of receptors coexist in the same dopaminergic neurone. They are both furosemide-sensitive and may represent different GABA(A)R subunit assemblies.

Serotonin Induces a Cyclic AMP-Mediated Outwardly Rectifying Slow K+ Current in a Single Identified Snail Neuron.

In the F2 neuron of the parietal ganglion of the snail Helix aspersa either bath or iontophoretic application of serotonin (5-HT) induces an outward current. This current has a long latency (10 - 60 s) and a slow time course, a 500 ms iontophoretic application of 5-HT evoking a response lasting 3 - 5 min. This slow outward current reverses at -80 mV, a value equal to EK. After doubling the extracellular K+ concentration the reversal potential of the 5-HT response is shifted by 19 mV, as predicted by the Nernst equation. The I-V curves reveal that the 5-HT-induced slow outward current is outwardly rectifying. This 5-HT response is blocked by intracellular Cs+ and tetraethylammonium (TEA+) and by extracellular TEA+ and Ba2+, but is not affected by the removal of extracellular Ca2+ or the intracellular injection of ethyleneglycol-bis-(beta-amino-ethylether)-N,N,N',N'-tetra-acetic acid (EGTA). These results indicate that the outwardly rectifying slow outward current induced by 5-HT in the F2 neuron is carried by K+ and is Ca2+-independent. In the single isolated F2 neuron, 5-HT induces a 2.5-fold stimulation of the adenylate cyclase activity. In addition, both the intracellular injection of 3',5'-adenosine monophosphate (cAMP) and the application of forskolin mimic the effect of 5-HT on the F2 neuron. The phosphodiesterase inhibitor isobutylmethylxanthine also induces a slow outward current and potentiates the 5-HT response. The intracellular injection of a synthetic 20-residue peptide inhibitor of the cAMP-dependent protein kinase blocks the slow outward K+ current induced by 5-HT. These results show that in the F2 neuron, 5-HT elicits a slow K+ current via the stimulation of adenylate cyclase, an increase in intracellular cAMP and the activation of the cAMP-dependent kinase which probably phosphorylates a population of outwardly rectifying K+ channels or some protein/s associated with these channels.