Vermifiltration as a green solution to promote digestate reuse in agriculture in small-scale farms.

Digestates from low-tech digesters need to be post-treated to ensure their safe agricultural reuse. This study evaluated, for the first time, vermifiltration as a post-treatment for the digestate from a low-tech digester implemented in a small-scale farm, treating cattle manure and cheese whey under psychrophilic conditions. Vermifiltration performance was monitored in terms of solids, organic matter, nutrients, and pathogens removal efficiency. In addition, the growth of earthworms (Eisenia foetida) and their role in the process was evaluated. Finally, the vermicompost and the effluent of the vermifilter were characterized in order to assess their potential reuse in agriculture. Vermifilters showed high removal efficiency of chemical oxygen demand (55-90%), total solids (60-80%), ammonium nitrogen (83-97%), and phosphate-P (28-49%). Earthworms effectively grew and reproduced on digestate (i.e. earthworms number increased by 183%), enhancing the vermifiltration performance, while reducing clogging and odour-related issues. Both the vermicompost and effluent produced complied with legislation limits established for soil improvers and wastewater for fertigation, respectively. Indeed, there was an absence of pathogens and non-detectable heavy metals concentrations. Vermifiltration may be thus considered a suitable post-treatment option for the digestate from low-tech digesters, allowing for its safe agricultural reuse and boosting the circular bioeconomy in small-scale farms.

Selective changes in cytosolic ß-adrenergic cAMP signals and L-type Calcium Channel regulation by Phosphodiesterases during cardiac hypertrophy.

Background In cardiomyocytes, phosphodiesterases (PDEs) type 3 and 4 are the predominant enzymes that degrade cAMP generated by ß-adrenergic receptors (ß-ARs), impacting notably the regulation of the L-type Ca2+ current (ICa,L). Cardiac hypertrophy (CH) is accompanied by a reduction in PDE3 and PDE4, however, whether this affects the dynamic regulation of cytosolic cAMP and ICa,L is not known. Methods and Results CH was induced in rats by thoracic aortic banding over a time period of five weeks and was confirmed by anatomical measurements. Left ventricular myocytes (LVMs) were isolated from CH and sham-operated (SHAM) rats and transduced with an adenovirus encoding a Förster resonance energy transfer (FRET)-based cAMP biosensor or subjected to the whole-cell configuration of the patch-clamp technique to measure ICa,L. Aortic stenosis resulted in a 46% increase in heart weight to body weight ratio in CH compared to SHAM. In SHAM and CH LVMs, a short isoprenaline stimulation (Iso, 100 nM, 15 s) elicited a similar transient increase in cAMP with a half decay time (t1/2off) of ~50 s. In both groups, PDE4 inhibition with Ro 20-1724 (10 µM) markedly potentiated the amplitude and slowed the decline of the cAMP transient, this latter effect being more pronounced in SHAM (t1/2off ~ 250 s) than in CH (t1/2off ~ 150 s, P < 0.01). In contrast, PDE3 inhibition with cilostamide (1 µM) had no effect on the amplitude of the cAMP transient and a minimal effect on its recovery in SHAM, whereas it potentiated the amplitude and slowed the decay in CH (t1/2off ~ 80 s). Iso pulse stimulation also elicited a similar transient increase in ICa,L in SHAM and CH, although the duration of the rising phase was delayed in CH. Inhibition of PDE3 or PDE4 potentiated ICa,L amplitude in SHAM but not in CH. Besides, while only PDE4 inhibition slowed down the decline of ICa,L in SHAM, both PDE3 and PDE4 contributed in CH. Conclusion These results identify selective alterations in cytosolic cAMP and ICa,L regulation by PDE3 and PDE4 in CH, and show that the balance between PDE3 and PDE4 for the regulation of ß-AR responses is shifted toward PDE3 during CH.

Evaluation of heavy metal contamination levels in river sediments and their risk to human health in urban areas: A case study in the Matanza-Riachuelo Basin, Argentina.

Anthropogenic contamination of Cd, Cr, Cu, Ni, Pb and Zn in riverbed sediments of the Pampean area (Matanza-Riachuelo Basin, MRB), Argentina, was evaluated using various indices: Geo-accumulation Index (Igeo), Contamination Factor (Cf), Metal Pollution Index (MPI), Pollution Load Index (PLI) and Contamination Degree (Cdeg). A database based on previous samplings of heavy metal concentrations at different sites in the Upper, Middle and Lower MRB was used. The Igeo and Cf values are calculated using selected local background (Pristine Pampean loess, LZB), and global average shale values (ASTW). Paired Igeo and Cf results with LZB and ASTW fall in dissimilar classes, especially in respect of Cd. In turn, MPI, PLI and Cdeg show similar results, as they do not have the same degree of confidence as Igeo and Cf. The Upper Basin, mainly rural, shows the following profile of sediment contamination: Cd > Ni > Cu â‰« Pb > Zn > Cr. The Middle Basin, which is at the periphery of a very industrialised and urban area, displays the following contamination profile for both Igeo and Cf: Cu > Pb > Zn > Ni > Cd > Cr. Conversely, in the Lower Basin, where land-use is urban/industrial, the levels of metal contamination are higher and appear in this order: Cr > Pb > Cu > Zn > Ni > Cd. Cadmium, Cu, Ni and Zn have shown no significant oral and/or dermal HQ nor carcinogenic risk but the potential risk posed by Cr and Pb must be taken into account. Maximum and mean values of Cr total carcinogenic risk (TCR) as well as Ni TCR also point to a health risk to children in the Lower Basin Area. All the data analysed confirm that industrial and urban growth without land-use planning, and a poor environmental legislation until the beginning of the 21st century, have not provided the necessary framework for sustainable development in the MRB.

Switch-like PKA responses in the nucleus of striatal neurons.

Although it is known that protein kinase A (PKA) in the nucleus regulates gene expression, the specificities of nuclear PKA signaling remain poorly understood. Here, we combined computational modeling and live-cell imaging of PKA-dependent phosphorylation in mouse brain slices to investigate how transient dopamine signals are translated into nuclear PKA activity in cortical pyramidal neurons and striatal medium spiny neurons. We observed that the nuclear PKA signal in striatal neurons featured an ultrasensitive responsiveness, associated with fast all-or-none responses, which is not consistent with the commonly accepted theory of a slow and passive diffusion of catalytic PKA in the nucleus. Our numerical model suggests that a positive feed-forward mechanism inhibiting nuclear phosphatase activity - possibly mediated by DARPP-32 (also known as PPP1R1B) - could be responsible for this non-linear pattern of nuclear PKA response, allowing for a better detection of the transient dopamine signals that are often associated with reward-mediated learning.

Phosphodiesterase 1 Bridges Glutamate Inputs with NO- and Dopamine-Induced Cyclic Nucleotide Signals in the Striatum.

The calcium-regulated phosphodiesterase 1 (PDE1) family is highly expressed in the brain, but its functional role in neurones is poorly understood. Using the selective PDE1 inhibitor Lu AF64196 and biosensors for cyclic nucleotides including a novel biosensor for cGMP, we analyzed the effect of PDE1 on cAMP and cGMP in individual neurones in brain slices from male newborn mice. Release of caged NMDA triggered a transient increase of intracellular calcium, which was associated with a decrease in cAMP and cGMP in medium spiny neurones in the striatum. Lu AF64196 alone did not increase neuronal cyclic nucleotide levels, but blocked the NMDA-induced reduction in cyclic nucleotides indicating that this was mediated by calcium-activated PDE1. Similar effects were observed in the prefrontal cortex and the hippocampus. Upon corelease of dopamine and NMDA, PDE1 was shown to down-regulate the D1-receptor mediated increase in cAMP. PDE1 inhibition increased long-term potentiation in rat ventral striatum, showing that PDE1 is implicated in the regulation of synaptic plasticity. Overall, our results show that PDE1 reduces cyclic nucleotide signaling in the context of glutamate and dopamine coincidence. This effect could have a therapeutic value for treating brain disorders related to dysfunctions in dopamine neuromodulation.

Involvement of Phosphodiesterase 2A Activity in the Pathophysiology of Fragile X Syndrome.

The fragile X mental retardation protein (FMRP) is an RNA-binding protein involved in translational regulation of mRNAs that play key roles in synaptic morphology and plasticity. The functional absence of FMRP causes the fragile X syndrome (FXS), the most common form of inherited intellectual disability and the most common monogenic cause of autism. No effective treatment is available for FXS. We recently identified the Phosphodiesterase 2A (Pde2a) mRNA as a prominent target of FMRP. PDE2A enzymatic activity is increased in the brain of Fmr1-KO mice, a recognized model of FXS, leading to decreased levels of cAMP and cGMP. Here, we pharmacologically inhibited PDE2A in Fmr1-KO mice and observed a rescue both of the maturity of dendritic spines and of the exaggerated hippocampal mGluR-dependent long-term depression. Remarkably, PDE2A blockade rescued the social and communicative deficits of both mouse and rat Fmr1-KO animals. Importantly, chronic inhibition of PDE2A in newborn Fmr1-KO mice followed by a washout interval, resulted in the rescue of the altered social behavior observed in adolescent mice. Altogether, these results reveal the key role of PDE2A in the physiopathology of FXS and suggest that its pharmacological inhibition represents a novel therapeutic approach for FXS.

Contrasting patterns of ERK activation in the tail of the striatum in response to aversive and rewarding signals.

The caudal part of the striatum, also named the tail of the striatum (TS), defines a fourth striatal domain. Determining whether rewarding, aversive and salient stimuli regulate the activity of striatal spiny projections neurons (SPNs) of the TS is therefore of paramount importance to understand its functions, which remain largely elusive. Taking advantage of genetically encoded biosensors (A-kinase activity reporter 3) to record protein kinase A signals and by analyzing the distribution of dopamine D1R- and D2R-SPNs in the TS, we characterized three subterritories: a D2R/A2aR-lacking, a D1R/D2R-intermingled and a D1R/D2R-SPNs-enriched area (corresponding to the amygdalostriatal transition). In addition, we provide evidence that the distribution of D1R- and D2R-SPNs in the TS is evolutionarily conserved (mouse, rat, gerbil). The in vivo analysis of extracellular signal-regulated kinase (ERK) phosphorylation in these TS subterritories in response to distinct appetitive, aversive and pharmacological stimuli revealed that SPNs of the TS are not recruited by stimuli triggering innate aversive responses, fasting, satiety, or palatable signals whereas a reduction in ERK phosphorylation occurred following learned avoidance. In contrast, D1R-SPNs of the intermingled and D2R/A2aR-lacking areas were strongly activated by both D1R agonists and psychostimulant drugs (d-amphetamine, cocaine, 3,4-methyl enedioxy methamphetamine, or methylphenidate), but not by hallucinogens. Finally, a similar pattern of ERK activation was observed by blocking selectively dopamine reuptake. Together, our results reveal that the caudal TS might participate in the processing of specific reward signals and discrete aversive stimuli. Cover Image for this issue: doi: 10.1111/jnc.14526. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.

Microsaccadic behavior when developing a complex dynamical activity.

Microsaccade are sensitive to changes of perceptual inputs as well as modulations of cognitive states. There are just a few works analyzing microsaccade while subjects are processing complex information and fewer when doing predictions about upcoming events. To evaluate whether contextual predictability would change microsaccadic behavior, we evaluated microsaccade of twenty one persons when reading 40 regular sentences and 40 proverbs. Analysis of microsaccade during reading proverbs and regular sentences revealed that microsaccade rate on words before maxjump, during maxjump and words after maxjump varied depending on the kind of sentence and on the word predictability. Maxjump was defined as the word with the largest difference between the cloze predictability of two consecutive words. Low and high predictable words demanded less or more microsaccade on words previous, during and on maxjump depending of the semantic context and of the readers' predictions of upcoming words.In summary, the present study shows that microsaccade' rate evidenced significant differences when reading proverbs and regular sentences. Hence, evaluation of microsaccade during reading sentences with different contextual predictability might provide information about specific effect of cue attention on complex task.

Detection of phasic dopamine by D1 and D2 striatal medium spiny neurons.

KEY POINTS: Brief dopamine events are critical actors of reward-mediated learning in the striatum; the intracellular cAMP-protein kinase A (PKA) response of striatal medium spiny neurons to such events was studied dynamically using a combination of biosensor imaging in mouse brain slices and in silico simulations. Both D1 and D2 medium spiny neurons can sense brief dopamine transients in the sub-micromolar range. While dopamine transients profoundly change cAMP levels in both types of medium spiny neurons, the PKA-dependent phosphorylation level remains unaffected in D2 neurons. At the level of PKA-dependent phosphorylation, D2 unresponsiveness depends on protein phosphatase-1 (PP1) inhibition by DARPP-32. Simulations suggest that D2 medium spiny neurons could detect transient dips in dopamine level. ABSTRACT: The phasic release of dopamine in the striatum determines various aspects of reward and action selection, but the dynamics of the dopamine effect on intracellular signalling remains poorly understood. We used genetically encoded FRET biosensors in striatal brain slices to quantify the effect of transient dopamine on cAMP or PKA-dependent phosphorylation levels, and computational modelling to further explore the dynamics of this signalling pathway. Medium-sized spiny neurons (MSNs), which express either D1 or D2 dopamine receptors, responded to dopamine by an increase or a decrease in cAMP, respectively. Transient dopamine showed similar sub-micromolar efficacies on cAMP in both D1 and D2 MSNs, thus challenging the commonly accepted notion that dopamine efficacy is much higher on D2 than on D1 receptors. However, in D2 MSNs, the large decrease in cAMP level triggered by transient dopamine did not translate to a decrease in PKA-dependent phosphorylation level, owing to the efficient inhibition of protein phosphatase 1 by DARPP-32. Simulations further suggested that D2 MSNs can also operate in a 'tone-sensing' mode, allowing them to detect transient dips in basal dopamine. Overall, our results show that D2 MSNs may sense much more complex patterns of dopamine than previously thought.

Iron responsive-like elements in the parasite Entamoeba histolytica.

In Entamoeba histolytica, iron modulates virulence and gene expression via unknown regulatory mechanisms. The existence of a posttranscriptional iron regulatory system parallel with the iron-responsive element (IRE)/iron regulatory protein (IRP) system in the protozoan Trichomonas vaginalis has recently been reported. Due to their evolutionary closeness and the importance of iron for growth and virulence in these protozoa, we hypothesized the existence of an IRE/IRP-like mechanism in E. histolytica. To determine the presence of IRE-like elements in some mRNAs from this parasite, we performed in silico analyses of the 5'- and 3'-UTRs of mRNAs encoding virulence factors and cytoskeleton, ribosomal and metabolism proteins. The Zuker mfold software predicted IRE-like secondary structures in 52 of the 135 mRNAs analysed. However, only nine structures shared sequence similarity with the apical loop sequence (CAGUGN) of the previously reported human IRE-ferritin, whereas the GUU/UUG protozoan-specific motif was detected in 23 stem-loop structures. A new motif, AUU/AUUU, was also observed in 23 structures, suggesting the possible existence of an amoeba-specific motif. Additionally, cross-linking and RNA electrophoretic mobility shift assays showed specific RNA-protein interactions, using as a model two amoebic IRE-like elements from iron-regulated mRNAs and HeLa, T. vaginalis and E. histolytica cytoplasmic proteins. Our data suggest the presence of a posttranscriptional iron regulatory IRE/IRP-like mechanism in E. histolytica.

Volumetric alterations in the nucleus accumbens and caudate nucleus in bulimia nervosa: a structural magnetic resonance imaging study.

OBJECTIVE: Bulimia nervosa (BN) is an eating disorder characterized by recurrent episodes of binge eating and inappropriate compensatory behaviors (such as purging, fasting, or excessive exercise) to prevent weight gain. BN has been associated with deficits in inhibitory control processes. The basal ganglia specifically, the nucleus accumbens (NAc) and the caudate nucleus (CN) are part of the frontostriatal circuits involved in inhibitory control. The main goal of this study was to investigate the presence of morphological alterations in the NAc and the CN in a sample of patients diagnosed with BN. METHOD: Forty-one female participants, 21 diagnosed with BN and 20 healthy matched controls (HC), underwent a structural magnetic resonance imaging (MRI) acquisition and clinical assessment. The NAc and the CN were manually segmented using the software Slicer 3D. RESULTS: The results reveal a significant volumetric decrease in the CN and a preserved NAc volume in BN compared to the control group. DISCUSSION: These findings suggest a contributory role of the caudate nucleus part of the dorsal striatum in the psychopathology of BN.

Diagnosis of mild Alzheimer disease through the analysis of eye movements during reading.

Reading requires the integration of several central cognitive subsystems, ranging from attention and oculomotor control to word identification and language comprehension. Reading saccades and fixations contain information that can be correlated with word properties. When reading a sentence, the brain must decide where to direct the next saccade according to what has been read up to the actual fixation. In this process, the retrieval memory brings information about the current word features and attributes into working memory. According to this information, the prefrontal cortex predicts and triggers the next saccade. The frequency and cloze predictability of the fixated word, the preceding words and the upcoming ones affect when and where the eyes will move next. In this paper we present a diagnostic technique for early stage cognitive impairment detection by analyzing eye movements during reading proverbs. We performed a case-control study involving 20 patients with probable Alzheimer's disease and 40 age-matched, healthy control patients. The measurements were analyzed using linear mixed-effects models, revealing that eye movement behavior while reading can provide valuable information about whether a person is cognitively impaired. To the best of our knowledge, this is the first study using word-based properties, proverbs and linear mixed-effect models for identifying cognitive abnormalities.

[Spending with unnecessary complementary tests for hypertension and diabetes in health services]. / Gastos com exames complementares desnecessários para hipertensos e diabéticos nos serviços de saúde.

The aims of this study were to analyze unnecessary laboratory exams for patients with hypertension and diabetes and to check the expenditures involved.This is an exploratory-descriptive, cross-sectional study with a quantitative approach.We used data from medical records of 293 patients registered in primary units - the Family Health Center (NSF3); secondary: School Health Center (CSE); and tertiary: Hospital das Clinicas (HC) from 2006 to 2009 in a city in Southeastern Brazil. We identified a total of 9,522 laboratory tests, of which 5.97% were unnecessary. Of these, about 58% were requested by NSF3 and 42% by CSE. Results suggest there is a lack of integration among different levels of health care, which result in misallocation of resources and unnecessary spending. Descriptors: Health expenditures. Hypertension. Diabetes mellitus. Family health. Health services.

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.

Integration of mesophilic biogas plant in the animal slaughter process under real limitations: Techno-economic evaluation of a colombian bovine slaughterhouse.

Anaerobic digestion (AD) has been a widely tested alternative for the management and valorization of wastewater from the animal slaughter process. However, the integration of AD in slaughterhouses depends on technical and economic aspects. In Colombian slaughterhouses AD integration is limited by the availability of land. In the present study, a techno-economic evaluation of the AD of offal wastewater (OWW) stream in a laboratory scale mesophilic tubular digester was carried out. The digester was operated at organic loading rates (OLR) of 0.28, 0.50, 1.0, 1.5 and 2.0 kg VS/m3 d. Boilers and a CHP (combined heat and power) system were considered for energy integration of biogas. For the economic study, the cost structure of a Colombian slaughterhouse was considered. The AD of OWW at 2.0 kg VS/m3 d OLR was unstable with risk of inhibition. Increasing the OLR from 0.28 to 1.5 kg VS/m3 d caused a reduction in the specific biogas production (SBP) from 0.474 to 0.069 m3/kg VS However, the biogas production rate (BPR) remained constant at around 0.105 m3/m3dig d for OLRs > 0.28 kg VSm3 d. Therefore, OWW anaerobic digestion in low-cost mesophilic biogas plants is technically feasible with OLRs between 0.28 and 1.5 kg VS/m3 d. The implementation of boilers is economically favorable for OLR ≥ 1.0 kg VS/m3 d. Nevertheless, feasibility is very sensitive to variations in the cost structure. The implementation of CHP was feasible in the range of OLRs evaluated and its viability is not affected by changes in assumed costs.

Post-treatment and agricultural reuse of digestate from low-tech digesters: A comparative life cycle assessment.

The objective of this study was to analyse the environmental impacts of the post-treatment and agricultural reuse of digestate from a low-tech digester implemented in a small-scale farm in Colombia using the Life Cycle Assessment methodology. The scenarios considered were: 1) digestate post-treatment with a sand filter and its reuse in agriculture; 2) digestate post-treatment with a vermifilter and the production of compost, and 3) untreated digestate directly applied on the agricultural land (current scenario). Moreover, an economic analysis was also addressed. Results showed that the vermifilter was the most environmentally friendly scenario. It considerably reduced (by up to 9 times) the environmental impacts compared to the other scenarios. From an economic point of view, the implementation of the vermifilter generated an increase in farmers' income (up to 70 $ year-1) since it avoids buying synthetic fertilizer. Finally, the implementation of a vermifilter for the post-treatment and agricultural reuse of digestate from low-tech digesters showed to have both environmental and economic benefits. This technology can help to promote the circular bioeconomy in small-scale farms, reducing poverty and improving the standard of living in rural areas.

Feedback control through cGMP-dependent protein kinase contributes to differential regulation and compartmentation of cGMP in rat cardiac myocytes.

RATIONALE: We have shown recently that particulate (pGC) and soluble guanylyl (sGC) cyclases synthesize cGMP in different compartments in adult rat ventricular myocytes (ARVMs). OBJECTIVE: We hypothesized that cGMP-dependent protein kinase (PKG) exerts a feedback control on cGMP concentration contributing to its intracellular compartmentation. METHODS AND RESULTS: Global cGMP levels, cGMP-phosphodiesterase (PDE) and pGC enzymatic activities were determined in purified ARVMs. Subsarcolemmal cGMP signals were monitored in single cells by recording the cGMP-gated current (I(CNG)) in myocytes expressing the wild-type rat olfactory cyclic nucleotide-gated (CNG) channel. Whereas the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) (100 µmol/L) produced little effect on I(CNG), the response increased 2-fold in the presence of the PKG inhibitors KT5823 (50 nmol/L) or DT-2 (2 µmol/L). The effect of KT5823 was abolished in the presence of the nonselective cyclic nucleotide PDE inhibitor 3-isobutyl-1-methylxantine (IBMX) (100 µmol/L) or the selective cGMP-PDE5 inhibitor sildenafil (100 nmol/L). PKG inhibition also potentiated the effect of SNAP on global cGMP levels and fully blocked the increase in cGMP-PDE5 activity. In contrast, PKG inhibition decreased by ≈50% the I(CNG) response to ANP (10 and 100 nmol/L), even in the presence of IBMX. Conversely, PKG activation increased the I(CNG) response to ANP and amplified the stimulatory effect of ANP on pGC activity. CONCLUSIONS: PKG activation in adult cardiomyocytes limits the accumulation of cGMP induced by NO donors via PDE5 stimulation but increases that induced by natriuretic peptides. These findings support the paradigm that cGMP is not uniformly distributed in the cytosol and identifies PKG as a key component in this process.

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.