In Vivo Assessment of Coronary Flow and Cardiac Function After Bolus Adenosine Injection in Adenosine Receptor Knockout Mice

Bolus injections of adenosine and the A2A adenosine receptor (AR) selective agonist (regadenoson) are used clinically as a substitute for a stress test in people who cannot exercise. Using isolated tissue preparations, our lab has shown that coronary flow and cardiac effects of adenosine are mostly regulated by the AR subtypes A1, A2A, and A2B. In this study, we used ultrasound imaging to measure the in vivo effects of adenosine on coronary blood flow (left coronary artery) and cardiac function in anesthetized wild-type, A1 knockout (KO), A2AKO, A2BKO, A3KO, A1, and A3 double KO (A1/3 DKO) and A2A and A2B double KO (A2A/2B DKO) mice in real time. Echocardiographic and Doppler studies were performed using a Visualsonic Vevo 2100 ultrasound system. Coronary blood flow (CBF) baseline data were obtained when animals were anesthetized with 1% isoflourane. Diameter (D) and velocity time integral (VTI) were measured on the left coronary arteries (CBF = ((p/4) 9 D2 9 VTI 9 HR)/1000). CBF changes were the highest within 2 min of injection (about 10 mg/kg). Heart rate, cardiac output, and stroke volume were measured by tracing the left ventricle long axis. Our data support a role for the A2 AR in CBF and further support our conclusions of previous studies from isolated tissues. Adenosine-mediated decreases in cardiac output and stroke volume may be A2B and/or A3 AR-mediated; however, the A1 and A2 ARs also play roles in overall cardiac function. These data further provide a powerful translational tool in studying the cardiovascular effects of adenosine in disease states

In Vivo Assessment of Coronary Flow and Cardiac Function After Bolus Adenosine Injection in Adenosine Receptor Knockout Mice

Bolus injections of adenosine and the A2A adenosine receptor (AR) selective agonist (regadenoson) are used clinically as a substitute for a stress test in people who cannot exercise. Using isolated tissue preparations, our lab has shown that coronary flow and cardiac effects of adenosine are mostly regulated by the AR subtypes A1, A2A, and A2B. In this study, we used ultrasound imaging to measure the in vivo effects of adenosine on coronary blood flow (left coronary artery) and cardiac function in anesthetized wild-type, A1 knockout (KO), A2AKO, A2BKO, A3KO, A1, and A3 double KO (A1/3 DKO) and A2A and A2B double KO (A2A/2B DKO) mice in real time. Echocardiographic and Doppler studies were performed using a Visualsonic Vevo 2100 ultrasound system. Coronary blood flow (CBF) baseline data were obtained when animals were anesthetized with 1% isoflourane. Diameter (D) and velocity time integral (VTI) were measured on the left coronary arteries (CBF = ((p/4) 9 D2 9 VTI 9 HR)/1000). CBF changes were the highest within 2 min of injection (about 10 mg/kg). Heart rate, cardiac output, and stroke volume were measured by tracing the left ventricle long axis. Our data support a role for the A2 AR in CBF and further support our conclusions of previous studies from isolated tissues. Adenosine-mediated decreases in cardiac output and stroke volume may be A2B and/or A3 AR-mediated; however, the A1 and A2 ARs also play roles in overall cardiac function. These data further provide a powerful translational tool in studying the cardiovascular effects of adenosine in disease states

Frequency-Dependent Cannabinoid Receptor-Independent Modulation of Glycine Receptors by Endocannabinoid 2-AG

Endocannabinoids are known as retrograde messengers, being released from the postsynaptic neuron and acting on specific presynaptic G-protein-coupled cannabinoid (CB) receptors to decrease neurotransmitter release. Also, at physiologically relevant concentrations cannabinoids can directly modulate the function of voltage-gated and receptor-operated ion channels. Using patch-clamp recording we analyzed the consequences of the direct action of an endocannabinoid, 2-arachidonoylglycerol (2-AG), on the functional properties of glycine receptor channels (GlyRs) and ionic currents in glycinergic synapses. At physiologically relevant concentrations (0.1–1 μM), 2-AG directly affected the functions of recombinant homomeric α1H GlyR: it inhibited peak amplitude and dramatically enhanced desensitization. The action of 2-AG on GlyR-mediated currents developed rapidly, within ∼300 ms. Addition of 1 μM 2-AG strongly facilitated the depression of glycine-induced currents during repetitive (4–10 Hz) application of short (2 ms duration) pulses of glycine to outside-out patches. In brainstem slices from CB1 receptor knockout mice, 2-AG significantly decreased the extent of facilitation of synaptic currents in hypoglossal motoneurons during repetitive (10–20 Hz) stimulation. These observations suggest that endocannabinoids can modulate postsynaptic metaplasticity of glycinergic synaptic currents in a CB1 receptor-independent manner

GPR3 Receptor, a Novel Actor in the Emotional-Like Responses

GPR3 is an orphan G protein-coupled receptor endowed with constitutive Gs signaling activity, which is expressed broadly in the central nervous system, with maximal expression in the habenula. We investigated the consequences of its genetic deletion in several behavioral paradigms and on neurotransmission. Compared to wild-type, hippocampal neurons from Gpr3−/− mice displayed lower basal intracellular cAMP levels, consistent with the strong constitutive activity of GPR3 in transiently transfected cells. Behavioral analyses revealed that Gpr3−/− mice exhibited a high level of avoidance of novel and unfamiliar environment, associated with increased stress reactivity in behavioral despair paradigms and aggressive behavior in the resident-intruder test. On the contrary, no deficit was found in the learning ability to avoid an aversive event in active avoidance task. The reduced ability of Gpr3−/− mice to cope with stress was unrelated to dysfunction of the hypothalamic-pituitary-adrenal axis, with Gpr3−/− mice showing normal corticosterone production under basal or stressful conditions. In contrast, dramatic alterations of monoamine contents were found in hippocampus, hypothalamus and frontal cortex of Gpr3−/− mice. Our results establish a link between tonic stimulation of the cAMP signaling pathway by GPR3 and control of neurotransmission by monoamines throughout the forebrain. GPR3 qualifies as a new player in the modulation of behavioral responses to stress and constitutes a novel promising pharmacological target for treatment of emotional disorders

Presynaptic TRPV1 vanilloid receptor function is age- but not CB1 cannabinoid receptor-dependent in the rodent forebrain

Neocortical and striatal TRPV1 (vanilloid or capsaicin) receptors (TRPV1Rs) are excitatory ligand-gated ion channels, and are implicated in psychiatric disorders. However, the purported presynaptic neuromodulator role of TRPV1Rs in glutamatergic, serotonergic or dopaminergic terminals of the rodent forebrain remains little understood. With the help of patch-clamp electrophysiology and neurochemical approaches, we mapped the age-dependence of presynaptic TRPV1R function, and furthermore, we aimed at exploring whether the presence of CB1 cannabinoid receptors (CB1Rs) influences the function of the TRPV1Rs, as both receptor types share endogenous ligands. We found that the major factor which affects presynaptic TRPV1R function is age: by post-natal day 13, the amplitude of capsaicin-induced release of dopamine and glutamate is halved in the rat striatum, and two weeks later, capsaicin already loses its effect. However, TRPV1R receptor function is not enhanced by chemical or genetic ablation of the CB1Rs in dopaminergic, glutamatergic and serotonergic terminals of the mouse brain. Altogether, our data indicate a possible neurodevelopmental role for presynaptic TRPV1Rs in the rodent brain, but we found no cross-talk between TRPV1Rs and CB1Rs in the same nerve terminal

Functional and RNA Expression Profile of Adenosine Receptor Subtypes in Mouse Mesenteric Arteries

Concentration–response curves (CRCs) of adenosine receptor (AR) agonists, NECA (nonspecific), CCPA (A1 specific), CGS-216870 (A2A specific), BAY 60-6583 (A2B specific), and Cl-IB-MECA (A3 specific) for mesenteric arteries (MAs) from 4 AR knockout (KO) mice (A1, A2A, A2B, and A3) and their wild type (WT) were constructed. The messenger RNA expression of MAs from KO mice and WT were also studied. Adenosine (10−5 to 10−4 M) and NECA (10−6 to 10−5 M) induced relaxation in all mice except A2B KO mice, which only showed constriction by adenosine at 10−6 to 10−4 and NECA at 10−8 to 10−5 M. The CCPA induced a significant constriction at 10−8 and 10−7 M in all mice, except A1KO. BAY 60-6583 induced relaxation (10−7 to 10−5 M) in WT and no response in A2BKO except at 10−5 M. The CRCs for BAY 60-6583 in A1, A2A, and A3 KO mice shifted to the left when compared with WT mice, suggesting an upregulation of A2B AR. No responses were noted to CGS-21680 in all mice. Cl-IB-MECA only induced relaxation at concentration greater than 10−7 M, and no differences were found between different KO mice. The CRC for Bay 60-6583 was not significantly changed in the presence of 10−5 M of L-NAME, 10−6 M of indomethacin, or both. Our data suggest that A2B AR is the predominant AR subtype and the effect may be endothelial independent, whereas A1 AR plays a significant modulatory role in mouse MAs

Transcriptomic effects of adenosine 2A receptor deletion in healthy and endotoxemic murine myocardium

Influences of adenosine 2A receptor (A2AR) activity on the cardiac transcriptome and genesis of endotoxemic myocarditis are unclear. We applied transcriptomic profiling (39 K Affymetrix arrays) to identify A2AR-sensitive molecules, revealed by receptor knockout (KO), in healthy and endotoxemic hearts. Baseline cardiac function was unaltered and only 37 A2AR-sensitive genes modified by A2AR KO (≥1.2-fold change, \u3c5 \u3e% FDR); the five most induced are Mtr, Ppbp, Chac1, Ctsk and Cnpy2 and the five most repressed are Hp, Yipf4, Acta1, Cidec and Map3k2. Few canonical paths were impacted, with altered Gnb1, Prkar2b, Pde3b and Map3k2 (among others) implicating modified G protein/cAMP/PKA and cGMP/NOS signalling. Lipopolysaccharide (LPS; 20 mg/kg) challenge for 24 h modified \u3e4100 transcripts in wild-type (WT) myocardium (≥1.5-fold change, FDR \u3c 1 %); the most induced are Lcn2 (+590); Saa3 (+516); Serpina3n (+122); Cxcl9 (+101) and Cxcl1 (+89) and the most repressed are Car3 (−38); Adipoq (−17); Atgrl1/Aplnr (−14); H19 (−11) and Itga8 (−8). Canonical responses centred on inflammation, immunity, cell death and remodelling, with pronounced amplification of toll-like receptor (TLR) and underlying JAK-STAT, NFκB and MAPK pathways, and a ‘cardio-depressant’ profile encompassing suppressed ß-adrenergic, PKA and Ca2+ signalling, electromechanical and mitochondrial function (and major shifts in transcripts impacting function/injury including Lcn2, S100a8/S100a9, Icam1/Vcam and Nox2 induction, and Adipoq, Igf1 and Aplnr repression). Endotoxemic responses were selectively modified by A2AR KO, supporting inflammatory suppression via A2AR sensitive shifts in regulators of NFκB and JAK-STAT signalling (IκBζ, IκBα, STAT1, CDKN1a and RRAS2) without impacting the cardio-depressant gene profile. Data indicate A2ARs exert minor effects in un-stressed myocardium and selectively suppress NFκB and JAK-STAT signalling and cardiac injury without influencing cardiac depression in endotoxemia

Intervenção sobre uma coleção fotográfica

O presente relatório descreve as atividades realizadas durante o estágio curricular no âmbito do Mestrado em Fotografia, perfil de Conservação, da Escola Superior de Tecnologia de Tomar / Instituto Politécnico de Tomar (ESTT/IPT). O estágio decorreu nas instalações do Arquivo Histórico Ultramarino (AHU), integrado no Instituto de Investigação Científica Tropical (IICT), e teve como objetivo a intervenção de conservação sobre uma coleção de fotografias em depósito no AHU, que contou com vários tipos de suportes (como vidro, películas fotográficas, provas em papel e álbuns fotográficos). As várias fases de trabalho foram realizadas durante nove meses, e consistiram no diagnóstico, higienização, estabilização, acondicionamento, digitalização, catalogação e disponibilização online. Este estágio visa a preservação e divulgação de um núcleo fotográfico que reúne valências do ponto de vista histórico, cultural e patrimonial

Role for Neuronal Nitric-Oxide Synthase in Cannabinoid-Induced Neurogenesis

ABSTRACT Cannabinoids, acting through the CB1 cannabinoid receptor (CB1R), protect the brain against ischemia and related forms of injury. This may involve inhibiting the neurotoxicity of endogenous excitatory amino acids and downstream effectors, such as nitric oxide (NO). Cannabinoids also stimulate neurogenesis in the adult brain through activation of CB1R. Because NO has been implicated in neurogenesis, we investigated whether cannabinoid-induced neurogenesis, like cannabinoid neuroprotection, might be mediated through alterations in NO production. Accordingly, we measured neurogenesis in dentate gyrus (DG) and subventricular zone (SVZ) of CB1R-knockout (KO) and wild-type mice, some of whom were treated with the cannabi- or the NO synthase (NOS) inhibitor 7-nitroindazole (7-NI). NOS activity was increased by ϳ25%, whereas bromodeoxyuridine (BrdU) labeling of newborn cells in DG and SVZ was reduced by ϳ50% in CB1R-KO compared with wild-type mice. 7-NI increased BrdU labeling in both DG and SVZ and to a greater extent in CB1R-KO than in wild-type mice. In addition, R(ϩ)-Win 55212-2 and 7-NI enhanced BrdU incorporation into neuron-enriched cerebral cortical cultures to a similar maximal extent and in nonadditive fashion, consistent with a shared mechanism of action. Double-label confocal microscopy showed coexpression of BrdU and the neuronal lineage marker doublecortin (Dcx) in DG and SVZ of untreated and 7-NI-treated CB1R-KO mice, and 7-NI increased the number of Dcx-and BrdU/Dcx-immunoreactive cells in SVZ and DG. Thus, cannabinoids appear to stimulate adult neurogenesis by opposing the antineurogenic effect of NO. Cannabinoids, which include naturally occurring plantderived compounds [e.g., ⌬ 9 -tetrahydrocannabinol (THC)], endogenous signaling molecules found in animal brains (e.g., anandamide and 2-arachidonoylglycerol), and synthetic drugs [e.g., R(ϩ)-Win 55212-2)], act on receptors in the brain to modify neuronal function. In addition to their effects on normal physiological functions such as blood pressure, immunity, pain perception, appetite, and cognition, cannabinoids can also regulate the severity of brain injury. We reported previously that administration of cannabinoids acting on the CB1 cannabinoid receptor (CB1R) reduces neuronal death from cerebral ischemi

Involvement of NADPH oxidase in A2A adenosine receptor-mediated increase in coronary flow in isolated mouse hearts

Adenosine increases coronary flow mainly through the activation of