Activity-dependent regulation of GABA release at immature mossy fibers-CA3 synapses: role of the Prion protein

In adulthood, mossy fibers (MFs), the axons of granule cells of the dentate gyrus (DG), release glutamate onto CA3 principal cells and interneurons. In contrast, during the first week of postnatal life MFs release -aminobutyric acid (GABA), which, at this early developmental stage exerts a depolarizing and excitatory action on targeted cells. The depolarizing action of GABA opens voltage-dependent calcium channels and NMDA receptors leading to calcium entry and activation of intracellular signaling pathways involved in several developmental processes, thus contributing to the refinement of neuronal connections and to the establishment of adult neuronal circuits. The release of GABA has been shown to be down regulated by several neurotransmitter receptors which would limit the enhanced excitability caused by the excitatory action of GABA. It is worth noting that the immature hippocampus exhibits spontaneous correlated activity, the so called giant depolarizing potentials or GDPs that act as coincident detector signals for enhancing synaptic activity, thus contributing to several developmental processes including synaptogenesis. GDPs render the immature hippocampus more prone to seizures. Here, I explored the molecular mechanisms underlying synaptic transmission and activity-dependent synaptic plasticity processes at immature GABAergic MF-CA3 synapses in wild-type rodents and in mice lacking the prion protein (Prnp0/0 mice). In the first paper, I studied the functional role of kainate receptors (KARs) in regulating GABA release from MF terminals. Presynaptic KARs regulate synaptic transmission in several brain areas and play a central role in modulating glutamate release at adult MF-CA3 synapses. I found that functional presynaptic GluK1 receptors are present on MF terminals where they down regulate GABA release. Thus, application of DNQX or UBP 302, a selective antagonist for GluK1 receptors, strongly increased the amplitude of MF-GABAA-mediated postsynaptic currents (GPSCs). This effect was associated with a decrease in failure rate and increase in PPR, indicating a presynaptic type of action. GluK1 receptors were found to be tonically activated by glutamate present in the extracellular space, since decreasing the extracellular concentration of glutamate with a glutamate scavenger system prevented their activation and mimicked the effects of KAR antagonists. The depressant effect of GluK1 on GABA release was dependent on pertussis toxin (PTx)-sensitive G protein-coupled kainate receptors since it was prevented when hippocampal slices were incubated in the presence of a solution containing PTx. This effect was presynaptic since application of UBP 302 to cells patched with an intracellular solution containing GDP S still potentiated synaptic responses. In addition, the depressant effect of GluK1 on GABA release was prevented by U73122, which selectively inhibits phospholipase C, downstream to G protein activation. Interestingly, U73122, enhanced the probability of GABA release, thus unveiling the ionotropic type of action of kainate receptors. In line with this, we found that GluK1 receptors enhanced MF excitability by directly depolarizing MF terminals via calcium-permeable cation channels. We also explored the possible involvement of GluK1 in spike time-dependent (STD) plasticity and we found that GluK1 dynamically regulate the direction of STD-plasticity, since the pharmacological block of this receptor shifted spike-time dependent potentiation into depression. The mechanisms underlying STD-LTD at immature MF-CA3 synapses have been investigated in detail in the second paper. STD-plasticity is a Hebbian form of learning which consists in bi-directional modifications of synaptic strength according to the temporal order of pre and postsynaptic spiking. Interestingly, we found that at immature mossy fibers (MF)-CA3 synapses, STD-LTD occurs regardless of the temporal order of stimulation (pre versus post or viceversa). However, as already mentioned, while STD-LTD induced by positive pairing (pre before post) could be shifted into STD-LTP after blocking presynaptic GluK1 receptors, STD-LTD induced by negative pairing (post before pre) relied on the activation of CB1 receptors. At P3 but not at P21, endocannabinoids released by the postsynaptic cell during spiking-induced membrane depolarization retrogradely activated CB1 receptors, probably expressed on MF terminals and persistently depressed GABA release in the rat hippocampus. Thus, bath application of selective CB1 receptor antagonists prevented STD-LTD. Pharmacological tools allow identifying anandamide as the endogenous ligand responsible of activity-dependent depressant effect. To further assess whether STD-LTD is dependent on the activation of CB1 receptors, similar experiments were performed on WT-littermates and CB1-KO mice. While in WT mice the pairing protocol produced a persistent depression of MF-GPSCs as in rats, in CB1-KO mice failed to induce LTD. Consistent with these data, in situ hybridization experiments revealed detectable levels of CB1 mRNA in the granule cell layer of P3 but not of P21mice. These experiments strongly suggest that at immature MF-CA3 synapses STD-LTD is mediated by CB1 receptors, probably transiently expressed, during a critical time window, on MF terminals. In the third paper, I studied synaptic transmission and activity dependent synaptic plasticity at immature MF-CA3 synapses in mice devoid of the prion protein (Prnp0/0). The prion protein (PrPC) is a conserved glycoprotein widely expressed in the brain and involved in several neuronal processes including neurotransmission. If converted to a conformationally altered form, PrPSc can cause neurodegenerative diseases, such as Creutzfeldt-Jakob disease in humans. Previous studies aimed at characterizing Prnp0/0 mice have revealed only mild behavioral changes, including an impaired spatial learning, accompanied by electrophysiological and biochemical alterations. Interestingly, PrPC is developmentally regulated and in the hippocampus its expression parallels the maturation of MF. Here, we tested the hypothesis that at immature (P3-P7) MF-CA3 synapses, PrPC interferes with synaptic plasticity processes. To this aim, the rising phase of Giant Depolarizing Potentials (GDPs), a hallmark of developmental networks, was used to stimulate granule cells in the dentate gyrus in such a way that GDPs were coincident with afferent inputs. In WT animals, the pairing procedure induced a persistent increase in amplitude of MF-GPSCs. In contrast, in Prnp0/0 mice, the same protocol produced a long-term depression (LTD). LTP was postsynaptic in origin and required the activation of cAMP-dependent PKA signaling while LTD was presynaptic and was reliant on G protein-coupled GluK1 receptor and protein lipase C downstream to G protein activation. In addition, at emerging CA3-CA1 synapses of PrPC-deficient mice, stimulation of Schaffer collateral failed to induce LTP, known to be PKA-dependent. Finally, we also found that LTD in Prnp0/0 mice was mediated by GluK1 receptors, since UBP 302 blocked its induction. These data suggest that in the immature hippocampus PrPC controls the direction of synaptic plasticity

Fluoxetine impairs GABAergic signaling in hippocampal slices from neonatal rats

Fluoxetine (Prozac), an antidepressant known to selectively inhibit serotonin reuptake, is widely used to treat mood disorders in women suffering from depression during pregnancy and postpartum period. Several lines of evidence suggest that this drug, which crosses the human placenta and is secreted into milk during lactation, exerts its action not only by interfering with serotoninergic but also with GABAergic transmission. GABA is known to play a crucial role in the construction of neuronal circuits early in postnatal development. The immature hippocampus is characterized by an early type of network activity, the so-called Giant Depolarizing Potentials (GDPs), generated by the synergistic action of glutamate and GABA, both depolarizing and excitatory. Here we tested the hypothesis that fluoxetine may interfere with GABAergic signaling during the first postnatal week, thus producing harmful effects on brain development. At micromolar concentrations fluoxetine severely depressed GDPs frequency (IC50 22 \u3bcM) in a reversible manner and independently of its action on serotonin reuptake. This effect was dependent on a reduced GABAergic (but not glutamatergic) drive to principal cells most probably from parvalbumin-positive fast spiking neurons. Cholecystokinin-positive GABAergic interneurons were not involved since the effects of the drug persisted when cannabinoid receptors were occluded with WIN55,212-2, a CB1/CB2 receptor agonist. Fluoxetine effects on GABAergic transmission were associated with a reduced firing rate of both principal cells and interneurons further suggesting that changes in network excitability account for GDPs disruption. This may have critical consequences on the functional organization and stabilization of neuronal circuits early in postnatal development. \ua9 2013 Cherubini and Caiati

Il sistema socio-economico-ambientale come strumento dello sviluppo sostenibile

In the past economics ignored the limits of natural resources. In the last few years a new methodological approach comes into economics improving the comprehension of the economic phenomena linked to the satisfaction of the needs of modern man that basic considers the quality of life. The objective of this research is to define the theoretical and methodological aspects of a sustainable economy pointing out the reasons and the grounds on which to build an environmental policy. This one has to consider either the economic matters or the environmental sensitivity of people. This research is divided into three parts. The first one defines the outlines of the sustainable economy and its characteristic features. The second one emphasizes the necessity of replacing the concept of sustainable development instead of development and growth. At last, the third part identifies the basic lines into which the environmental policy has to be realized defining its reasons, its foundations and its valuation systems

FEASIBILITY OF A NEW NON INVASIVE METHOD FOR THE EVALUATION OF CORONARY BLOOD FLOW IN CORONARIES: TRANSTHORACIC CONVERGENT COLOR DOPPLER MODE ALONG WITH PHARMACOLOGICALLY INDUCED HEART RATE LOWERING

This approach greatly improved the success rate of BF Doppler recording in coronaries, making CC-Doppler TTE suitable for accelerated stenotic flow and flow reserve assessment not only in the left anterior descending but also in the circumflex branch

A Novel Clinical Perspective on New Masses after Lead Extraction (Ghosts) by Means of Intracardiac Echocardiography

Background: A lead-reactive fibrous capsule (FC) identified by ultrasounds as an atrial or ventricular lead thickness of more than 1 mm above the vendor-declared lead diameter (TL) and its local fibrotic attachment to the cardiac wall (FAC) have never been investigated in vivo, so their relationship with post-extraction masses (ghost) is not known. Methods: Intracardiac echocardiography (ICE) was performed twice during the same extraction procedure in 40 consecutive patients: before and immediately after infected lead extraction Results: The ghost detection rate was high: 60% (24/40 patients); ICE could identify both TL and FAC, TL being noted in 25/40 (62%) patients and FAC in 12/40 patients (30%). Both TL and FAC were significantly associated with ghosts (p< 0.001 andp= 0.002, respectively), but TL had a higher prediction power. The specificity was similar: 94% (15/16) and 100% (16/16), respectively, but TL showed a much higher sensitivity: 100%, (24/24) vs 50% (12/24) (p= 0.016). The ghost group did not show a higher event rate in the follow-up (mean follow-up time = 20 +/- 17 months). Conclusion: ICE is able to evaluate both TL and FAC in vivo; ghosts are mostly benign remnants of fibrotic lead capsule cut off during extraction and retained inside the heart by FAC

Predictors of exercise capacity in dilated cardiomyopathy with focus on pulmonary venous flow recorded with transesophageal eco-doppler

The aim of this study was to clarify the relative contribution of elevated left ventricle (LV) filling pressure (FP) estimated by pulmonary venous (PV) and mitral flow, transesophageal Doppler recording (TEE), and other extracardiac factors like obesity and renal insufficiency (KI) to exercise capacity (ExC) evaluated by cardiopulmonary exercise testing (CPX) in patients with dilated cardiomyopathy (DCM). During the CPX test, 119 patients (pts) with DCM underwent both peak VO2 consumption and then TEE with color-guided pulsed-wave Doppler recording of PVF and transmitral flow. In 78 patients (65%), peak VO2 was normal or mildly reduced (>14 mL/kg/min) (group 1) while it was markedly reduced (≤14 mL/kg/min) in 41 (group 2). In univariate analysis, systolic fraction (S Fract), a predictor of elevated pre-a LV diastolic FP, appeared to be the best diastolic parameter predicting a significantly reduced peak VO2. Logistic regression analysis identi-fied five parameters yielding a unique, statistically significant contribution in predicting reduced ExC: creatinine clearance < 52 mL/min (odds ratio (OR) = 7.4, p = 0.007); female gender (OR = 7.1, p = 0.004); BMI > 28 (OR = 5.8, p = 0.029), age > 62 years (OR = 5.5, p = 0.03), S Fract < 59% (OR = 4.9, p = 0.02). Conclusion: KI was the strongest predictor of reduced ExC. The other modifiable factors were obesity and severe LV diastolic dysfunction expressed by blunted systolic venous flow. Contrar-ily, LV ejection fraction was not predictive, confirming other previous studies. This has important clinical implications

Control of GABA Release at Mossy Fiber-CA3 Connections in the Developing Hippocampus

In this review some of the recent work carried out in our laboratory concerning the functional role of GABAergic signalling at immature mossy fibres (MF)-CA3 principal cell synapses has been highlighted. While in adulthood MF, the axons of dentate gyrus granule cells release onto CA3 principal cells and interneurons glutamate, early in postnatal life they release GABA, which exerts into targeted cells a depolarizing and excitatory action. We found that GABAA-mediated postsynaptic currents (MF-GPSCs) exhibited a very low probability of release, were sensitive to L-AP4, a group III metabotropic glutamate receptor agonist, and revealed short-term frequency-dependent facilitation. Moreover, MF-GPSCs were down regulated by presynaptic GABAB and kainate receptors, activated by spillover of GABA from MF terminals and by glutamate present in the extracellular medium, respectively. Activation of these receptors contributed to the low release probability and in some cases to synapses silencing. By pairing calcium transients, associated with network-driven giant depolarizing potentials or GDPs (a hallmark of developmental networks thought to represent a primordial form of synchrony between neurons), generated by the synergistic action of glutamate and GABA with MF activation increased the probability of GABA release and caused the conversion of silent synapses into conductive ones suggesting that GDPs act as coincident detector signals for enhancing synaptic efficacy. Finally, to compare the relative strength of CA3 pyramidal cell output in relation to their MF glutamatergic or GABAergic inputs in adulthood or in postnatal development, respectively, a realistic model was constructed taking into account different biophysical properties of these synapses

ACCELERATED STENOTIC FLOW BY ENHANCED TRANSTHORACIC DOPPLER ECHOCARDIOGRAPHY IS SUPERIOR TO THE ASCVD RISK SCORE IN PREDICTING OBSTRUCTIVE CORONARY ATHEROSCLEROSIS IN PATIENTS WITH ATYPICAL ANGINA

Background: Atypical angina (AA) has only an intermediate probability of coronary obstructive atherosclerosis (COA). Accelerated stenotic flow (ASF) by enhanced Doppler echo (E-Doppler TTE) in the whole left main (LMCA) and left anterior descending coronary artery (LAD) is a highly feasible and reliable approach to detect both mild and critical coronary stenosis. The ASCVD risk score is a practical, non-invasive way to risk-stratify patients for COA. The relative diagnostic potential of the 2 methods in predicting COA is unknown in pts with AA. Methods: Eighty-six pts (age 30 -75 years) with AA scheduled for Angiography (CA)/IVUS (intracoronary Doppler) underwent E-Doppler TTE and ASCVD risk score assessment. ASF was expressed as % increment of velocity. COA was defined as either coronary plaque in the LAD/LMCA detected by IVUS (76 pts) or diffuse lumen irregularities in LAD along with stenosis in the other coronaries at CA (8 pts). Results: COA was present in 59 pts (69%) and absent in 27 (31%). The ASCVD score was 14±11: 36 pts were at low risk (ASCVD<10) and the other 50 at moderate/high risk. E-Doppler TTE showed a better performance than ASCVD, with 85% sensitivity and 100% specificity (cutoff ASF 23 %) versus 66% and 59% (cutoff ASCVD score 10%), confirmed by AUC comparison (graph). Conclusion: ASF had a better predictive power than the ASCVD score for COA in pts with AA. Moreover, E-Doppler TTE can reliably assess plaque severity and location in the LAD, making it a superior clinical tool compared to the ASCVD score

PrPC Controls via Protein Kinase A the Direction of Synaptic Plasticity in the Immature Hippocampus

The cellular form of prion protein PrPC is highly expressed in the brain, where it can be converted into its abnormally folded isoform PrPSc to cause neurodegenerative diseases. Its predominant synaptic localization suggests a crucial role in synaptic signaling. Interestingly, PrPC is developmentally regulated and its high expression in the immature brain could be instrumental in regulating neurogenesis and cell proliferation. Here, PrPC-deficient (Prnp0/0) mice were used to assess whether the prion protein is involved in synaptic plasticity processes in the neonatal hippocampus. To this aim, calcium transients associated with giant depolarizing potentials, a hallmark of developmental networks, were transiently paired with mossy fiber activation in such a way that the two events were coincident. While this procedure caused long-term potentiation (LTP) in wild-type (WT) animals, it caused long-term depression (LTD) in Prnp0/0 mice. Induction of LTP was postsynaptic and required the activation of cAMP-dependent protein kinase A (PKA) signaling. The induction of LTD was presynaptic and relied on G-protein-coupled GluK1 receptor and protein lipase C. In addition, at emerging CA3-CA1 synapses in WT mice, but not in Prnp0/0 mice, pairing Schaffer collateral stimulation with depolarization of CA1 principal cells induced LTP, known to be PKA dependent. Postsynaptic infusion of a constitutively active isoform of PKA catalytic subunit C\u3b1 into CA1 and CA3 principal cells in the hippocampus of Prnp0/0 mice caused a persistent synaptic facilitation that was occluded by subsequent pairing. These data suggest that PrPC plays a crucial role in regulating via PKA synaptic plasticity in the developing hippocampus. \ua9 2013 the authors

Coronary Flow and Reserve by Enhanced Transthoracic Doppler Trumps Coronary Anatomy by Computed Tomography in Assessing Coronary Artery Stenosis

We report the case of a 71-year-old patient with many risk factors for coronary atherosclerosis, who underwent computed coronary angiography (CTA), in accordance with the guidelines, for recent onset atypical chest pain. CTA revealed critical (>50% lumen diameter narrowing) stenosis of the proximal anterior descending coronary, and the patient was scheduled for invasive coronary angiography (ICA). Before ICA he underwent enhanced transthoracic echo-Doppler (E-Doppler TTE) for coronary flow detection by color-guided pulsed-wave Doppler recording of the left main (LMCA) and whole left anterior descending coronary artery (LAD,) along with coronary flow reserve (CFR) in the distal LAD calculated as the ratio, of peak flow velocity during i.v. adenosine (140 mcg/Kg/m) to resting flow velocity. E-Doppler TTE mapping revealed only mild stenosis (28% area narrowing) of the mid LAD and a CFR of 3.20, in perfect agreement with the color mapping showing no flow limiting stenosis in the LMCA and LAD. ICA revealed only a very mild stenosis in the mid LAD and mild atherosclerosis in the other coronaries (intimal irregularities). Thus, coronary stenosis was better predicted by E-Doppler TTE than by CTA. Coronary flow and reserve as assessed by E-Doppler TTE trumps coronary anatomy as assessed by CTA, without exposing the patient to harmful radiation and iodinated contrast medium