In this study the subcellular modifications undergone by cerebral cortex mitochondrial metabolism in chronic hypertension during aging were evaluated. The catalytic properties of regulatory energy-linked enzymes of Tricarboxylic Acid Cycle (TCA), Electron Transport Chain (ETC) and glutamate metabolism were assayed on non-synaptic mitochondria (FM, located in post-synaptic compartment) and on intra-synaptic mitochondria of pre-synaptic compartment, furtherly divided in “light” (LM) and “heavy” (HM) mitochondria, purified form cerebral cortex of normotensive Wistar Kyoto Rats (WKY) versus Spontaneously Hypertensive Rats (SHR) at 6, 12 and 18 months. During physiological aging, the metabolic machinery was differently expressed in pre- and post-synaptic compartments: LM and above all HM were more affected by aging, displaying lower ETC activities. In SHR at 6 months, FM and LM showed an uncoupling between TCA and ETC, likely as initial adaptive response to hypertension. During pathological aging, HM were particularly affected at 12 months in SHR, as if the adaptive modifications in FM and LM at 6 months granted a mitochondrial functional balance, while at 18 months all the neuronal mitochondria displayed decreased metabolic fluxes versus WKY. This study describes the effects of chronic hypertension on cerebral mitochondrial energy metabolism during aging through functional proteomics of enzymes at subcellular levels, i.e. in neuronal soma and synapses. In addition, this represents the starting point to envisage an experimental physiopathological model which could be useful also for pharmacological studies, to assess drug actions during the development of age-related pathologies that could coexist and/or are provoked by chronic hypertension.

Clonidine is an anti-hypertensive drug that inhibits the release of norepinephrine from pre-synaptic terminals binding to pre-synaptic α2-adrenoreceptors. Some studies suggest that this drug decreases brain energy expenditure, particularly in hypoxic-ischemic injury. However, data about clonidine effects on the functional parameters regulating brain energy metabolism are lacking. In this study, the effects of acute clonidine treatment (5 μg×kg−1 i.p., 30 min) were evaluated on the catalytic activity of regulatory energy-linked enzymes of Krebs’ cycle, Electron Transport Chain and glutamate metabolism of temporal cerebral cortex of 3-month-old male Sprague–Dawley rats. Enzyme activities were assayed on non-synaptic “free” mitochondria (FM) of neuronal perikaryon and partly of glial cells, and on intra-synaptic “light” (LM) and “heavy” mitochondria (HM), localized within synaptic terminals. This subcellular analysis differentiates clonidine effects on post-synaptic and pre-synaptic neuronal compartments. The results showed that clonidine increased citrate synthase, cytochrome oxidase and glutamate–oxaloacetate transaminase activities of FM. In LM, citrate synthase activity was decreased, while cytochrome oxidase and glutamate–oxaloacetate transaminase activities were increased; on the contrary, citrate synthase, cytochrome oxidase and glutamate dehydrogenase were all decreased in HM. Therefore, clonidine exerted different effects with respect to brain mitochondria, coherently with the in vivo energy requirements of each synaptic compartment: the drug increased energy-linked enzyme activities in post-synaptic compartment, while the metabolic variations were complex in the pre-synaptic one, being enzyme activities heterogeneously modified in LM and decreased in HM. This study highlights the relationships existing between the clonidine-induced neuroreceptorial effects and the energy metabolism in pre- and post- synaptic bioenergetics.

Energy metabolism is fundamental to maintain Central Nervous System homeostasis because of high requirement of adenosine triphosphate (ATP), that is necessary to sustain neuronal events. During aging, changes in brain bioenergetics may influence the recovery of cerebral tissue in coping with pathophysiological conditions and pharmacological treatments. For this reason, we have previously studied enzyme catalytic activities related to energy-yielding systems. In the present study, the maximum rates (Vmax) of some enzymatic activities related to energy consumption (ATPases) were evaluated on synaptic plasma membranes (S.P.M.) isolated from frontal cerebral cortex of male Wistar rats aged 2, 6, 12, 18 and 24 months, because of the key role of these enzymes in modulating presynaptic nerve ending homeostasis. The following enzyme activities were evaluated: Na+, K+, Mg2+-ATPase; ouabain-insensitive Mg2+-ATPase; Na+, K+-ATPase; specific Mg2+-ATPase; Ca2+, Mg2+-ATPase; acetylcholinesterase (AChE). The present results show that both the activities of (i) ATPases and (ii) AChE were significantly decreased during aging. Comparing these observations with those previously done on rat striatum on the same functional parameters and in the same experimental settings, ATPases activities were influenced by the age factor in different ways, suggesting that the frontal cerebral cortex independently adapt to the different age-dependent biochemical situations at each single age. Overall, this experimental approach is therefore important to add pieces of information for the understanding of the correlation between aging and brain energy metabolism, and could be a suitable model to assess also drug effects, differentiating between different cerebral areas.

Glutamate is involved in cerebral ischemic injury, but its role has not been completely clarified and studies are required to understand how to minimize its detrimental effects, contemporarily boosting the positive ones. In fact, glutamate is not only a neurotransmitter, but primarily a key metabolite for brain bioenergetics. Thus, we investigated the relationships between glutamate and brain energy metabolism in an in vivo model of complete cerebral ischemia of 15 min and during post‐ischemic recovery after 1, 24, 48, 72, and 96 h in 1‐year‐old adult and 2‐year‐old aged rats. The maximum rates (Vmax) of glutamate dehydrogenase (GlDH), glutamate‐oxaloacetate transaminase, and glutamate‐pyruvate transaminase were assayed in somatic mitochondria (FM) and in intra‐synaptic ‘Light’ mitochondria and intra‐synaptic ‘Heavy’ mitochondria ones purified from cerebral cortex, distinguishing post‐ and pre‐synaptic compartments. During ischemia, none of the enzymes were modified in adult animals. In aged ones, glutamate‐oxaloacetate transaminase was increased in FM and GlDH in intra‐synaptic ‘Heavy’ mitochondria, stimulating glutamate catabolism. During post‐ischemic recovery, FM did not show modifications at both ages while, in intra‐synaptic mitochondria of adult animals, glutamate catabolism was increased after 1 h of recirculation and decreased after 48 and 72 h, whereas it remained decreased up to 96 h in aged rats. These results, with those previously published about Krebs’ cycle and Electron Transport Chain (Villa et al., [2013] Neurochem. Int. 63, 765–781), demonstrate that: (i) Vmax of energy‐linked enzymes are different in the various cerebral mitochondria, which (ii) respond differently to ischemia and post‐ischemic recovery, also (iii) with respect to aging.

Alterations in mitochondrial functions have been hypothesized to participate in the pathogenesis of depression, because brain bioenergetic abnormalities have been detected in depressed patients by neuroimaging in vivo studies. However, this hypothesis is not clearly demonstrated in experimental studies: some suggest that antidepressants are inhibitors of mitochondrial metabolism, while others observe the opposite. In this study, the effects of 21-day treatment with desipramine (15 mg/kg) and fluoxetine (10 mg/kg) were examined on the energy metabolism of rat hippocampus, evaluating the catalytic activity of regulatory enzymes of mitochondrial energy-yielding metabolic pathways. Because of the micro-heterogeneity of brain mitochondria, we have distinguished between (a) non-synaptic mitochondria (FM) of neuronal perikaryon (post-synaptic compartment) and (b) intra-synaptic light (LM) and heavy (HM) mitochondria (pre-synaptic compartment). Desipramine and fluoxetine changed the catalytic activity of specific enzymes in the different types of mitochondria: (a) in FM, both drugs enhanced cytochrome oxidase and glutamate dehydrogenase, (b) in LM, the overall bioenergetics was unaffected and (c) in HM only desipramine increased malate dehydrogenase and decreased the activities of Electron Transport Chain Complexes. These results integrate the pharmacodynamic features of desipramine and fluoxetine at subcellular level, overcoming the previous conflicting data about the effects of antidepressants on brain energy metabolism, mainly referred to whole brain homogenates or to bulk of cerebral mitochondria. With the differentiation in non-synaptic and intra-synaptic mitochondria, this study demonstrates that desipramine and fluoxetine lead to adjustments in the mitochondrial bioenergetics respect to the energy requirements of pre- and post-synaptic compartments.

Functional proteomics was used to characterize age-related changes in energy metabolism of different neuronal pathways within the cerebellar cortex of Wistar rats aged 2, 6, 12, 18, and 24 months. The “large” synaptosomes, derived from the glutamatergic mossy fibre endings which make synaptic contact with the granule cells of the granular layer, and the “small” synaptosomes, derived from the pre-synaptic terminals of granule cells making synaptic contact with the dendrites of Purkinje cells, were isolated by a combined differential/gradient centrifugation technique. Because most brain disorders are associated with bioenergetic changes, the maximum rate (V max ) of selected enzymes of glycolysis, Krebs’ cycle, glutamate and amino acids metabolism, and acetylcholine catabolism were evaluated. The results show that “large” and “small” synaptosomes possess specific and independent metabolic features. This study represents a reliable model to study in vivo (1) the physiopathological molecular mechanisms of some brain diseases dependent on energy metabolism, (2) the responsiveness to noxious stimuli, and (3) the effects of drugs, discriminating their action sites at subcellular level on specific neuronal pathways.

Synaptic energy state and mitochondrial dysfunction are crucial factors in many brain pathologies. l-acetylcarnitine, a natural derivative of carnitine, improves brain energy metabolism, and has been proposed for the Therapy of many neurological and psychiatric diseases. The effects of the drug on the maximum rate (Vmax) of enzymatic activities related to hippocampal synaptic energy utilization were evaluated, in the perspective of its employment for Dementias and Depression Therapy. Two types of synaptic plasma membranes (SPM1 and SPM2) were isolated from the hippocampus of rats treated with l-acetylcarnitine (30 and 60mg/kg i.p., 28 days, 5 days/week). Acetylcholinesterase (AChE); Na(+), K(+), Mg(2+)-ATP-ase; ouabain-insensitive Mg(2+)-ATP-ase; Na(+), K(+)-ATP-ase; Ca(2+), Mg(2+)-ATP-ase activities were evaluated. In control animals, enzymatic activities were differently expressed in SPM1 , being the evaluated enzymatic activities higher in SPM2. Subchronic treatment with l-acetylcarnitine (i) did not modify AChE on both SPMs; (ii) increased Na(+), K(+), Mg(2+)-ATP-ase, ouabain-insensitive Mg(2+)-ATP-ase and Na(+), K(+)-ATP-ase at the dose of 30 and 60mg/kg on SPM1 and SPM2; (iii) increased Ca(2+), Mg(2+)-ATP-ase activity on both SPMs at the dose of 60mg/kg. These results have been discussed considering the pathophysiology and treatment of Dementias and Depression because, although referred to normal healthy animals, they support the notion that l-acetylcarnitine may have positive effects in these pathologies.

Stroke is a leading cause of death and disability, but most of the therapeutic approaches failed in clinical trials. The energy metabolism alterations, due to marked ATP decline, are strongly related to stroke and, at present, their physiopathological roles are not fully understood. Thus, the aim of this study was to evaluate the effects of aging on ischemia-induced changes in energy mitochondrial transduction and the consequences on overall brain energy metabolism in an in vivo experimental model of complete cerebral ischemia of 15min duration and during post-ischemic recirculation after 1, 24, 48, 72 and 96h, in 1year "adult" and 2year-old "aged" rats. The maximum rate (Vmax) of citrate synthase, malate dehydrogenase, succinate dehydrogenase for Krebs' cycle; NADH-cytochrome c reductase and cytochrome oxidase for electron transfer chain (ETC) were assayed in non-synaptic "free" mitochondria and in two populations of intra-synaptic mitochondria, i.e., "light" and "heavy" mitochondria. The catalytic activities of enzymes markedly differ according to: (a) mitochondrial type (non-synaptic, intra-synaptic), (b) age, (c) acute effects of ischemia and (d) post-ischemic recirculation at different times. Enzyme activities changes are injury maturation events and strictly reflect the bioenergetic state of the tissue in each specific experimental condition respect to the energy demand, as shown by the comparative evaluation of the energy-linked metabolites and substrates content. Remarkably, recovery of mitochondrial function was more difficult for intra-synaptic mitochondria in "aged" rats, but enzyme activities of energy metabolism tended to normalize in all mitochondrial populations after 96h of recirculation. This observation is relevant for Therapy, indicating that mitochondrial enzymes may be important metabolic factors for the responsiveness of ischemic penumbra towards the restore of cerebral functions.

The effect of aging on hippocampus is often confounded by diseases that commonly occur in the elderly. In this research, functional proteomics was used to characterize age-related changes in energy metabolism of different neuronal pathways within the hippocampus of Wistar rats aged 2, 6, 12, 18, and 24 months. The "large" synaptosomes, derived from glutamatergic mossy fiber endings connecting granule cells of dentate gyrus with apical dendrites of CA3 pyramidal cells, and the "small" synaptosomes, derived from the cholinergic small nerve endings of septo-hippocampal fibers, whose projections reach CA1 pyramidal cells, were isolated. Because most brain disorders are associated with bioenergetic changes, the maximum rate (Vmax) of selected enzymes of glycolysis, Krebs cycle, glutamate and amino acids metabolism, and acetylcholine catabolism were evaluated. The results show that "large" and "small" synaptosomes possess specific and independent metabolic features coherently with the selective vulnerability of the respective hippocampal subfields to Alzheimer's disease and cerebral ischemia. This study represents a reliable model to study in vivo (i) the physiopathological molecular mechanisms of some brain diseases dependent on energy metabolism, (ii) the responsiveness to noxious stimuli, and (iii) the effects of drugs, discriminating their action sites at subcellular level.

The effect of aging and CDP-choline treatment (20 mg kg(-1) body weight i.p. for 28 days) on the maximal rates (V-max) of representative mitochondrial enzyme activities related to Krebs' cycle (citrate synthase, alpha-ketoglutarate dehydrogenase, malate dehydrogenase), glutamate and related amino acid metabolism (glutamate dehydrogenase, glutamate-oxaloacetate- and glutamate-pyruvate transaminases) were evaluated in non-synaptic and intra-synaptic "light" and "heavy" mitochondria from frontal cerebral cortex of male Wistar rats aged 4, 12, 18 and 24 months.During aging, enzyme activities vary in a complex way respect to the type of mitochondria, i.e. non-synaptic and intra-synaptic. This micro-heterogeneity is an important factor, because energy-related mitochondrial enzyme catalytic properties cause metabolic modifications of physiopathological significance in cerebral tissue in vivo, also discriminating pre- and post-synaptic sites of action for drugs and affecting tissue responsiveness to noxious stimuli.Results show that CDP-choline in vivo treatment enhances cerebral energy metabolism selectively at 18 months, specifically modifying enzyme catalytic activities in non-synaptic and intra-synaptic "light" mitochondrial sub-populations. This confirms that the observed changes in enzyme catalytic activities during aging reflect the bioenergetic state at each single age and the corresponding energy requirements, further proving that in vivo drug treatment is able to interfere with the neuronal energy metabolism. (C) 2012 Published by Elsevier Ltd.

The maximum rates of adenosine triphosphatase (ATPase) systems related to energy consumption were systematically evaluated in synaptic plasma membranes isolated from the striata of male Wistar rats aged 2, 6, 12, 18, and 24 months, because of their key role in presynaptic nerve ending homeostasis. The following enzyme activities were evaluated: sodium-potassium-magnesium adenosine triphosphatase (Na(+), K(+), Mg(2+)-ATPase); ouabain-insensitive magnesium adenosine triphosphatase (Mg(2+)-ATPase); sodium-potassium adenosine triphosphatase (Na(+), K(+)-ATPase); direct magnesium adenosine triphosphatase (Mg(2+)-ATPase); calcium-magnesium adenosine triphosphatase (Ca(2+), Mg(2+)-ATPase); and acetylcholinesterase. The results showed that Na(+), K(+)-ATPase decreased at 18 and 24 months, Ca(2+), Mg(2+)-ATPase and acetylcholinesterase decreased from 6 months, while Mg(2+)-ATPase was unmodified. Therefore, ATPases vary independently during aging, suggesting that the ATPase enzyme systems are of neuropathological and pharmacological importance. This could be considered as an experimental model to study regeneration processes, because of the age-dependent modifications of specific synaptic plasma membranes. ATPases cause selective changes in some cerebral functions, especially bioenergetic systems. This could be of physiopathological significance, particularly in many central nervous system diseases, where, during regenerative processes, energy availability is essential.

The maximum rates (V max) of some enzymatic activities related to energy consumption (ATP-ases) were evaluated in two types of synaptic plasma membranes (SPM) isolated from cerebral cortex of rats subjected to in vivo treatment with l-acetylcarnitine at two different doses (30 and 60 mg kg−1 i.p., 28 days, 5 days/week). The following enzyme activities were evaluated: acetylcholinesterase (AChE); Na+, K+, Mg2+-ATP-ase; ouabain insensitive Mg2+-ATP-ase; Na+, K+-ATP-ase; direct Mg2+-ATP-ase; Ca2+, Mg2+-ATP-ase; Low- and High-affinity Ca2+-ATP-ase. Sub-chronic treatment with l-acetylcarnitine increased Na+, K+-ATP-ase activity on SPM 2 and Ca2+, Mg2+-ATP-ase activity on both SPM fractions. These results suggest (1) that the sensitivity to drug treatment is different between the two populations of SPM, confirming the micro-heterogeneity of these sub-fractions, probably originating from different types of synapses, (2) the specificity of the molecular site of action of the drug on SPM and (3) its interference on ion homeostasis at synaptic level.

The Italian Strong Motion Database, ITACA, was developed within projects S6 and S4, funded in the framework of the agreements between the Italian Department of Civil Protection (Dipartimento della Protezione Civile, DPC) and the Istituto Nazionale di Geofisica e Vulcanologia (INGV), starting from 2005. The alpha version of the database was released in 2007 and subsequently upgraded to version 1.0 after: (i) including the most recent strong motion data (from 2005 to 2007) recorded in Italy, in addition to the 2008 Parma earthquake, M 5.4, and the M ≥  4.0, 2009 Abruzzo seismic events; (ii) processing the raw strong motion data using an updated procedure; (iii) increasing the number of stations with a measured shear wave velocity profile; (iv) improving the utilities to retrieve time series and ground motion parameters; (v) implementing a tool for selecting time series in agreement with design-response spectra; (vi) compiling detailed station reports containing miscellaneous information such as photo, maps and site parameters; (vii) developing procedures for the automatic generation of station reports and for the updating of the header files. After such improvements, ITACA 1.0 was published at the web site http://itaca.mi.ingv.it, in 2010. It presently contains 3,955 three-component waveforms, comprising the most complete catalogue of the Italian accelerometric records in the period 1972–2007 (3,562 records) and the strongest events in the period 2008–2009. Records were mainly acquired by DPC through its Accelerometric National Network (RAN) and, in few cases, by local networks and temporary stations or networks. This paper introduces the published version of the Italian Strong Motion database (ITACA version 1.0) together with main improvements and new functionalities.

This paper describes the analyses of the single-station ambient-vibration measurements performed on the Italian accelerometric network to detect site resonance phenomena potentially affecting earthquake recordings. The use of low cost, high quality microtremor measurement can be helpful to discriminate among soil classes, since several classification schemes based on resonance frequencies were proposed in the last decades. Operatively, in the framework of the Italian Strong Motion Database project (DPC-INGV 2007–2009 S4; http://esse4.mi.ingv.it), soil resonance frequencies have been evaluated from more than 200 ambient vibration measurements in correspondence of accelerometric stations included in ITACA (http://itaca.mi.ingv.it/ItacaNet/). The noise recordings have been analyzed using the same numerical protocol in order to standardize the results. Particular attention has been paid to evaluate the quality of measurements and to develop an on-purpose mathematical tool to automatically estimate the peaks in the horizontal-to-vertical spectral ratio (HVSR) curve. The reliability of the resonance frequencies from HVSR has been tested by comparing estimates provided by independent methods (modeling or earthquake recordings). The test confirmed the reliability of the microtremor HVSR for assessing the resonance frequencies of the examined sites.

The normal-faulting earthquake of 6 April 2009 in the Abruzzo Region of central Italy caused heavy losses of life and substantial damage to centuries-old buildings of significant cultural importance and to modern reinforced-concrete-framed buildings with hollow masonry infill walls. Although structural deficiencies were significant and widespread, the study of the characteristics of strong motion data from the heavily affected area indicated that the short duration of strong shaking may have spared many more damaged buildings from collapsing. It is recognized that, with this caveat of short-duration shaking, the infill walls may have played a very important role in preventing further deterioration or collapse of many buildings. It is concluded that better new or retrofit construction practices that include reinforced-concrete shear walls may prove helpful in reducing risks in such seismic areas of Italy, other Mediterranean countries, and even in United States, where there are large inventories of deficient structures.

Geological, geophysical and geotechnical investigations, for the characterization of the strong-motion recording sites managed by the Italian Civil Protection, have been carried out in the framework of the project “Italian strong-motion database in the period 1972–2004”. The project aimed at creating an updated database of strong-motion data acquired in Italy by different institutions in the time span 1972–2004, and at improving the quality of disseminated data. This article illustrates the state of the recording site characterization before the beginning of the project, explains the criteria adopted to select the sites where geophysical/geotechnical investigation have been performed and describes the results of the promoted field surveys.

The Italian strong-motion database was created during a joint project between Istituto Nazionale di Geofisica e Vulcanologia (INGV, Italian Institute for Geophysics and Vulcanology) and Dipartimento della Protezione Civile (DPC, Italian Civil Protection). The aim of the project was the collection, homogenization and distribution of strong motion data acquired in Italy in the period 1972–2004 by different institutions, namely Ente Nazionale per l’Energia Elettrica (ENEL, Italian electricity company), Ente per le Nuove tecnologie, l’Energia e l’Ambiente (ENEA, Italian energy and environment organization) and DPC. Recently the strong-motion data relative to the 23th December 2009, Parma (Mw = 5.4 and Mw = 4.9) and to the April 2009 L’Aquila sequences (13 earthquakes with 4.1 ≤ Mw ≤ 6.3) were included in the Italian Accelerometric Archive (ITACA) database (beta release). The database contains 7,038 waveforms from analog and digital instruments, generated by 1.019 earthquakes with magnitude up to 6.9 and can be accessed on-line at the web site http://itaca.mi.ingv.it. The strong motion data are provided in the unprocessed and processed versions. This article describes the steps followed to process the acceleration time series recorded by analogue and digital instruments. The procedures implemented involve: baseline removal, instrumental correction, band pass filtering with acausal filters, integration of the corrected acceleration in order to obtain velocity and displacement waveforms, computation of acceleration response spectra and strong motion parameters. This procedure is applied to each accelerogram and it is realised to preserve the low frequency content of the records.

The Italian Strong Motion Network is a permanent monitoring system run by the Italian national emergency management department (Dipartimento della Protezione Civile, DPC). The network is known as RAN (Rete Accelerometrica Nazionale). An extensive project for updating and improving the technology of RAN instruments as well as the number of recording points was performed in the last 10 years. A wide site selection survey was carried out from eastern Sicily along the Italian peninsula, covering high seismic risk areas. The recording station density and the choice of high-quality digital strong motion instruments ensure reliability of the RAN network in the long-term. At the end of 2008, the free field sites selection and instruments installation, planned in the project, were quite completed. In planning and drawing the new RAN, special attention has been devoted to the robustness of the transmission systems, and to the distribution of new stations in order to ensure plenty of data during a seismic emergency. We spent special care both in the estimation of the RAN site responses and in the diffusion of the strong motion data. In order to better identify damaged earthquake areas, improved ground motion parameters need to be set. Such parameters will also assist future progress for engineering seismic design techniques as well as disaster mitigation.