Mitochondrial bioenergetics and cytometric characterization of a synaptosomal preparation from mouse brain cortex.

Mitochondrial function at synapses can be assessed in isolated nerve terminals. Synaptosomes are structures obtained in vitro by detaching the nerve endings from neuronal bodies under controlled homogenization conditions. Several protocols have been described for the preparation of intact synaptosomal fractions. Herein a fast and economical method to obtain synaptosomes with optimal intrasynaptic mitochondria functionality was described. Synaptosomal fractions were obtained from mouse brain cortex by differential centrifugation followed by centrifugation in a Ficoll gradient. The characteristics of the subcellular particles obtained were analyzed by flow cytometry employing specific tools. Integrity and specificity of the obtained organelles were evaluated by calcein and SNAP-25 probes. The proportion of positive events of the synaptosomal preparation was 75 ± 2 % and 48 ± 7% for calcein and Synaptosomal-Associated Protein of 25 kDa (SNAP-25), respectively. Mitochondrial integrity was evaluated by flow cytometric analysis of cardiolipin content, which indicated that 73 ± 1% of the total events were 10 N-nonylacridine orange (NAO)-positive. Oxygen consumption, ATP production and mitochondrial membrane potential determinations showed that mitochondria inside synaptosomes remained functional after the isolation procedure. Mitochondrial and synaptosomal enrichment were determined by measuring synaptosomes/ homogenate ratio of specific markers. Functionality of synaptosomes was verified by nitric oxide detection after glutamate addition. As compared with other methods, the present protocol can be performed briefly, does not imply high economic costs, and provides an useful tool for the isolation of a synaptosomal preparation with high mitochondrial respiratory capacity and an adequate integrity and function of intraterminal mitochondria.

Plasticity of GluN1 at Ventral Hippocampal Synapses in the Infralimbic Cortex.

Although the infralimbic cortex (IL) is not thought to play a role in fear acquisition, recent experiments found evidence that synaptic plasticity is occurring at ventral hippocampal (vHPC) synapses in IL during auditory fear acquisition as measured by changes in the N-methyl-D-aspartate (NMDA) receptor-mediated currents in male rats. These electrophysiological data suggest that fear conditioning changes the expression of NMDA receptors on vHPC-to-IL synapses. To further evaluate the plasticity of NMDA receptors at this specific synapse, we injected AAV particles expressing channelrhodopsin-EYFP into the vHPC of male and female rats to label vHPC projections with EYFP. To test for NMDA receptor changes in vHPC-to-IL synapses after fear learning, we used fluorescence-activated cell sorting (FACS) to quantify synaptosomes isolated from IL tissue punches that were positive for EYFP and the obligatory GluN1 subunit. More EYFP+/GluN1+ synaptosomes with greater average expression of GluN1 were isolated from male rats exposed to auditory fear conditioning (AFC) than those exposed to context and tones only or to contextual fear conditioning (CFC), suggesting that AFC increased NMDA receptor expression in males. In a second experiment, we found that pairing the tones and shocks was required to induce the molecular changes and that fear extinction did not reverse the changes. In contrast, females showed similar levels of EYFP+/GluN1+ synaptosomes in all behavioral groups. These findings suggest that AFC induces synaptic plasticity of NMDA receptors in the vHPC-to-IL projection in males, while female rats rely on different synaptic mechanisms.

Alcohol hangover induces nitric oxide metabolism changes by impairing NMDA receptor-PSD95-nNOS pathway.

Alcohol hangover is defined as the combination of mental and physical symptoms experienced the day after a single episode of heavy drinking, starting when blood alcohol concentration approaches zero. We previously evidenced increments in free radical generation and an imbalance in antioxidant defences in non-synaptic mitochondria and synaptosomes during hangover. It is widely known that acute alcohol exposure induces changes in nitric oxide (NO) production and blocks the binding of glutamate to NMDAR in central nervous system. Our aim was to evaluate the residual effect of acute ethanol exposure (hangover) on NO metabolism and the role of NMDA receptor-PSD95-nNOS pathway in non-synaptic mitochondria and synaptosomes from mouse brain cortex. Results obtained for the synaptosomes fraction showed a 37% decrease in NO total content, a 36% decrease in NOS activity and a 19% decrease in nNOS protein expression. The in vitro addition of glutamate to synaptosomes produced a concentration-dependent enhancement of NO production which was significantly lower in samples from hangover mice than in controls for all the glutamate concentrations tested. A similar patter of response was observed for nNOS activity being decreased both in basal conditions and after glutamate addition. In addition, synaptosomes exhibited a 64% and 15% reduction in NMDA receptor subunit GluN2B and PSD-95 protein expression, respectively. Together with this, glutamate-induced calcium entry was significant decreased in synaptosomes from alcohol-treated mice. On the other hand, in non-synaptic mitochondria, no significant differences were observed in NO content, NOS activity or nNOS protein expression. The expression of iNOS remained unaltered in synaptosomes and non-synaptic mitochondria. Here we demonstrated that hangover effects on NO metabolism are strongly evidenced in synaptosomes probably due to a disruption in NMDAR/PSD-95/nNOS pathway.

Age-Dependent Decline in Synaptic Mitochondrial Function Is Exacerbated in Vulnerable Brain Regions of Female 3xTg-AD Mice.

Synaptic aging has been associated with neuronal circuit dysfunction and cognitive decline. Reduced mitochondrial function may be an early event that compromises synaptic integrity and neurotransmission in vulnerable brain regions during physiological and pathological aging. Thus, we aimed to measure mitochondrial function in synapses from three brain regions at two different ages in the 3xTg-AD mouse model and in wild mice. We found that aging is the main factor associated with the decline in synaptic mitochondrial function, particularly in synapses isolated from the cerebellum. Accumulation of toxic compounds, such as tau and Aß, that occurred in the 3xTg-AD mouse model seemed to participate in the worsening of this decline in the hippocampus. The changes in synaptic bioenergetics were also associated with increased activation of the mitochondrial fission protein Drp1. These results suggest the presence of altered mechanisms of synaptic mitochondrial dynamics and their quality control during aging and in the 3xTg-AD mouse model; they also point to bioenergetic restoration as a useful therapeutic strategy to preserve synaptic function during aging and at the early stages of Alzheimer's disease (AD).

Gyroxin, a toxin from Crotalus durissus terrificus snake venom, induces a calcium dependent increase in glutamate release in mice brain cortical synaptosomes.

Gyroxin is a thrombin-like toxin obtained from the venom of the South American rattlesnake, Crotalus durissus terrificus. Literature has reported "gyroxin syndrome" characterized, in mice, as series of aberrant motor behavior, known as barrel rotation, mainly after intraperitoneal administration. Despites several studies, a physiological mechanism of "gyroxin syndrome" are still not completely understood. In this context, alterations on the central nervous system (CNS), especially causing neurotoxic events, are pointed out as likely candidates. Then, we decided to investigate whether gyroxin induces alterations in glutamate release, one of the most important neurotransmitter involved in neurotoxicity. For that, we performed all experiments, in vitro, using a model of mice brain cortical synaptosomes. Notably, our results indicate that the administration of gyroxin on purified presynaptic brain cortical terminals resulted in an extracellular Ca2+- dependent raise in glutamate release. Indeed, our results also showed that gyroxin increases intrasynaptosomal calcium (Ca2+) levels through acting on voltage gated calcium channels (VGCC), specifically N and P/Q subtypes. Moreover, our data show that gyroxin increases exocytosis rate. Interestingly, these data suggest that gyroxin might induce neurotoxicity by increasing glutamate levels. However, future investigations are needed in order to elucidate the nature of the following events.

Cross-talk between dopamine D1 and corticotropin releasing factor type 2 receptors leads to occlusion of their ERK1/2 signaling.

One manner in which G protein-coupled receptors potentiate, increase, and change their functionality is through the formation of heteromers in a specific cellular context. Previously, we have shown that dopamine D1 receptor (D1R) and the corticotropin releasing factor receptor type-2α (CRF2α) heteromerize in HEK293T cells, enabling D1R to mobilize intracellular calcium in response to D1R agonists. In this study, we further investigated the pharmacological properties of the CRF2α-D1R heteromer and the consequences of the heteromerization in their signaling and subcellular localization when both receptors are co-expressed in HEK293T cells. Using immunoprecipitation assays, we observed that the addition of 10 µM dopamine in the incubation medium significantly decreased the amount of CRF2α on the cell surface of cells expressing both receptors. The presence of agonists of both receptors increased the interaction between CRF2α and D1R as assessed by co-immunoprecipitation. However, the presence of agonists of both receptors resulted in a lesser efficient activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase. Using a synaptosomal preparation of rat prefrontal cortex devoid of post-synaptic elements, we found that CRF2α and D1R co-localize in synaptic terminals of the rat medial prefrontal cortex and that the simultaneous activation of both receptors also occluded phosphorylation of extracellular signal-regulated kinase. These results strengthen the idea that the heteromer CRF2a-D1R is an entity functionally different from each receptor that composes it and suggests that its formation is enhanced by CRF and dopamine co-transmission, as occurs in stress and addiction.

Neuroprotective and MAOB inhibitory effects of a series of caffeine-8-thioglycolic acid amides

The effects of new derivatives of caffeine-8-thioglycolic acid (100 µM) on isolated rat brain synaptosomes, human neuroblastoma cell line SH-SY5Y and human recombinant MAOB enzyme (hMAOB) (1 µM) were evaluated. Most of the compounds, administered alone, didn't show statistically significant neurotoxic effects on SH-SY5Y, when compared to the control (non-treated cells). Of all studied structures JTA-2Ox, JTA-11, JTA-12 and JTA-13 decreased cell viability. In combination with 6-hydroxydopamine (6-OHDA) (100 µM), only JTA-1 and JTA-2 revealed neuroprotective effects, stronger than those of caffeine. All compounds administered alone revealed, neurotoxic effects on synaptosomes, as compared to non-treated synaptosomes. JTA-1, JTA-2 and JTA-3 showed lowest neurotoxic effects and were investigated in a model of 6-OHDA-induced oxidative stress. In this model of neurotoxicity, only JTA-1 and JTA-2 showed statistically significant neuroprotective effect, by preserving the synaptosomal viability and the level of reduced glutathione. Inhibition of hMAOB, was revealed by JTA-1 and JTA-2. They inhibited the enzyme by 23% and 25% respectively, thus approaching the selegiline activity, which was 42%. The possible mechanisms of neuroprotection of JTA-1 and JTA-2 might be a result from the inhibition of hMAOB, which catalyze the production of neurotoxic p-quinone from 6-OHDA.

Alcohol hangover effects on brain cortex non-synaptic mitochondria and synaptosomes bioenergetics.

Alcohol hangover (AH) has been associated with oxidative stress and mitochondrial dysfunction. We herein postulate that AH-induced mitochondrial alterations can be due to a different pattern of response in synaptosomes and non-synaptic (NS) mitochondria. Mice received intraperitoneal (i.p.) injections of ethanol (3.8 g/kg) or saline and were sacrificed 6 h afterward. Brain cortex NS mitochondria and synaptosomes were isolated by Ficoll gradient. Oxygen consumption rates were measured in NS mitochondria and synaptosomes by high-resolution respirometry. Results showed that NS-synaptic mitochondria from AH animals presented a 26% decrease in malate-glutamate state 3 respiration, a 64% reduction in ATP content, 28-37% decrements in ATP production rates (malate-glutamate or succinate-dependent, respectively), and 44% inhibition in complex IV activity. No changes were observed in mitochondrial transmembrane potential (ΔΨ) or in UCP-2 expression in NS-mitochondria. Synaptosome respiration driving proton leak (in the presence of oligomycin), and spare respiratory capacity (percentage ratio between maximum and basal respiration) were 30% and 15% increased in hangover condition, respectively. Synaptosomal ATP content was 26% decreased, and ATP production rates were 40-55% decreased (malate-glutamate or succinate-dependent, respectively) in AH mice. In addition, a 24% decrease in ΔΨ and a 21% increase in UCP-2 protein expression were observed in synaptosomes from AH mice. Moreover, mitochondrial respiratory complexes I-III, II-III, and IV activities measured in synaptosomes from AH mice were decreased by 18%, 34%, and 50%, respectively. Results of this study reveal that alterations in bioenergetics status during AH could be mainly due to changes in mitochondrial function at the level of synapses.

Toxicity of beta-amyloid protein during aging: Influence of synaptosomal energy metabolism / Toxicidad del beta-amiloide durante el envejecimiento: influencia del metabolismo energético sinaptosomal

Background: Recent evidence suggests that early neurodegenerative events associated with Alzheimer's disease (AD) probably begin in the synaptic terminal, where it has been reported a large accumulation of ß-amyloid protein (Aß), one of the main factors described in the development of AD. We analyzed the influence of energy metabolism on the toxic effects of Aß during aging on synaptosomes from neocortex and hippocampus of rats exposed to inhibitors of glycolytic and mitochondrial metabolism and we evaluated the protective effects of some antioxidant compounds. Methods: Synaptosomes were obtained by differential centrifugation in sucrose gradients and their redox activity was determined with the MTT assay. Results: The mitochondrial activity of synaptosomes from young rats was not altered by the presence of Aß; the ones obtained from old rats showed an increase in susceptibility to Aß; this activity was greater in the synaptic terminals of the hippocampus. Conclusions: These results provide experimental support for the hypothesis that certain risk factors, such as energy metabolism dysfunction or the aging process itself, may increase vulnerability to Aß. Hippocampal region is more susceptible to Aß and its effect increases with age in relation to the neocortex, which would agree with the damage gradient reported in the AD.

Introducción: evidencia reciente sugiere que eventos neurodegenerativos tempranos asociados con la enfermedad de Alzheimer (EA) probablemente se inicien en la terminal sináptica, en donde se observa una gran acumulación de la proteína ß-amiloide (Aß), uno de los factores involucrados en el desarrollo de la EA. Estudiamos la influencia del metabolismo energético en los efectos tóxicos de la Aß en el envejecimiento en sinaptosomas de neocorteza e hipocampo de ratas expuestas a inhibidores del metabolismo glucolítico y mitocondrial, y evaluamos los efectos protectores de algunos antioxidantes. Métodos: los sinaptosomas se obtuvieron por centrifugación diferencial en gradientes de sacarosa y su actividad óxido-reductura se determinó con la técnica de MTT. Resultados: la actividad mitocondrial de los sinaptosomas de ratas jóvenes no se alteró por la presencia de la Aß; los de ratas viejas mostraron un aumento en la susceptibilidad a la Aß, el efecto fue mayor en las terminales sinápticas del hipocampo. Conclusiones: los resultados sustentan la hipótesis de que ciertos factores de riesgo, como las disfunciones del metabolismo energético o el proceso de envejecimiento, pueden incrementar la vulnerabilidad a la Aß y su efecto se incrementa con la edad en relación con la neocorteza, lo cual concordaría con el gradiente de daño reportado en la EA.

Thallium-Induced Toxicity in Rat Brain Crude Synaptosomal/Mitochondrial Fractions is Sensitive to Anti-excitatory and Antioxidant Agents.

The mechanisms by which the heavy metal thallium (Tl+) produces toxicity in the brain remain unclear. Herein, isolated synaptosomal/mitochondrial P2 crude fractions from adult rat brains were exposed to Tl+ (5-250 µM) for 30 min. Three toxic endpoints were evaluated: mitochondrial dysfunction, lipid peroxidation, and Na+/K+-ATPase activity inhibition. Concentration-response curves for two of these endpoints revealed the optimum concentration of Tl+ to induce damage in this preparation, 5 µM. Toxic markers were also estimated in preconditioned synaptosomes incubated in the presence of the N-methyl-D-aspartate receptor antagonist kynurenic acid (KYNA, 50 µM), the cannabinoid receptor agonist WIN 55,212-2 (1 µM), or the antioxidant S-allyl-L-cysteine (SAC, 100 µM). All these agents prevented Tl+ toxicity, though SAC did it with lower efficacy. Our results suggest that energy depletion, oxidative damage, and Na+/K+-ATPase activity inhibition account for the toxic pattern elicited by Tl+ in nerve terminals. In addition, the efficacy of the drugs employed against Tl+ toxicity supports an active role of excitatory/cannabinoid and oxidative components in the toxic pattern elicited by the metal.

2-Arachidonoylglycerol metabolism is differently modulated by oligomeric and fibrillar conformations of amyloid beta in synaptic terminals.

Alzheimer's disease (AD) is the most prevalent disorder of senile dementia mainly characterized by amyloid-beta peptide (Aß) deposits in the brain. Cannabinoids are relevant to AD as they exert several beneficial effects in many models of this disease. Still, whether the endocannabinoid system is either up- or down-regulated in AD has not yet been fully elucidated. Thus, the aim of the present paper was to analyze endocannabinoid 2-arachidonoylglycerol (2-AG) metabolism in cerebral cortex synaptosomes incubated with Aß oligomers or fibrils. These Aß conformations were obtained by "aging" the 1-40 fragment of the peptide under different agitation and time conditions. A diminished availability of 2-AG resulting from a significant decrease in diacylglycerol lipase (DAGL) activity was observed in the presence of large Aß1-40 oligomers along with synaptosomal membrane damage, as judged by transmission electron microscopy and LDH release. Conversely, a high availability of 2-AG resulting from an increase in DAGL and lysophosphatidic acid phosphohydrolase activities occurred in the presence of Aß1-40 fibrils although synaptosomal membrane disruption was also observed. Interestingly, neither synaptosomal mitochondrial viability assayed by MTT reduction nor membrane lipid peroxidation assayed by TBARS formation measurements were altered by Aß1-40 oligomers or fibrils. These results show a differential effect of Aß1-40 peptide on 2-AG metabolism depending on its conformation.

Free radical production and antioxidant status in brain cortex non-synaptic mitochondria and synaptosomes at alcohol hangover onset.

Alcohol hangover (AH) is the pathophysiological state after a binge-like drinking. We have previously demonstrated that AH induced bioenergetics impairments in a total fresh mitochondrial fraction in brain cortex and cerebellum. The aim of this work was to determine free radical production and antioxidant systems in non-synaptic mitochondria and synaptosomes in control and hangover animals. Superoxide production was not modified in non-synaptic mitochondria while a 17.5% increase was observed in synaptosomes. A similar response was observed for cardiolipin content as no changes were evidenced in non-synaptic mitochondria while a 55% decrease in cardiolipin content was found in synaptosomes. Hydrogen peroxide production was 3-fold increased in non-synaptic mitochondria and 4-fold increased in synaptosomes. In the presence of deprenyl, synaptosomal H2O2 production was 67% decreased in the AH condition. Hydrogen peroxide generation was not affected by deprenyl addition in non-synaptic mitochondria from AH mice. MAO activity was 57% increased in non-synaptic mitochondria and 3-fold increased in synaptosomes. Catalase activity was 40% and 50% decreased in non-synaptic mitochondria and synaptosomes, respectively. Superoxide dismutase was 60% decreased in non-synaptic mitochondria and 80% increased in synaptosomal fractions. On the other hand, GSH (glutathione) content was 43% and 17% decreased in synaptosomes and cytosol. GSH-related enzymes were mostly affected in synaptosomes fractions by AH condition. Acetylcholinesterase activity in synaptosomes was 11% increased due to AH. The present work reveals that AH provokes an imbalance in the cellular redox homeostasis mainly affecting mitochondria present in synaptic terminals.

Controversial alkoxyl and peroxyl radical scavenging activity of the tryptophan metabolite 3-hydroxy-anthranilic acid.

3-Hydroxy-anthranilic acid (3-OHAA), a tryptophan metabolite produced in the kynurenine pathway, is an efficient antioxidant towards peroxyl radicals (ROO) derived from the AAPH (2,2'-azobis(2-amidinopropane) dihydrochloride) thermolysis. However, self-reactions of ROO can give rise to alkoxyl radicals (RO), which could strongly affect the fate of scavenging reactions. In the present work, we studied the influence of RO in the scavenging activity of 3-OHAA in three different systems: i) Monitoring of the direct reaction between 3-OHAA and AAPH-derived free radicals (kinetic studies); ii) Evaluation of the protective effect of 3-OHAA on the AAPH-induced consumption of fluorescein; and, iii) Inhibition, given by 3-OHAA, of the AAPH-initiated lipid peroxidation of both, rat brain synaptosomes and homogenate preparations (assessed by chemiluminescence). For such purposes, the fraction of free radicals (f) trapped per 3-OHAA molecule was determined in each system. Kinetic results show that the oxidation of 3-OHAA follows a process dominated by ROO with a zero order kinetic limit in 3-OHAA, and a fraction (fri) equal to 0.88. From the induction times, elicited by 3-OHAA in the kinetic profiles of fluorescein consumption, a fraction (fT) of 0.28 was determined. 3-OHAA also generated induction times in the kinetic profiles of light emission during the AAPH-initiated lipid peroxidation of rat brain synaptosomes and homogenates. From such induction times, fractions of 0.61 and 0.63 were determined for rat brain synaptosomes (fsyn) and homogenates (fhom), respectively. These results show that during the incubation of 3-OHAA and AAPH, a low fraction of ROO self-reacts to generate RO. Nevertheless, when 3-OHAA is employed to protect particular targets, such as fluorescein, rat brain synaptosomes and homogenates, reactions of ROO and/or RO should be considered.

Synaptosomal bioenergetic defects are associated with cognitive impairment in a transgenic rat model of early Alzheimer's disease.

Synaptic bioenergetic deficiencies may be associated with early Alzheimer's disease (AD). To explore this concept, we assessed pre-synaptic mitochondrial function in hemizygous (+/-)TgMcGill-R-Thy1-APP rats. The low burden of Aß and the wide array of behavioral and cognitive impairments described in 6-month-old hemizygous TgMcGill-R-Thy1-APP rats (Tg(+/-)) support their use to investigate synaptic bioenergetics deficiencies described in subjects with early Alzheimer's disease (AD). In this report, we show that pre-synaptic mitochondria from Tg(+/-) rats evidence a decreased respiratory control ratio and spare respiratory capacity associated with deficits in complex I enzymatic activity. Cognitive impairments were prevented and bioenergetic deficits partially reversed when Tg(+/-) rats were fed a nutritionally complete diet from weaning to 6-month-old supplemented with pyrroloquinoline quinone, a mitochondrial biogenesis stimulator with antioxidant and neuroprotective effects. These results provide evidence that, as described in AD brain and not proven in Tg mice models with AD-like phenotype, the mitochondrial bioenergetic capacity of synaptosomes is not conserved in the Tg(+/-) rats. This animal model may be suitable for understanding the basic biochemical mechanisms involved in early AD.

Hypothyroidism Enhanced Ectonucleotidases and Acetylcholinesterase Activities in Rat Synaptosomes can be Prevented by the Naturally Occurring Polyphenol Quercetin.

Thyroid hormones have an influence on the functioning of the central nervous system. Furthermore, the cholinergic and purinergic systems also are extensively involved in brain function. In this context, quercetin is a polyphenol with antioxidant and neuroprotective properties. This study investigated the effects of (MMI)-induced hypothyroidism on the NTPDase, 5'-nucleotidase, adenosine deaminase (ADA), and acetylcholinesterase (AChE) activities in synaptosomes of rats and whether the quercetin can prevent it. MMI at a concentration of 20 mg/100 mL was administered for 90 days in the drinking water. The animals were divided into six groups: control/water (CT/W), control/quercetin 10 mg/kg, control/quercetin 25 mg/kg, methimazole/water (MMI/W), methimazole/quercetin 10 mg/kg (MMI/Q10), and methimazole/quercetin 25 mg/kg (MMI/Q25). On the 30th day, hormonal dosing was performed to confirm hypothyroidism, and the animals were subsequently treated with 10 or 25 mg/kg quercetin for 60 days. NTPDase activity was not altered in the MMI/W group. However, treatment with quercetin decreased ATP and ADP hydrolysis in the MMI/Q10 and MMI/Q25 groups. 5'-nucleotidase activity increased in the MMI/W group, but treatments with 10 or 25 mg/kg quercetin decreased 5'-nucleotidase activity. ADA activity decreased in the CT/25 and MMI/Q25 groups. Furthermore, AChE activity was reduced in all groups with hypothyroidism. In vitro tests also demonstrated that quercetin per se decreased NTPDase, 5'-nucleotidase, and AChE activities. This study demonstrated changes in the 5'-nucleotidase and AChE activities indicating that purinergic and cholinergic neurotransmission are altered in this condition. In addition, quercetin can alter these parameters and may be a promising natural compound with important neuroprotective actions in hypothyroidism.

Brain cortex mitochondrial bioenergetics in synaptosomes and non-synaptic mitochondria during aging.

Alterations in mitochondrial bioenergetics have been associated with brain aging. In order to evaluate the susceptibility of brain cortex synaptosomes and non-synaptic mitochondria to aging-dependent dysfunction, male Swiss mice of 3 or 17 months old were used. Mitochondrial function was evaluated by oxygen consumption, mitochondrial membrane potential and respiratory complexes activity, together with UCP-2 protein expression. Basal respiration and respiration driving proton leak were decreased by 26 and 33 % in synaptosomes from 17-months old mice, but spare respiratory capacity was not modified by aging. Succinate supported state 3 respiratory rate was decreased by 45 % in brain cortex non-synaptic mitochondria from 17-month-old mice, as compared with young animals, but respiratory control was not affected. Synaptosomal mitochondria would be susceptible to undergo calcium-induced depolarization in 17 months-old mice, while non-synaptic mitochondria would not be affected by calcium overload. UCP-2 was significantly up-regulated in both synaptosomal and submitochondrial membranes from 17-months old mice, compared to young animals. UCP-2 upregulation seems to be a possible mechanism by which mitochondria would be resistant to suffer oxidative damage during aging.

Corticotropin-releasing factor type-2 receptor and corticotropin-releasing factor-binding protein coexist in rat ventral tegmental area nerve terminals originated in the lateral hypothalamic area.

There is significant functional evidence showing that corticotropin-releasing factor type-2 receptor (CRF2R) and corticotropin-releasing factor-binding protein (CRF-BP) regulate glutamatergic synapses onto ventral tegmental area (VTA) dopaminergic neurons. It has been shown that CRF requires CRF-BP to potentiate N-methyl-D-aspartate receptors in dopaminergic neurons through CRF2R, and that increases glutamate release in cocaine-treated rats through the activation of CRF2R only by agonists with high affinity to CRF-BP. Furthermore, this CRF-mediated increase in VTA glutamate is responsible for stress-induced relapse to cocaine-seeking behaviour. However, there is a lack of anatomical evidence to explain the mechanisms of CRF actions in VTA. Thus, it was studied whether CRF2R and CRF-BP are expressed in VTA nerve terminals, using a synaptosomal preparation devoid of postsynaptic elements. The current results show that both proteins are co-expressed in glutamatergic and γ-aminobutyric acid (GABA)ergic VTA synaptosomes. A main glutamatergic input to the VTA that has been associated to addictive behaviour is originated in the lateral hypothalamic area (LHA). Thus, this study was focused in the LHA-VTA input using orexin as a marker of this input. The results show that CRF2R and CRF-BP mRNA and protein are expressed in the LHA, and that both proteins are present in orexin-positive VTA synaptosomes. The results showing that CRF2R and CRF-BP are expressed in the LHA-VTA input give anatomical support to suggest that this input plays a role in stress-induced relapse to cocaine-seeking behaviour.

The effect of WIN 55,212-2 suggests a cannabinoid-sensitive component in the early toxicity induced by organic acids accumulating in glutaric acidemia type I and in related disorders of propionate metabolism in rat brain synaptosomes.

Several physiological processes in the CNS are regulated by the endocannabinoid system (ECS). Cannabinoid receptors (CBr) and CBr agonists have been involved in the modulation of the N-methyl-D-aspartate receptor (NMDAr) activation. Glutaric (GA), 3-hydroxyglutaric (3-OHGA), methylmalonic (MMA) and propionic (PA) acids are endogenous metabolites produced and accumulated in the brain of children affected by severe organic acidemias (OAs) with neurodegeneration. Oxidative stress and excitotoxicity have been involved in the toxic pattern exerted by these organic acids. Studying the early pattern of toxicity exerted by these metabolites is crucial to explain the extent of damage that they can produce in the brain. Herein, we investigated the effects of the synthetic CBr agonist WIN 55,212-2 (WIN) on early markers of GA-, 3-OHGA-, MMA- and PA-induced toxicity in brain synaptosomes from adult (90-day-old) and adolescent (30-day-old) rats. As pre-treatment, WIN exerted protective effects on the GA- and MMA-induced mitochondrial dysfunction, and prevented the reactive oxygen species (ROS) formation and lipid peroxidation induced by all metabolites. Our findings support a protective and modulatory role of cannabinoids in the early toxic events elicited by toxic metabolites involved in OAs.

The effects of JM-20 on the glutamatergic system in synaptic vesicles, synaptosomes and neural cells cultured from rat brain.

JM-20 (3-ethoxycarbonyl-2-methyl-4-(2-nitrophenyl)-4,11-dihydro-1H-pyrido[2,3-b][1,5]benzodiazepine) is a novel benzodiazepine dihydropyridine hybrid molecule, which has been shown to be a neuroprotective agent in brain disorders involving glutamate receptors. However, the effect of JM-20 on the functionality of the glutamatergic system has not been investigated. In this study, by using different in vitro preparations, we investigated the effects of JM-20 on (i) rat brain synaptic vesicles (L-[(3)H]-glutamate uptake, proton gradient built-up and bafilomycin-sensitive H(+)-ATPase activity), (ii) rat brain synaptosomes (glutamate release) and (iii) primary cultures of rat cortical neurons, astrocytes and astrocyte-neuron co-cultures (L-[(3)H]-glutamate uptake and glutamate release). We observed here that JM-20 impairs H(+)-ATPase activity and consequently reduces vesicular glutamate uptake. This molecule also inhibits glutamate release from brain synaptosomes and markedly increases glutamate uptake in astrocytes alone, and co-cultured neurons and astrocytes. The impairment of vesicular glutamate uptake by inhibition of the H(+)-ATPase caused by JM-20 could decrease the amount of the transmitter stored in synaptic vesicles, increase the cytosolic levels of glutamate, and will thus down-regulate neurotransmitter release. Together, these results contribute to explain the anti-excitotoxic effect of JM-20 and its strong neuroprotective effect observed in different in vitro and in vivo models of brain ischemia.

Antioxidant effects of JM-20 on rat brain mitochondria and synaptosomes: mitoprotection against Ca²âº-induced mitochondrial impairment.

Because mitochondrial oxidative stress and impairment are important mediators of neuronal damage in neurodegenerative diseases and in brain ischemia/reperfusion, in the present study, we evaluated the antioxidant and mitoprotective effect of a new promising neuroprotective molecule, JM-20, in mitochondria and synaptosomes isolated from rat brains. JM-20 inhibited succinate-mediated H2O2 generation in both mitochondria and synaptosomes incubated in depolarized (high K(+)) medium at extremely low micromolar concentration and with identical IC50 values of 0.91 µM. JM-20 also repressed glucose-induced H2O2 generation stimulated by rotenone or by antimycin A in synaptosomes incubated in high sodium-polarized medium at extremely low IC50 values of 0.395 µM and 2.452 µM, respectively. JM-20 was unable to react directly with H2O2 or with superoxide anion radicals but displayed a cathodic reduction peak at -0.71V, which is close to that of oxygen (-0.8V), indicating high electron affinity. JM-20 also inhibited uncoupled respiration in mitochondria or synaptosomes and was a more effective inhibitor in the presence of the respiratory substrates glutamate/malate than in the presence of succinate. JM-20 also prevented Ca(2+)-induced mitochondrial permeability transition pore opening, membrane potential dissipation and cytochrome c release, which are key pathogenic events during stroke. This molecule also prevented Ca(2+) influx into synaptosomes and mitochondria; the former effect was a consequence of the latter because JM-20 inhibition followed the patterns of carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP), which is a classic mitochondrial uncoupler. Because the mitochondrion is considered an important source and target of neuronal cell death signaling after an ischemic insult, the antioxidant and protective effects of JM-20 against the deleterious effects of Ca(2+) observed at the mitochondrial level in this study may endow this molecule with the ability to succeed in mitochondrion-targeted strategies to combat ischemic brain damage.