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Vascular dementia (VD) is a major cognitive disorder originated from a blood flow disruption in the brain. This process leads to chronic cerebral ischemia that deeply affects neuronal tissues and lipid homeostasis. The understanding of cerebral lipid dynamics during chronic ischemia can reveal biomarkers and novel pharmacological targets for the treatment of VD. In this study, we used the Desorption Electrospray Ionization - imaging mass spectrometry (DESI-IMS) technique to map lipids in the rat brain tissues after bilateral common carotid artery occlusion (BCCAO) rat model of chronic cerebral hypoperfusion. The brain imaging enabled the detection of differences in lipids from ischemic and non-ischemic brains. The analysis demonstrated that arachidonic acid (ARA), docosahexaenoic acid (DHA), dihomo-γ-linolenic acid, hydroxyeicosatetraenoic (HETE)-Ala and glycerophosphoethanolamine levels were significantly reduced in the hippocampus and cortex of animals submitted to BCCAO model when compared to control animals. Decanoic acid was increased after 30 days of BCCAO model. Partial least squares discriminant analysis (PLS-DA) could discriminate between BCCAO group and the control group, in which γ-linolenic acid (m/z 277) ion and stearic acid (m/z 283) had the highest discrimination potential. Taken together, these findings indicate that lipid dynamics are altered in chronic ischemia-induced by BCCAO in rats and indicate potential biomarkers and pharmacological targets for VD., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

GABA is an inhibitory neurotransmitter that appears to be associated with the action of volatile anesthetics. These anesthetics potentiate GABA-induced postsynaptic currents by synaptic GABAA receptors, although recent evidence suggests that these agents also significantly affect extrasynaptic GABA receptors. However, the effect of volatile anesthetics on the extracellular concentration of GABA in the central nervous system has not been fully established. In the present study, rat brain cortical slices loaded with [(3)H]GABA were used to investigate the effect of halothane and sevoflurane on the extracellular accumulation of this neurotransmitter. The accumulation of [(3)H]GABA was significantly increased by sevoflurane (0.058, 0.11, 0.23, 0.46, and 0.93 mM) and halothane (0.006, 0.012, 0.024, 0.048, 0072, and 0.096 mM) with an EC50 of 0.26 mM and 35 μM, respectively. TTX (blocker of voltage-dependent Na(+) channels), EGTA (an extracellular Ca(2+) chelator) and BAPTA-AM (an intracellular Ca(2+) chelator) did not interfere with the accumulation of [(3)H]GABA induced by 0.23 mM sevoflurane and 0.048 mM halothane. SKF 89976A, a GABA transporter type 1 (GAT-1) inhibitor, reduced the sevoflurane- and halothane-induced increase in the accumulation of GABA by 57 and 63 %, respectively. Incubation of brain cortical slices at low temperature (17 °C), a condition that inhibits GAT function and reduces GABA release through reverse transport, reduced the sevoflurane- and halothane-induced increase in the accumulation of [(3)H]GABA by 82 and 75 %, respectively, relative to that at normal temperature (37 °C). Ouabain, a Na(+)/K(+) ATPase pump inhibitor, which is known to induce GABA release through reverse transport, abolished the sevoflurane and halothane effects on the accumulation of [(3)H]GABA. The effect of sevoflurane and halothane did not involve glial transporters because β-alanine, a blocker of GAT-2 and GAT-3, did not inhibit the effect of the anesthetics. In conclusion, the present study suggests that sevoflurane and halothane increase the accumulation of GABA by inducing the reverse transport of this neurotransmitter. Therefore, volatile anesthetics could interfere with neuronal excitability by increasing the action of GABA on synaptic and extrasynaptic GABA receptors.

Membrane rafts are domains enriched in sphingolipids, glycolipids and cholesterol that are able to compartmentalize cellular processes. Noteworthy, many proteins have been assigned to membrane rafts including those related to the control of the synaptic vesicle release machinery, which is a important step for neurotransmission between synapses. In this work, we have investigated the role of cholesterol in key steps of glutamate release in isolated nerve terminals (synaptosomes) from rat brain cortices. Incubation of synaptosomes with methyl-β-cyclodextrin (MβCD) induced glutamate release in a dose-dependent fashion. HγCD, a cyclodextrin with low affinity for cholesterol, had no significant effect on spontaneous glutamate release. When we evaluated the effects of MβCD on glutamate release induced by depolarizing stimuli, we observed that MβCD treatment inhibited the KCl-evoked glutamate release. The glutamate release induced by MβCD was not altered by treatment with EGTA nor with EGTA-AM. The KCl-evoked glutamate release was no further inhibited when synaptosomes were incubated with MβCD in the absence of calcium. We therefore investigated whether the cholesterol removal by MβCD changes intrasynaptosomal sodium and calcium levels. Our results suggested that the cholesterol removal effect on spontaneous and evoked glutamate release might be upstream to sodium and calcium entry through voltage-activated channels. We therefore tested if MβCD would have a direct effect on synaptic vesicle exocytosis and we showed that cholesterol removal by MβCD induced spontaneous exocytosis and inhibited synaptic vesicle exocytosis evoked by depolarizing stimuli. Lastly, we investigated the effect of protein kinase inhibitors on the spontaneous exocytosis evoked by MβCD and we observed a statistically significant reduction of synaptic vesicles exocytosis. In conclusion, our work shows that cholesterol removal facilitates protein kinase activation that favors spontaneous synaptic vesicles and consequently glutamate release in isolated nerve terminals., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Experimental data suggest that halothane anesthesia is associated with significant changes in dopamine (DA) concentration in some brain regions but the mechanism of this effect is not well known. Rat brain cortical slices were labeled with [(3)H]DA to further characterize the effects of halothane on the release of this neurotransmitter from the central nervous system. Halothane induced an increase on the release of [(3)H]DA that was dependent on incubation time and anesthetic concentration (0.012, 0.024, 0.048, 0.072 and 0.096 mM). This effect was independent of extracellular or intracellular calcium. In addition, [(3)H]DA release evoked by halothane was not affected by TTX (blocker of voltage-dependent Na(+) channels) or reserpine (a blocker of vesicular monoamine transporter). These data suggest that [(3)H]DA release induced by halothane is non-vesicular and would be mediated by the dopamine transporter (DAT) and norepinephrine transporter (NET). GBR 12909 and nomifensine, inhibitors of DAT, decreased the release of [(3)H]DA evoked by halothane. Nisoxetine, a blocker of NET, reduced the release of [(3)H]DA induced by halothane. In addition, GBR 12909, nisoxetine and, halothane decrease the uptake of [(3)H]DA into rat brain cortical slices. A decrease on halothane-induced release of [(3)H]DA was also observed when the brain cortical slices were incubated at low temperature and low extracellular sodium, which are known to interfere with the carrier-mediated release of the neurotransmitter. Ouabain, a Na(+)/K(+) ATPase pump inhibitor, which induces DA release through reverse transport, decreased [(3)H]DA release induced by halothane. It is suggested that halothane increases [(3)H]DA release in brain cortical slices that is mediated by DAT and NET present in the plasma membrane.

Dopamine is a neurotransmitter that exerts major control on important brain functions and some lines of studies suggest that dopaminergic neurotransmission may be a potential target for volatile anesthetics. In the present study, rat brain cortical slices were labeled with [(3)H]dopamine to investigate the effects of sevoflurane on the release of this neurotransmitter. [(3)H]dopamine release was significantly increased in the presence of sevoflurane (0.46 mM) and this effect was independent of extracellular or intracellular calcium. In addition, [(3)H]dopamine release evoked by sevoflurane was not affected by TTX (blocker of voltage-dependent sodium channels) or reserpine (a blocker of the vesicular monoamine transporter). These data suggest that the dopamine release induced by sevoflurane is non-vesicular, independent of exocytosis and, would be mediated by the dopamine transporter (DAT). GBR12909 and nomifensine, inhibitors of DAT, decreased the release of [(3)H]dopamine evoked by sevoflurane. The same effect was also observed when the brain cortical slices were incubated at low temperature and low extracellular sodium. Ouabain, a Na(+)/K(+) ATPase pump inhibitor, which is known to induce dopamine release through reverse transport, decreased [(3)H]dopamine release induced by sevoflurane. In conclusion, the present study suggests that sevoflurane increases [(3)H]dopamine release in brain cortical slices that is mediated by DAT located at the plasma membrane.

1. We have investigated the effect of the volatile anesthetic sevoflurane on acetylcholine (ACh) release from rat brain cortical slices. 2. The release of [3H]-ACh into the incubation fluid was studied after labeling the tissue ACh with [methyl-3H]-choline chloride. 3. We observed that sevoflurane induced an increase on the release of ACh that was dependent on incubation time and anesthetic concentration. The sevoflurane-induced ACh release was not blocked by tetrodotoxin (TTX) and therefore was independent of sodium channels. In addition, the sevoflurane effect was not blocked by ethylene glycol-bis(beta-aminoethyl ether (EGTA) or cadmium (Cd2+), thus independent of extracellular calcium. 4. The sevoflurane-induced ACh release was inhibited by 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid (BAPTA-AM), suggesting the involvement of intracellular calcium-sensitive stores in the process. Dantrolene, an inhibitor of ryanodine receptors, had no effect but 2-aminoethoxydiphenylborate (2-APB), a membrane-permeable inhibitor of inositol 1,4,5-triphosphate receptor inhibited the sevoflurane-induced release of ACh. 5. It is concluded that sevoflurane-induced release of ACh in brain cortical slices involves the mobilization of calcium from IP3-sensitive calcium stores.

1. The effect of ouabain on the release of [3H]acetylcholine ([3H]ACh) in rat brain cortical slices was investigated. 2. The ouabain-induced release of [3H]ACh was calcium-independent and not blocked by EGTA. 3. BAPTA-AM, a chelator of intracellular calcium, inhibited the ouabain effect suggesting the involvement of intracellular calcium stores. 4. Vesamicol, a drug that blocks the storage of acetylcholine in synaptic vesicles inhibited by 73% the ouabain-induced release of [3H] ACh, suggesting exocytotic release of the neurotransmitter. 5. Dantrolene and tetracaine, inhibitors of ryanodine and InP3 receptors, inhibited by 57 and 66% respectively, the ouabain-elicited release of [3H]ACh in brain cortical slices. 6. Confocal microscopy and calcium imaging showed that ouabain increased the levels of [Ca2+]i in cholinergic SN56 cells and that this increase was concentrated in the cell soma. 7. In conclusion, we suggested that ouabain causes Ca2+ release from intracellular stores that can increase [3H] ACh exocytosis from rat brain cortical slices.

Abstract: Dopamine may alter the phosphorylation state of DARPP-32 that plays a central role in the dopaminergic neurons biology. Studies have shown that DARPP-32/protein phosphatase 1 cascade is a major target for psychostimulants drugs. Methylphenidate is a psychostimulant that acts blocking the dopamine transporter has been used as an effective treatment for Attention Deficit Hyperactivity Disorder. We investigated if methylphenidate could alter DARPP-32 expression in five brain regions (striatum, hippocampus, prefrontal cortex, cortex and cerebellum) in young and adult rats. Our results showed that methylphenidate treatment is able to alter DARPP-32 expression in rat brain. Acute methylphenidate treatment has reduced hippocampal DARPP-32 protein levels in old rats, while chronic methylphenidate treatment has decreased them in old rat hippocampus and young rat cerebellum. It was found an increased cortical expression after chronic methylphenidate administration in old rats. Our results provide the first experimental demonstration that methylphenidate induces changes in total DARPP-32 expression that are posology- and age-related in some rat brain areas, although further studies are needed to shed more light on the mechanisms behind these findings. [Copyright &y& Elsevier]

Investigates the effect of ouabain on the release of [[sup 3]H]acetylcholine ([[sup 3]H]ACh) in rat brain cortical slices. Evidence that the ouabain-induced release of ([[sup 3]H]ACh) was calcium-independent and not blocked by EGTA; Suggestion that ouabain causes Ca[sup 2+] release from intracellular stores that can increase ([[sup 3]H]ACh) exocytosis from rat brain cortical slices.

Abstract: The role of calcium channels blockers in ischemic condition has been well documented. The PhTx3 neurotoxic fraction of the spider Phoneutria nigriventer venom is a broad-spectrum calcium channel blocker that inhibits glutamate release, calcium uptake and also glutamate uptake in synaptosomes. In the present study we describe the effect of PhTx3 (1.0μg/mL), ω-conotoxin GVIA (1.0μmol/L) and ω-conotoxin MVIIC (100nmol/L) on neuroprotection of hippocampal slices and SN56 cells subjected to ischemia by oxygen deprivation and low glucose insult (ODLG). After the insult, cell viability in the slices and SN56 cells was assessed by confocal microscopy and epifluorescence, using live/dead kit containing calcein-AM and ethidium homodimer. Confocal images of CA1 region of the rat hippocampal slices subjected to ischemia insult and treated with ω-conotoxin GVIA, ω-conotoxin MVIIC and PhTx3 showed a percentage of dead cells of 68%, 54% and 18%, respectively. The SN56 cells subjected to ischemia were almost completely protected from damage by PhTx3 while with ω-conotoxin GVIA or ω-conotoxin MVIIC the cell protection was only partial. Thus, PhTx3 provided robust ischemic neuroprotection showing potential as a novel class of agents that targets multiple components and exerts neuroprotection in in vitro model of brain ischemia. [Copyright &y& Elsevier]