Rat Cortex Synaptosomes Research Articles

ATR-IR and EPR spectroscopy for detecting the alterations in cortical synaptosomes induced by aluminium stress.

Aluminium (Al) is reported to promote free radical production, decrease the antioxidant enzyme status and disturb the enzyme activity involved in acetylcholine metabolism leading to cognitive dysfunction that are strongly associated with Alzheimer's disease (AD) pathogenesis. This work aimed at investigating the effect of Al-toxicity on synaptosomal membrane biophysical properties and lipid peroxidation during 65 days. We utilized ATR-IR spectroscopy to study the changes in membrane biochemical structure and biophysical properties of isolated rat cortical synaptosomes, and EPR spin trapping and labeling to follow NADPH oxidase activity and changes of membrane order parameter, respectively. The results showed increase in membrane fluidity and disorder in early 21d of AlCl3 treatment, while after 42d the membrane rigidity, packing, and order increased. The late (65d) an increase in the amount of unsaturated fatty acids, the accumulation of lipid peroxide end products, and ROS production were detected in rat cortex synaptosomes mediated by Al toxicity and oxidative stress (OS). A dramatic increase was also detected in Ca2+ level, synaptic membrane polarity, and EPR-detected order S-parameter. These outcomes strongly suggest that the synaptosomal membrane phospholipids underwent free radical attacks mediated by AlCl3 due to greater NOX activity, and the release of synaptic vesicles into synaptic cleft might be hindered. The adopted spectroscopic techniques have shed light on the biomolecular structure and membrane biophysical changes of isolated cortical synaptosomes for the first time, allowing researchers to move closer to a complete understanding of pathological tissues.

Benzo[b]tiophen-3-ol derivatives as effective inhibitors of human monoamine oxidase: design, synthesis, and biological activity

A series of benzo[b]thiophen-3-ols were synthesised and investigated as potential human monoamine oxidase (hMAO) inhibitors in vitro as well as ex vivo in rat cortex synaptosomes by means of evaluation of 3,4-dihydroxyphenylacetic acid/dopamine (DOPAC/DA) ratio and lactate dehydrogenase (LDH) activity. Most of these compounds possessed high selectivity for the MAO-B isoform and a discrete antioxidant and chelating potential. Molecular docking studies of all the compounds underscored potential binding site interactions suitable for MAO inhibition activity, and suggested structural requirements to further improve the activity of this scaffold by chemical modification of the aryl substituents. Starting from this heterocyclic nucleus, novel lead compounds for the treatment of neurodegenerative disease could be developed.

Protective Effects Induced by Microwave-Assisted Aqueous Harpagophytum Extract on Rat Cortex Synaptosomes Challenged with Amyloid β-Peptide.

Harpagophytum procumbens is a plant species that displays anti-inflammatory properties in multiple tissues. The iridoid glycosides arpagoside, harpagide, and procumbide appear to be the most therapeutically important constituents. In addition, harpagoside treatment exerted neuroprotective effects both in vitro and in vivo. Considering these findings, the aim of the present work is to explore the possible protective role of the previously described microwave-assisted aqueous extract of H.procumbens on rat hypothalamic (Hypo-E22) cells, and in rat cortex challenged with amyloid β-peptide (1-40). In this context, we assayed the protective effects induced by H.procumbens by measuring the levels of malondialdehyde, 3-hydroxykynurenine (3-HK), brain-derived neurotrophic factor, and tumor necrosis factor-α, 3-HK. Finally, we evaluated the effects of H.procumbens treatment on cortex levels of dopamine, norepinephrine, and serotonin. H.procumbens extract was well tolerated by Hypo-E22 cells and upregulated brain-derived neurotrophic factor gene expression but down-regulated tumor necrosis factor-α gene expression. In addition, the extract reduced amyloid β-peptide stimulation of malondialdehyde and 3-HK and blunted the decrease of dopamine, norepinephrine, and serotonin, in the cortex. In this context, our work supports further studies for the evaluation and confirmation of Harpagophytum in the management of the clinical symptoms related to Alzheimer's disease. Copyright © 2017 John Wiley & Sons, Ltd.

Inhibition of Ca2+‐dependent glutamate release from cerebral cortex synaptosomes of rats with experimental autoimmune encephalomyelitis

Several pathological studies have revealed a prominent involvement of the cerebral cortex in patients with multiple sclerosis (MS). In order to better understand the events that lead to the progressive neuronal dysfunction in MS, herein we explore the contribution of the glutamatergic release in cerebral cortex synaptosomes isolated from rats with experimental autoimmune encephalomyelitis, an animal model reproducing many features of MS. We found that the Ca(2+)-dependent but not the Ca(2+)-independent glutamate release induced by KCl and 4-aminopyridine was significantly decreased during the acute stage of the disease. This inhibited release coincides with the onset of the clinical signs and after 24 h tends to recover the level of the control animals. The results also showed an inhibition of the glutamate release stimulated by ionomycin. When the animals were totally recovered from clinical signs, the neurotransmitter release stimulated by the different inductors was similar to the controls. Examination of the cytosolic Ca(2+) using fura-2-acetoxymethyl ester revealed that the inhibition of glutamate release could not be attributed to a reduction in voltage-dependent Ca(2+) influx. However, this inhibition was concomitant with a lower phosphorylation of synapsin I at P-site1. Our results show that the inhibition observed on the Ca(2+)-dependent neurotransmitter release from cerebral cortex synaptosomes in experimental autoimmune encephalomyelitis is specific and correlates with the beginning of the clinical disease. Moreover, they suggest an alteration in the metabolism of proteins involved in the vesicular glutamate release more than a deregulation in the influx of cytosolic Ca(2+).

A snake venom phospholipase A 2 with high affinity for muscarinic acetylcholine receptors acts on guinea pig ileum

A group of small proteins, designated as muscarinic toxins (MTs), have been isolated from the venom of African green mamba ( Dendroaspis angusticeps) and documented to bind selectively to individual muscarinic acetylcholine receptor (mAChR) subtypes. These components have less been reported to be isolated from other snake venoms. In this study, we have isolated a snake factor with high affinity for mAChR from the venom of Naja atra (Chinese cobra) by column chromatography on Sephadex G-50, Sephadex G-150, CM-Sepharose Fast Flow and Poros ®CM 4.6/100 Perfusion Chromatography Column. The final preparation was homogeneous as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and HPLC. The isolated active component, which was designated muscarinic protein (MP), was found to displace [ 3H]quinuclidinyl benzilate binding to rat cortex synaptosomes in a dose-dependent manner, and the K i value estimated was 10.1 nM. The isolated MP was determined to have a molecular weight of 13.3 kDa and an N-terminal amino acid sequence of NLYQFKNMIQCTVPSR, which is highly homologous with phospholipase A 2 from the venoms of genus Naja. The N. atra MP could hydrolyze phosphatidylcholine in a dose-dependent manner. In guinea-pig ileum, MP produced an onset and dose-dependent contraction, which could be reversed by atropine indicating the involvement of mAChR. The EC 50 value of MP for guinea-pig ileum contraction was estimated as 30 nM, and the maximum contraction caused by MP was approximately 43% of that obtained from carbachol. These results seem to suggest that the snake venom phospholipase A 2 may not only have high affinity for mAChRs but also have the ability to activate mAChRs. However, it is possible that the toxin caused the contraction in the guinea-pig ileum by inducing acetylcholine release via another mechanism.

No modulatory effect of neurokinin-1 receptor antagonists on serotonin uptake in human and rat brain synaptosomes

Some studies have demonstrated antidepressant activity of neurokinin-1-receptor antagonists (NK-1-RA) in major depressive disorder. However, the underlying mechanisms of this antidepressant effect are largely unknown. Preclinical studies in rats and mice have suggested that NK-1-RA do increase the neuronal release of serotonin (5-HT). This, however, seems to be compensated by an increased 5-HT reuptake, indicating that NK-1-RA have no inhibitory effect on the 5-HT transporter in rodents. Given the possibility that modulation of neurotransmitter release and reuptake may differ between species, with major differences found between rodents and humans, we investigated for the first time the possible modulatory effect of NK-1-RA on 5-HT uptake in human brain synaptosomes and compared it with the situation in rat cortex. We found that the specific human NK-1-RA L-733060, in contrast to the SSRI fluvoxamine (IC 50 = 10 − 7.96 M) did not inhibit 5-HT uptake in human brain synaptosomes and did not modulate fluvoxamine-induced 5-HT uptake inhibition at 1 μM. Furthermore, substance P as well as Sar 9Met(O 2) 11SP, as the major agonists at the NK-1-R, did not modulate 5-HT uptake in human brain synaptosomes. Similar results were found in rat cortex synaptosomes by using the rat-specific NK-1-RA WIN51708. These results show that in humans, as in rodents, inhibition of the 5-HT transporter is probably not the underlying mechanism of the assumed antidepressant activity of NK-1-RA.

Hypothyroidism changes adenine nucleotide hydrolysis in synaptosomes from hippocampus and cerebral cortex of rats in different phases of development

The influence of the thyroid hormones on the normal function of the mammalian central nervous system depends on the brain region and on the developmental stage. Adenine nucleotides and their products also affect the brain function; ATP is an excitatory neurotransmitter, and adenosine has inhibitory effects on neurotransmission. Thus, this study aimed to evaluate the effects of hypothyroidism on the hydrolysis of ATP to adenosine in hippocampal and cortical synaptosomes and blood serum of rats during different phases of development. Rats aged 60 and 420 days old were divided into three groups: control, sham-operated and hypothyroid. Hypothyroidism was induced in these rats by thyroidectomy and methimazole (0.05%) added to their drinking water for 14 days. Neonatal hypothyroidism was induced by adding 0.02% methimazole in the drinking water from day 9 of gestation, and continually until 14 days old. Hypothyroidism increased the AMP hydrolysis in both hippocampus and cerebral cortex synaptosomes of rats in all aged tested. In blood serum, thyroid hormones deficiency increased the AMP hydrolysis in 14-day-old rats and the hydrolysis of ATP, ADP and AMP in 60-day-old rats; however, no alteration was observed in 420-day-old rats. Thus, our results suggest the involvement of the 5′-nucleotidase in synaptic function control in hypothyroidism throughout brain development.

3H]-noradrenaline secretion from rat cortex synaptosomes perforated with Staphylococcus aureusα-toxin

Rat cortex synaptosomes have been successfully perforated with high concentrations (≥400 U/ml) of Staphylococcus aureus α-toxin. The free Ca 2+-concentration dependence of [ 3H]-noradrenaline release was similar to that observed for PC 12 and chromaffin cells. Release from the α-toxin perforated synaptosomes was not significantly inhibited by ω-conotoxin GVIA. Initially, Ca 2+-dependent release was independent of MgATP (for 0.5 min), but became increasingly dependent on MgATP with time. Lactate dehydrogenase efflux from α-toxin-perforated synaptosomes was not different than efflux from control synaptosomes, and an antibody to N-ethylmaleimide-sensitive fusion protein did not enter the synaptosomes. [ 3H]-noradrenaline release was temperature and α-toxin-concentration dependent. Ca 2+-dependent release was more resistant to rundown from α-toxin- than from streptolysin-O-perforated synaptosomes. This preparation of perforated synaptosomes should be useful for studies of regulated exocytosis from nerve endings.

Huntingtin is a cytoplasmic protein associated with vesicles in human and rat brain neurons

The gene defective in Huntington's disease encodes a protein, huntingtin, with unknown function. Antisera generated against three separate regions of huntingtin identified a single high molecular weight protein of approximately 320 kDa on immunoblots of human neuroblastoma extracts. The same protein species was detected in human and rat cortex synaptosomes and in sucrose density gradients of vesicle-enriched fractions, where huntingtin immunoreactivity overlapped with the distribution of vesicle membrane proteins (SV2, transferrin receptor, and synaptophysin). Immunohistochemistry in human and rat brain revealed widespread cytoplasmic labeling of huntingtin within neurons, particularly cell bodies and dendrites, rather than the more selective pattern of axon terminal labeling characteristic of many vesicle-associated proteins. At the ultrastructural level, immunoreactivity in cortical neurons was detected in the matrix of the cytoplasm and around the membranes of the vesicles. The ubiquitous cytoplasmic distribution of huntingtin in neurons and its association with vesicles suggest that huntingtin may have a role in vesicle trafficking.

On the usefulness of Fura‐2 measurements of intrasynaptosomal calcium levels in rat cortical synaptosomes to study mechanisms of presynaptic function

Levels of [Ca2+]i in rat cortex synaptosomes were measured using the Ca2+ indicator Fura-2. Ca2+ influx was induced by veratridine in a concentration-dependent manner (1-10 microM). The resulting increase in [Ca2+]i was inhibited by tetrodotoxin (TTX). K+ (18 mM) increased the [Ca2+]i which was not influenced by TTX. K(+)-channel blockers such as 4-aminopyridine, alpha- and delta-dendrotoxin pre se were ineffective. The veratridine-induced Ca2+ influx in synaptosomes was reduced by L-type Ca(2+)-channel blockers, such as felodipine, nifedipine and PN-200-110, verapamil and diltiazem. omega-Conotoxin, and N-type Ca(2+)-channel blocker, did not inhibit the veratridine-stimulated [Ca2+]i increase. Bay K 8644, and L-channel agonist, stimulated an increase of [Ca2+]i in synaptosomes which was not sensitive to TTX. R-N6-Phenyl-isopropyl-adenosine (R-PIA) and clonidine, agonists at adenosine A1-receptors and alpha 2-adrenoceptors, respectively, did not influence the veratridine-stimulated [Ca2+]i increase. R-PIA did not interact with Bay K 8644-stimulated [Ca2+]i increase in synaptosomes. The results for all the substances used show major differences between the effects on Ca2+ influx in synaptosomes and on the electrically evoked neurotransmitter release in slice preparations. Thus, the synaptosome preparation is not a generally applicable experimental model for the study of Ca2+ mechanisms of presynaptic neuromodulation.

GABA B autoreceptors in rat cortex synaptosomes: response under different depolarizing and ionic conditions

Rat cerebral cortex synaptosomes prelabeled with [ 3H]γ-aminobutyric acid ([ 3H]GABA) were exposed in superfusion to various concentrations of KCl (9–50 mM). The evoked release of [ 3H]GABA reached a plateau at about 35 mM KCl. The K +-induced release was Ca 2+-dependent, particularly at the lowest K + concentrations. The GABA B agonist (−)-baclofen concentration dependently inhibited the release of [ 3H]GABA evoked by K +; this effect decreased with increasing K + concentration and disappeared at 35 mM KCl. The GABA A agonist muscimol (1–100 μM) was totally ineffective to inhibit the release of [ 3H]GABA. Veratrine (1–30 μM) induced the release of [ 3H]GABA and the effect was tetrodotoxin-sensitive. (−)-Baclofen, but not muscimol, decreased the veratrine-induced [ 3H]GABA release; the GABA B agonist was particularly effective in presence of low concentrations of veratrine (1–3 μM) but the effect disappeared when 30 μM of the alkaloid was used. The inhibitory effect of (−)-baclofen on the release of [ 3H]GABA evoked by 15 mM KCl was dependent on the concentration of Ca 2+: the effect increased as the concentration of Ca 2+ was raised, reaching a plateau at 0.6 mM Ca 2+. Exogenous GABA, in presence of the GABA uptake blocker SK&F 89976A, inhibited the release of [ 3H]GABA evoked by K +; this effect was antagonized by phaclofen. The data support the idea that terminal GABA autoreceptors in the rat cerebral cortex are of the GABA B type.

Studies on [ 3H] GABA and endogenous GABA release in rat cerebral cortex suggest the presence of autoreceptors of the GABA B type

The presence of autoreceptors for γ-aminobutyric acid (GABA) in the CNS was reinvestigated using rat cortex synaptosomes prelabeled with [ 3H]GABA and exposed to GABA by superfusion in the presence of a new GABA uptake inhibitor, N-(4,4-diphenyl-3-butenyl)-nipecotic acid (SK&F 89976A). This compound itself did not increase the basal or the depolarization-evoked release of [ 3H]GABA. GABA reduced in a concentration-dependent way the release of [ 3H]GABA evoked by 15 mM K +. The effect was not antagonized by bicuculline, picrotoxin or by the new GABA A antagonist SR 95531. The GABA A agonist muscimol did not affect [ 3H]GABA release. This was reduced by (−)baclofen (but not by the (+) isomer) and the concentration-inhibition curve of (−)baclofen was superimposable on to that of GABA. Also the K +evoked release of endogenous GABA was stereoselectively and concentration dependently inhibited by the (−) enantiomer of baclofen. It is concluded that the release of GABA from rat cortical nerve endings may be inhibited through the activation of autoreceptors which appear to belong to the GABA B type.

Studies on a possible functional coupling between presynaptic acetylcholinesterase and high-affinity choline uptake in the rat brain.

The relationships between presynaptic acetylcholinesterase (AChE) and high-affinity choline uptake (HACU) were investigated using a monolayer of rat cortex synaptosomes in superfusion conditions. The following sets of experiments were performed: determination of [3H]choline ([3H]Ch) uptake during superfusion with [3H]Ch; determination of [3H]Ch uptake during superfusion with acetylcholine (ACh) tritiated in the Ch moiety; evaluation of ACh hydrolysis during superfusion with ACh labelled in the acetate moiety; and comparison of the uptake of [3H]Ch generated by hydrolysis of [3H]ACh with that occurring during superfusion with [3H]Ch. Intact ACh was not taken up by superfused synaptosomes. The uptake of [3H]Ch during superfusion with 1 or 0.1 microM [N-methyl-3H]ACh was two-thirds of that occurring during superfusion with the same concentrations of [3H]Ch. The amount of [3H]Ch produced by hydrolysis during 16 min of superfusion was 1/25 of the amount passing through the synaptosomal monolayer during 16 min of superfusion with [3H]Ch. The results indicate that presynaptic AChE and HACU are located in close proximity to each other on the cholinergic terminal membrane, an observation suggesting the possibility of a functional coupling between the two mechanisms.

Noradrenaline uptake inhibitors do not reduce the presynaptic action of clonidine on 3H-noradrenaline release in superfused synaptosomes.

The interaction between clonidine, as an agonist at the alpha 2-autoreceptors regulating noradrenaline release, and inhibitors of noradrenaline neuronal uptake was investigated in superfused synaptosomes, i.e. in conditions preventing accumulation of the released transmitter in the vicinity of presynaptic autoreceptors. Desipramine or cocaine did not release the inhibitory action of clonidine on the release of 3H-noradrenaline evoked by 15 mM KCl from rat cortex synaptosomes, even when the concentration ratio between uptake inhibitor and clonidine was very high. The present results do not support the hypothesis of an interaction between imidazolines and noradrenaline uptake inhibitors at the level of alpha 2-autoreceptors, or that of a functional coupling between presynaptic alpha 2-autoreceptors and noradrenaline uptake mechanism.

Effects of β-bungarotoxin and tityustoxin on accumulation of putative amino acid neurotransmitters by rat cortex synaptosomes

14C-labelled glutamate and γ-aminobutyrate, preloaded into purified synaptosomes from rat cortex, were released by β-bungarotoxin and depolarization with 55 mM K + or tityustoxin; in all cases the induced release was higher in the presence of Ca 2+. Thus, these neurotoxins exert similar effects in vitro on central synapses, which release neuroactive amino acids, to those previously observed in the peripheral nervous system. Accumulation of these two putative transmitters by synaptosomes was de creased by either toxin, probably due to their disruption of the membrane potential. β-Bungarotoxin, a homogeneous protein which exhibits phospholipase activity in the presence of deoxycholate and Ca 2+, but not Sr 2+, augmented the respiration rate of synaptic mitochondria. Although K +-stimulated release was observed in the absence of Ca 2 + and the presence of Sr 2+, the action of the toxin on the release of amino acids and lactate dehydrogenase was diminished. These results, therefore, support the view that phospholipase activity of β-bungarotoxin may play an important part in increasing the release of transmitters observed both in vivo and in vitro. The effects of tityustoxin on both the accumulation and release of the putative transmitters were abolished by tetrodo toxin; K +-induced depolarisation greatly decreased its potentiating action on release, possibly suggesting that the binding of tityustoxin, like other similar scorpion toxins, is dependent on membrane potential.