Electro-pharmacological profiles of two brain mitoplast anion channels

We have characterized the conduction and blocking properties of two different chloride channels from brain mi- tochondrial inner membranes after incorporation into planar lipid bilayers. Our experiments revealed the existence of channels with a mean conductance of 158 ± 7 and 301 ± 8 pS in asymmetrical 200 mM cis/50 mM trans KCl solutions. We determined that the channels were ten times more permeable for Cl− than for K+, calculated from the reversal potential using the Goldman-Hodgkin-Katz equation. The channels were bell-shaped voltage depend- ent, with maximum open probability 0.9 at ± 20 mV. Two mitochondrial chloride channels were blocked after the addition of 10 µM DIDS. In addition, 158 pS chloride channel was blocked by 300 nM NPPB, acidic pH and 2.5 mM ATP, whereas the 301 pS chloride channel was blocked by 600 µM NPPB but not by acidic pH or ATP. Gating and conducting behaviors of these channels were unaffected by Ca2+. These results demonstrate that the inner membrane, is probably identical to IMAC and 301 pS Cl channel displays different properties than those classically described for mitochondrial anion channels

Pre- and post-treatment of α-Tocopherol on cognitive, synaptic plasticity, and mitochondrial disorders of the hippocampus in icv-streptozotocin-induced sporadic Alzheimer’s-like disease in male Wistar rat

ObjectiveMost dementia cases in the elderly are caused by Alzheimer’s disease (AD), a complex, progressive neurological disease. Intracerebroventricular (ICV) administration of streptozotocin (STZ) in rat’s results in aberrant brain insulin signaling, oxidative stress, and mitochondrial dysfunction that impair cognition change neural plasticity, and eventually lead to neuronal death. The current study aims to define the neuroprotective action of alpha-tocopherol in enhancing mitochondrial function and the function of synapses in memory-impaired rats brought on by icv-STZ.MethodsMale Wistar rats were pre-treated with (α-Tocopherol 150 mg/kg) orally once daily for 7 days before and 14 days after being bilaterally injected with icv-STZ (3 mg/kg), while sham group rats received the same volume of STZ solvent. After 2 weeks of icv-STZ infusion, rats were tested for cognitive performance using a behaviors test and then were prepared electrophysiology recordings or sacrificed for biochemical and histopathological assays.ResultsThe cognitive impairment was significantly minimized in the behavioral paradigms for those who had taken α-Tocopherol. In the hippocampus of icv-STZ rat brains, α-Tocopherol ocopherol effectively prevented the loss of glutathione levels and superoxide dismutase enzyme activity, lowered mitochondrial ROS and mitochondrial membrane potential, and also brought about a decrease in Aβ aggregation and neuronal death.ConclusionOur findings demonstrated that by lowering neurobehavioral impairments caused by icv-STZ, oxidative stress, and mitochondrial dysfunction, α-Tocopherol enhanced intracellular calcium homeostasis and corrected neurodegenerative defects in the brain. These findings examine the available approach for delaying AD connected to mitochondrial malfunction and plasticity issues

Differential Effect of Amyloid Beta1-40 on Short-term and Long-term Plasticity in Dentate Gyrus of a Rat Model of Alzheimer Disease

Introduction: Synaptic plasticity is inappropriately affected by neurodegenerative diseases, including Alzheimer Disease (AD). In this study, we examined the effect of intrahippocampal amyloid-beta (Aβ1-40) on dentate gyrus Long-term Potentiation (LTP) and presynaptic short-term plasticity in a rat model of AD.  Methods: The experimental groups in this research included the control with no treatment, sham-operated receiving the vehicle (normal saline), and Aβ-lesioned groups. For modeling AD, aggregated Aβ1-40 (10 μg/2 μl on each side) was injected into the hippocampal CA1. Three weeks later, Population Spike (PS) amplitude and slope ratios were determined at different Inter-pulse Intervals (IPI) of 10, 20, 30, and 50 ms as a valid indicator of the short-term presynaptic facilitation and/or depression. In addition, PS amplitude and slope were taken as an index of long-term synaptic plasticity after application of High-frequency Stimulation (HFS) to induce LTP in the medial perforant-dentate gyrus pathway.  Results: No significant differences were noted amongst the experimental groups regarding fEPSP slope and paired-pulse indices as indicators of short-term plasticity. In contrast, fEPSP slope and PS amplitude significantly decreased following the application of HFS in Aβ-injected group. In addition, there was no significant difference between the control and sham-operated groups regarding the mentioned parameters.  Conclusion: Findings of this study clearly demonstrated that microinjection of Aβ1-40 into the CA1 could impair LTP in dentate gyrus but could not modify short-term plasticity

Cognitive Impairments Induced by Repeated Sevoflurane Exposure During Pre-adolescence in Adult Male and Female Rats: Involvement of Biochemical, Histological and Neuroplasticity Approaches

Background: In some therapeutic interventions, repeated exposure to pre-adolescence anesthesia is necessary. According to research, exposure to general anesthetics during pre-adolescence can lead to cell death, cognitive and behavioral problems, and neurobehavioral difficulties as an adult. The current study aimed to provide detailed morphological and functional evaluations of the long-term impacts of repeated sevoflurane exposure in male and female rats. Materials and Methods: Seventy-two pre-adolescent rats were randomly divided into male and female control and inhaled sevoflurane groups (concentration of 2%) daily for 15 days. Animals received care for 20-30 days. The influence of repeated exposure to sevoflurane on cognitive functions was tested using the Morris Water Maze, novel object, and social interaction tests. As a measure of oxidative stress, superoxide dismutase (SOD) and glutathione levels were measured. Toluidine blue stain was utilized to evaluate the number of dark neurons in the hippocampus. Effects of sevoflurane on synaptic plasticity were compared in the performant pathway of the CA1 of the hippocampus. Results: Repeated sevoflurane exposure in pre-adolescence led to behavioral disorders in male and female adult rats; there was no significant difference in levels of superoxide dismutase and glutathione. We found a significant quantifiable increase in dark neurons. Electrophysiological recordings indicated impaired long-term potentiation and pair-pulse in adult animals that received repeat sevoflurane exposure. Conclusion: According to our findings, repeated exposure to sevoflurane during pre-adolescence can cause changes in the hippocampus and neuroplasticity in the adult brain. Results from this study may provide a new perspective on how repeated exposure to anesthesia can lead to toxic effects in pre-adolescent rats

Hepatocyte Growth Factor Attenuates the Severity of Status Epilepticus in Kainic Acid-induced Model of Temporal Lobe Epilepsy by Targeting Apoptosis and Astrogliosis

Introduction: Although pharmacotherapy is the most common treatment for epilepsy, proper seizure control is not achieved with current medications. This study evaluated the protective effects of the Hepatocyte Growth Factor (HGF) in a rat model of Temporal Lobe Epilepsy (TLE) and explored possible molecular mechanisms. Methods: A TLE rat model was determined using an intra-hippocampal kainic acid injection (4 μg). Intra-cerebrovascular injection of HGF (6 μg) was performed 30 min before kainic acid injection. Learning and memory impairment were investigated by behavioral tests. The Enzyme-Linked Immunosorbent (ELISA) was used to determine astrogliosis and DNA fragmentation. Changes in neuronal density and mossy fiber sprouting were evaluated by Nissl and Timm staining, respectively.  Results: Behavioral assessments indicated that kainate-treated rats presented spontaneous seizures. Moreover, their alternation percentage scores in the Y-Maze test were lower (P<0.001). Likewise, the passive avoidance test confirmed learning disability in Kainate-treated rats (P<0.001). HGF administration reduced the number of spontaneous seizures, alternation percentage score (P<0.001), and cognitive disturbances (P<0.001). The histopathological results also showed that a protected HGF administration contributed to the reduction of neuronal loss in the CA3 subregion of the hippocampus and inhibited the formation of aberrant Mossy Fiber Sprouting (MFS) (P<0.01). Furthermore, the ELISA data indicated a significant decrease in GFAP (P<0.01) and DNA fragmentation (P<0.05) following HGF administration. Conclusion: Our findings demonstrated the validity of HGF in protection against the progression of the kainate-induced TLE in rats. This measure improved learning, cognitive disturbances and inhibited apoptosis and astrogliosis

Evidence for a KATP Channel in Rough Endoplasmic Reticulum (rerKATP Channel) of Rat Hepatocytes.

We report in a previous study the presence of a large conductance K+ channel in the membrane of rough endoplasmic reticulum (RER) from rat hepatocytes incorporated into lipid bilayers. Channel activity in this case was found to decrease in presence of ATP 100 µM on the cytoplasmic side and was totally inhibited at ATP concentrations greater than 0.25 mM. Although such features would be compatible with the presence of a KATP channel in the RER, recent data obtained from a brain mitochondrial inner membrane preparation have provided evidence for a Maxi-K channel which could also be blocked by ATP within the mM concentration range. A series of channel incorporation experiments was thus undertaken to determine if the ATP-sensitive channel originally observed in the RER corresponds to KATP channel. Our results indicate that the gating and permeation properties of this channel are unaffected by the addition of 800 nM charybdotoxin and 1 µM iberiotoxin, but appeared sensitive to 10 mM TEA and 2.5 mM ATP. Furthermore, adding 100 µM glibenclamide at positive potentials and 400 µM tolbutamide at negative or positive voltages caused a strong inhibition of channel activity. Finally Western blot analyses provided evidence for Kir6.2, SUR1 and/or SUR2B, and SUR2A expression in our RER fractions. It was concluded on the basis of these observations that the channel previously characterized in RER membranes corresponds to KATP, suggesting that opening of this channel may enhance Ca2+ releases, alter the dynamics of the Ca2+ transient and prevent accumulation of Ca2+ in the ER during Ca2+ overload

The effect of glibenclamide on channel activity at +20, 0 and -20 mV.

<p>Representative unitary current recordings in control conditions (200/50 mMKCl; cis/trans) and after cis addition of 10, 25, 50 (A) or 100 μM glibenclamide (B) at different voltages. Histogram bars show current amplitudes and P_o of reconstituted channels in absence or presence of glibenclamide. The addition of 50 μM glibenclamide resulted in a decreased current jump amplitude and a reduction in P_o. 100 μM glibenclamide caused a total channel inhibition at +20 and 0 mV (n = 5), but was ineffective at -20 mV. The significant difference in the channel amplitude and P_o values are marked by asterisks (*P < 0.05 and ** P_o < 0.01, respectively, n = 5). Data are means ± S.E. Closed levels are indicated by –.</p

Effect of EGTA, charybdotoxin, and iberitoxin on channel activity.

<p>Representative unitary current recordings in control conditions (200/50 mMKCl; cis/trans) and after addition of 1 mM EGTA at -20 mV (A), 800 nM charybdotoxin (cis face) at 0 and -40 mV (B), and 1 μM iberiotoxin at +10 and -10 mV (C). Data are summarized in the bar graph presented in the right panel. There were no significant differences in open probability and current jump amplitude (n = 2, n = 4, n = 5, respectively), indicating that the K⁺ channel from RER does not correspond to a Max-K_Ca channel. The—indicates the closed levels.</p

Effect of tolbutamide on channel gating behavior at +20, 0 and -10 mV.

<p>Examples of single channel recordings in control conditions (200/50 mMKCl; cis/trans), and immediately after cis addition of tolbutamide 400 μM (n = 5). Tolbutamide caused a total voltage independent inhibition of channel activity. The—indicates the closed levels.</p

Western blot analysis of Kir6.2 and SURs in the rat microsome fractions.

<p>(A) The goat anti-Kir6.2 antibody led to the identification of a prominent ~55 kDa band in microsome fractions, a molecular weight compatible with a Kir6.2 K_ATP subunit. (B) ~150 kDa band was detected using the goat anti-SUR2A antibody, whereas the goat anti-human SUR2B and SUR1 antibodies led to the labeling of two bands of 150 and 100 kDa respectively. The 100 kDa may be resulting from proteolysis of the 150 kDa paptide. (C) Western blot indicating that of the Maxi-K_Ca channel α-subunit (138 kDa) is expressed in rat endoplasmic reticulum membranes.</p