Rat Cortical Slices Research Articles

P2‐084: T‐type voltage‐gated calcium channel activated by ST101 is a novel molecular target as cognitive enhancer

We have developed a novel cognitive enhancer, ST101 (spiro[imidazo[1,2- a]pyridine-3,2-indan]-2(3 H)-one; previously coded as ZSET1446, Sonexa Therapeutics, Inc. San Diego, CA). We previously reported that ST101 improves the impairment in hippocampal long-term potentiation (LTP) via activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) in olfactory bulbectomized (OBX) mice (Han et al., 2008). As yet, the mechanisms underlying the activation of memory-related protein kinases are not known. Rat somatosensory cortical and hippocampal slices were incubated with 0.01 to 100 nM ST101 in the perfusion chamber with or without voltage-gated calcium channel (VGCC) inhibitors and performed immunoblotting and electrophysiological analyses. Treatment of the cortical slices with a range of ST101 concentrations significantly increased CaMKII autophosphorylation with a bell-shaped dose-response curve, with the maximal effective concentration at 0.1 nM ST101. The enhanced CaMKII autophosphorylation with 0.1 nM ST101 treatment was significantly inhibited by pretreatment with 1 m M mibefradil, a T-type VGCC inhibitor, but not with N-type (w -conotoxin), P/Q-type (w -agatoxin) or L-type (nifedipine) VGCC inhibitors. Similarly, 0.1 nM ST101 significantly potentiated LTP, which was inhibited by 1 m M mibefadil treatment in cortical slices. Finally, whole-cell patch-clamp analysis of Neuro2A cells overexpressing recombinant human Ca V 3.1 (a 1G) T-channels confirmed that T-type VGCC currents are stimulated with 0.1 nM ST101 (1). These results indicate that T-type VGCCs are direct molecular targets for the novel cognitive enhancer ST101, a potential Alzheimer disease therapeutics.(1) Moriguchi et al. Journal of Neurochemistry (2012) The T-type voltage-gated calcium channel as a molecular target of the novel cognitive enhancer ST101: Enhancement of long-term potentiation and CaMKII autophosphorylation in rat cortical slices.(doi: 10.1111/j.1471-4159.2012.07667.x.)

Automated model optimization to study spike shape modulation in Layer 2/3 cortical pyramidal neurons

It has been proposed that, contrary to the “classical” view in which cortical action potentials are encoded as purely digital, all-or-none events, action potential (AP) shape may be used by the brain for representing and processing information [1-4]. Cortical pyramidal neurons of Layer 2/3 show prominent variations in AP waveform during sustained depolarizing responses, which may lead to different levels of synaptic output at proximal axonal synaptic terminals, and different patterns of invasion of the dendrites by back-propagating APs. A range of membrane ionic channels probably plays an important part in this phenomenon. We carried out electrical recording during conductance injection, combined with morphological reconstruction and multicompartmental modelling of Layer II/III pyramidal neurons in rat and mouse cortical slices, to investigate the mechanism of AP waveform modulation. Evolutionary optimization techniques [5-7] were implemented and a computational cluster architecture was designed for searching the parameter space of ion channel distributions and properties efficiently in parallel, to fit models to experimental data. As a test of the fitting process, we assessed its ability to detect point voltage-dependent conductances of different types, introduced at the soma by conductance injection (dynamic clamp). Using the available evidence, we describe how the inactivation kinetics of voltage-dependent potassium channels appears to play a particularly important role in the modulation of action potential waveform of these neurons.

Effect of Endothelin-1 on the Excitability of Rat Cortical and Hippocampal Slices In Vitro

Endothelin-1 (ET-1) is a neuroactive protein produced in most brain cell types and participates in regulation of cerebral blood flow and blood pressure. In addition to its vascular effects, ET-1 affects synaptic and nonsynaptic neuronal and glial functions. Direct application of ET-1 to the hippocampus of immature rats results in cerebral ischemia, acute seizures, and epileptogenesis. Here, we investigated whether ET-1 itself modifies the excitability of hippocampal and cortical circuitry and whether acute seizures observed in vivo are due to nonvascular actions of ET-1. We used acute hippocampal and cortical slices that were preincubated with ET-1 (20 μM) for electrophysiological recordings. None of the slices preincubated with ET-1 exhibited spontaneous epileptic activity. The slope of the stimulus intensity-evoked response (input-output) curve and shape of the evoked response did not differ between ET-1-pretreated and control groups, suggesting no changes in excitability after ET-1 treatment. The threshold for eliciting an evoked response was not significantly increased in either hippocampal or cortical regions when pretreated with ET-1. Our data suggest that acute seizures after intrahippocampal application of ET-1 in rats are likely caused by ischemia rather than by a direct action of ET-1 on brain tissue.

Advanced in vitro approach to study neurovascular coupling mechanisms in the brain microcirculation

An understanding of the signalling events underlying neurovascular coupling mechanisms in the brain is a crucial step in the development of novel therapeutic approaches for the treatment of cerebrovascular-associated disorders. In this study we present an enhanced in vitro brain slice preparation from male Wistar rat cortical slices that incorporates haemodynamic variables (flow and pressure) into parenchymal arterioles resulting in the development of myogenic tone (28% from maximum dilatation). Moreover, we characterized flow-induced vascular responses, resulting in various degrees of vasoconstrictions and the response to 10 mM K(+) or astrocytic activation with the mGluR agonist, t-ACPD (100 μM), resulting in vasodilatations of 33.6±4.7% and 38.6±4.6%, respectively. Using fluorescence recovery, we determined perfusate velocity to calculate diameter changes under different experimental pH conditions. Using this approach, we demonstrate no significant differences between diameter changes measured using videomicroscopy or predicted from the velocity values obtained using fluorescence recovery after photobleaching. The model is further validated by demonstrating our ability to cannulate arterioles in two brain regions (cortex and supraoptic nucleus of the hypothalamus). Altogether, we believe this is the first study demonstrating successful cannulation and perfusion of parenchymal arterioles while monitoring/estimating luminal diameter and pressure under conditions where flow rates are controlled.

Selective ozone concentrations may reduce the ischemic damage after a stroke

Stroke is one of the most debilitating diseases, and it is unfortunate that only a small percentage of patients can be treated with thrombolytic agents. Consequently, there is an urgent need of finding an alternative procedure for reoxygenating the so-called penumbra at the earliest time as possible for reducing morbidity and disability. A preliminary, preclinical study has been carried out by using rat hippocampal and cortical brain slices subjected to oxygen-glucose deprivation. Oxygen-ozone gaseous mixture appeared to be effective in reverting damage of brain tissues, supporting the evaluation of this approach in well-designed clinical trials in stroke patients.

The T‐type voltage‐gated calcium channel as a molecular target of the novel cognitive enhancer ST101: enhancement of long‐term potentiation and CaMKII autophosphorylation in rat cortical slices

In this study, we report that spiro[imidazo[1,2-a]pyridine-3,2-indan]-2(3H)-one (ST101; previously coded as ZSET1446) targets T-type voltage-gated calcium channels in mediating improved cognition in the CNS. We prepared rat somatosensory cortical and hippocampal slices, treated them with 0.01 to 100 nM ST101, and performed immunoblotting and electrophysiological analyses using various voltage-gated calcium channel (VGCC) inhibitors. Treatment of rat cortical slices with a range of ST101 concentrations significantly increased calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation following a bell-shaped dose-response curve, with 0.1 nM ST101 representing the maximally effective concentration. protein kinase Cα autophosphorylation was also significantly increased by 0.1 nM ST101 treatment. ST101 treatment had a moderate effect on CaMKII autophosphorylation but no effect on hippocampal protein kinase Cα autophosphorylation in slice preparations. Consistent with increased cortical CaMKII autophosphorylation, AMPA-type glutamate receptor subunit 1 (Ser-831) phosphorylation as a CaMKII post-synaptic substrate was significantly increased by treatment with 0.1-1 nM ST101, whereas phosphorylation of the pre-synaptic substrate synapsin I (Ser-603) remained unchanged. Notably, enhanced CaMKII autophosphorylation seen following 0.1 nM ST101 treatment was significantly inhibited by pre-treatment with 1 μM mibefradil, a T-type VGCC inhibitor, but not with N-type (ω-conotoxin), P/Q-type (ω-agatoxin) or L-type (nifedipine) VGCC inhibitors. Similarly, 0.1 nM ST101 significantly potentiated long-term potentiation in cortical but not hippocampal slices. Enhanced long-term potentiation in cortical slices was totally inhibited by 1 μM mibefadil treatment. Finally, whole-cell patch-clamp analysis of Neuro2A cells over-expressing recombinant human Ca(V) 3.1 (α1G) T-channels and treated with 0.1 nM ST101 showed significant increases in T-type VGCC currents. These results indicate that T-type VGCCs are direct molecular targets for the novel cognitive enhancer ST101, a potential Alzheimer disease therapeutic.

Optical analysis of developmental changes in synaptic potentiation in the neonatal rat corticostriatal projection

We applied voltage-sensitive dye imaging to neonatal rat cortical slice preparations and analyzed developmental changes in synaptic plasticity, long-term potentiation (LTP), in the corticostriatal projection. Coronal slice preparations were dissected from postnatal 1- to 21-day (P1–P21) rats, and the transmembrane voltage-related optical signals evoked by cortical stimulation were recorded using a 464ch optical recording system with the voltage-sensitive absorption dye. In the striatum, the optical signal was composed of a fast spike-like signal followed by a slow signal, which corresponded to an action potential and an excitatory postsynaptic potential (EPSP), respectively. The slow signal could be detected at the P1 stage, suggesting that the EPSP is already expressed in the corticostriatal projection at least at early stages after birth. On the other hand, the slow signal was potentiated with a single shot of tetanic stimulation and the potentiation lasted at least 1 h, which is considered to correspond to long-term potentiation. With ontogenetic examinations, we found that (1) the EPSP could be potentiated with tetanic stimulation from the P9 stage and that (2) after the LTP induction, the potentiation was maintained for a longer time in the postnatal 3W stage than in the 2W stage. These results suggest that characteristics of LTP change dynamically during postnatal development.

CEP-26401 (Irdabisant), a Potent and Selective Histamine H3 Receptor Antagonist/Inverse Agonist with Cognition-Enhancing and Wake-Promoting Activities

CEP-26401 [irdabisant; 6-{4-[3-((R)-2-methyl-pyrrolidin-1-yl)-propoxy]-phenyl}-2H-pyridazin-3-one HCl] is a novel, potent histamine H₃ receptor (H₃R) antagonist/inverse agonist with drug-like properties. High affinity of CEP-26401 for H₃R was demonstrated in radioligand binding displacement assays in rat brain membranes (K(i) = 2.7 ± 0.3 nM) and recombinant rat and human H₃R-expressing systems (K(i) = 7.2 ± 0.4 and 2.0 ± 1.0 nM, respectively). CEP-26401 displayed potent antagonist and inverse agonist activities in [³⁵S]guanosine 5'-O-(γ-thio)triphosphate binding assays. After oral dosing of CEP-26401, occupancy of H₃R was estimated by the inhibition of ex vivo binding in rat cortical slices (OCC₅₀ = 0.1 ± 0.003 mg/kg), and antagonism of the H₃R agonist R-α-methylhistamine- induced drinking response in the rat dipsogenia model was demonstrated in a similar dose range (ED₅₀ = 0.06 mg/kg). CEP-26401 improved performance in the rat social recognition model of short-term memory at doses of 0.01 to 0.1 mg/kg p.o. and was wake-promoting at 3 to 30 mg/kg p.o. In DBA/2NCrl mice, CEP-26401 at 10 and 30 mg/kg i.p. increased prepulse inhibition (PPI), whereas the antipsychotic risperidone was effective at 0.3 and 1 mg/kg i.p. Coadministration of CEP-26401 and risperidone at subefficacious doses (3 and 0.1 mg/kg i.p., respectively) increased PPI. These results demonstrate potent behavioral effects of CEP-26401 in rodent models and suggest that this novel H₃R antagonist may have therapeutic utility in the treatment of cognitive and attentional disorders. CEP-26401 may also have therapeutic utility in treating schizophrenia or as adjunctive therapy to approved antipsychotics.

Frequency-dependent autoinhibition of histamine release from rat cortical slices: a possible role for H3 receptor reserve

The inhibition of histamine release after depolarization of rat cerebral cortex slices by electrical stimulation and mediated by the postulated presynaptic autoreceptor (H3) depends strongly on the conditions of stimulation. Using electrically stimulated slices of the cortex a rightwards shift of the concentration-response curve of histamine (an H3 agonist) was observed on increasing the frequency of stimulation. The pA2 value of the H3 antagonist impromidine was, however, not altered at different stimulation frequencies; for a partial agonist only the maximal effect was influenced. These results indicate the existence of a receptor reserve at the H3 autoreceptor.

Uptake of Cisplatin and its Metabolites Into Rat and Rabbit Renal Cortical Slices

The uptake of cisplatin and its metabolites into rat and rabbit renal cortical slices was investigated. Under anaerobic conditions the cisplatin uptake rate was significantly reduced by pretreatment with dinitrophenol and by organic cationic compounds (cimetidine, verapamil and quinidine). Verapamil competitively inhibited the uptake of cisplatin (apparent inhibitory constant (Ki) = 204.5 ± 43.0 μM). There was very little uptake of the cisplatin metabolite formed by complexation with glutathione. In addition, both aquated cisplatin and a metabolite formed by complexation with thiosulphate were taken up to a very low extent at 37°C compared with unchanged cisplatin. These results suggest that the intact chemical structure of cisplatin may be required for transport of cisplatin into the kidney and cisplatin may be partly transported via organic cation transport systems.

Preferential Control of Basal Dendritic Protrusions by EphB2

The flow of information between neurons in many neural circuits is controlled by a highly specialized site of cell-cell contact known as a synapse. A number of molecules have been identified that are involved in central nervous system synapse development, but knowledge is limited regarding whether these cues direct organization of specific synapse types or on particular regions of individual neurons. Glutamate is the primary excitatory neurotransmitter in the brain, and the majority of glutamatergic synapses occur on mushroom-shaped protrusions called dendritic spines. Changes in the morphology of these structures are associated with long-lasting modulation of synaptic strength thought to underlie learning and memory, and can be abnormal in neuropsychiatric disease. Here, we use rat cortical slice cultures to examine how a previously-described synaptogenic molecule, the EphB2 receptor tyrosine kinase, regulates dendritic protrusion morphology in specific regions of the dendritic arbor in cortical pyramidal neurons. We find that alterations in EphB2 signaling can bidirectionally control protrusion length, and knockdown of EphB2 expression levels reduces the number of dendritic spines and filopodia. Expression of wild-type or dominant negative EphB2 reveals that EphB2 preferentially regulates dendritic protrusion structure in basal dendrites. Our findings suggest that EphB2 may act to specify synapse formation in a particular subcellular region of cortical pyramidal neurons.

Saffron extract and trans-crocetin inhibit glutamatergic synaptic transmission in rat cortical brain slices

Saffron, the dried stigmata of Crocus sativus L., is used in traditional medicine for a wide range of indications including cramps, asthma, and depression. To investigate the influence of hydro-ethanolic saffron extract (CSE) and trans-crocetin on synaptic transmission, postsynaptic potentials (PSPs) were elicited by focal electrical stimulation and recorded using intracellular placed microelectrodes in pyramidal cells from rat cingulate cortex. CSE (10–200 μg/ml) inhibited evoked PSPs as well as the isolated NMDA and non-NMDA component of PSPs. Glutamate (500 μM) added into the organ bath induced membrane depolarization. CSE decreased glutamate-induced membrane depolarization. Additionally, CSE at 100 μg/ml decreased NMDA (20 μM) and kainate (1 μM)-induced depolarization, whereas AMPA (1 μM)-induced depolarization was not affected. Trans-crocetin (1–50 μM) showed inhibition of evoked PSPs and glutamate-induced membrane depolarization comparable to CSE. Trans-crocetin at 10 μM decreased NMDA (20 μM)-induced membrane depolarization, but did not inhibit the isolated non-NMDA component of PSPs. We conclude that trans-crocetin is involved in the antagonistic effect of CSE on NMDA but not on kainate receptors.

Aberrant Neuronal Avalanches in Cortical Tissue Removed From Juvenile Epilepsy Patients

Some forms of epilepsy may arise as a result of pathologic interactions among neurons. Many forms of collective activity have been identified, including waves, spirals, oscillations, synchrony, and neuronal avalanches. All these emergent activity patterns have been hypothesized to show pathologic signatures associated with epilepsy. Here, the authors used 60-channel multielectrode arrays to record neuronal avalanches in cortical tissue removed from juvenile epilepsy patients. For comparison, they also recorded activity in rat cortical slices. The authors found that some human tissue removed from epilepsy patients exhibited prolonged periods of hyperactivity not seen in rat slices. In addition, they found a positive correlation between the branching parameter, a measure of network gain, and firing rate in human slices during periods of hyperactivity. This relationship was not present in rat slices. The authors suggest that this positive correlation between the branching parameter and the firing rate is part of a positive feedback loop and may contribute to some forms of epilepsy. These results also indicate that neuronal avalanches are abnormally regulated in slices removed from pediatric epilepsy patients.

Properties of Layer 6 Pyramidal Neuron Apical Dendrites

Layer 6 (L6) pyramidal neurons are the only neocortical pyramidal cell type whose apical dendrite terminates in layer 4 rather than layer 1. Like layer 5 pyramidal neurons, they participate in a feedback loop with the thalamus and project to other cortical areas. Despite their unique location in the cortical microcircuit, synaptic integration in dendrites of L6 neurons has never been investigated. Given that all other neocortical pyramidal neurons perform active integration of synaptic inputs via local dendritic spike generation, we were interested to establish the apical dendritic properties of L6 pyramidal neurons. We measured active and passive properties of the apical dendrites of L6 pyramidal neurons in the somatosensory region of rat cortical slices using dual patch-clamp recordings from somata and dendrites and calcium imaging. We found that L6 pyramidal neurons share many fundamental dendritic properties with other neocortical pyramidal neurons, including the generation of local dendritic spikes under the control of dendritic inhibition, voltage-dependent support of backpropagating action potentials, timing-dependent dendritic integration, distally located I(h) channels, frequency-dependent Ca(2+) spike activation, and NMDA spike electrogenesis in the distal apical dendrite. The results suggest that L6 pyramidal neurons integrate synaptic inputs in layer 4 similar to the way other neocortical pyramidal neurons integrate input to layer 1. Thus, L6 pyramidal neurons can perform a similar associational task operating on inputs arriving at the granular and subgranular layers.

Leu27 insulin-like growth factor-II, an insulin-like growth factor-II analog, attenuates depolarization-evoked GABA release from adult rat hippocampal and cortical slices

Accumulated evidence suggests that the single transmembrane domain insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M6P or IGF-II receptor) plays an important role in the intracellular trafficking of lysosomal enzymes and endocytosis-mediated degradation of insulin like growth factor (IGF-II). However, the role of this receptor in signal transduction following IGF-II binding remains controversial. In the present study, we revealed that Leu27IGF-II, an analog which binds preferentially to the IGF-II receptor, can attenuate K+-as well as veratridine-evoked GABA release from the adult rat hippocampal formation. Tetrodotoxin failed to alter the effects of Leu27IGF-II on GABA release, thus suggesting the lack of involvement of voltage-dependent Na+ channels. Interestingly, the effect is found to be sensitive to pertussis toxin (PTX), indicating the possible involvement of a Gi/o protein-dependent pathway in mediating the release of GABA from the hippocampal slices. Additionally, Leu27IGF-II was found to attenuate GABA release from frontal cortex but not from striatum. These results, together with the evidence that IGF-II receptors are localized on GABAergic neurons, raised the possibility that this receptor, apart from mediating intracellular trafficking, may also be involved in the regulation of endogenous GABA release by acting directly on GABAergic terminals.

Acute activation of the calcium-sensing receptor inhibits plasma renin activity in vivo.

In vitro, the renin-secreting juxtaglomerular cells express the calcium-sensing receptor, and its activation with the calcimimetic cinacalcet inhibits renin release. To test whether the activation of calcium-sensing receptor similarly inhibits plasma renin activity (PRA) in vivo, we hypothesized that the calcium-sensing receptor is expressed in juxtaglomerular cells in vivo, and acutely administered cinacalcet would inhibit renin activity in anesthetized rats. Since cinacalcet inhibits parathyroid hormone, which may stimulate renin activity, we sought to determine whether cinacalcet inhibits renin activity by decreasing parathyroid hormone. Lastly, we hypothesized that chronically administered cinacalcet would inhibit basal and stimulated renin in conscious rats. Calcium-sensing receptors and renin were localized in the same juxtaglomerular cells using immunofluorescence in rat cortical slices fixed in vivo. Cinacalcet was administered acutely via intravenous bolus in anesthetized rats and chronically in conscious rats by oral gavage. Acute administration of cinacalcet decreased basal renin activity from 13.6 ± 2.4 to 6.1 ± 1.1 ng ANG I·ml(-1)·h(-1) (P < 0.001). Likewise, cinacalcet decreased furosemide-stimulated renin from 30.6 ± 2.3 to 21.3 ± 2.3 ng ANG I·ml(-1)·h(-1) (P < 0.001). In parathyroidectomized rats, cinacalcet decreased renin activity from 9.3 ± 1.3 to 5.2 ± 0.5 ng ANG I·ml(-1)·h(-1) (P < 0.05) similar to sham-operated controls (13.5 ± 2.2 to 6.6 ± 0.8 ng ANG I·ml(-1)·h(-1), P < 0.05). Chronic administration of cinacalcet over 7 days had no significant effect on PRA under basal or stimulated conditions. In conclusion, calcium-sensing receptors are expressed in juxtaglomerular cells in vivo, and acute activation of these receptors with cinacalcet inhibits PRA in anesthetized rats, independent of parathyroid hormone.

V-ATPase Membrane Sector Associates with Synaptobrevin to Modulate Neurotransmitter Release

Acidification of synaptic vesicles by the vacuolar proton ATPase is essential for loading with neurotransmitter. Debated findings have suggested that V-ATPase membrane domain (V0) also contributes to Ca(2+)-dependent transmitter release via a direct role in vesicle membrane fusion, but the underlying mechanisms remain obscure. We now report a direct interaction between V0 c-subunit and the v-SNARE synaptobrevin, constituting a molecular link between the V-ATPase and SNARE-mediated fusion. Interaction domains were mapped to the membrane-proximal domain of VAMP2 and the cytosolic 3.4 loop of c-subunit. Acute perturbation of this interaction with c-subunit 3.4 loop peptides did not affect synaptic vesicle proton pump activity, but induced a substantial decrease in neurotransmitter release probability, inhibiting glutamatergic as well as cholinergic transmission in cortical slices and cultured sympathetic neurons, respectively. Thus, V-ATPase may ensure two independent functions: proton transport by a fully assembled V-ATPase and a role in SNARE-dependent exocytosis by the V0 sector.

Neuroprotective Effect of Taurine against Oxidative Stress-Induced Damages in Neuronal Cells

Taurine, 2-aminoethanesulfonic acid, is an abundant free amino acid present in brain cells and exerts many important biological functions such as anti-convulsant, modulation of neuronal excitability, regulation of learning and memory, anti-aggressiveness and anti-alcoholic effects. In the present study, we investigated to explore whether taurine has any protective actions against oxidative stress-induced damages in neuronal cells. ERK I/II regulates signaling pathways involved in nitric oxide (NO) and reactive oxygen species (ROS) production and plays a role in the regulation of cell growth, and apoptosis. We have found that taurine significantly inhibited AMPA induced cortical depolarization in the Grease Gap assays using rat cortical slices. Taurine also inhibited AMPA-induced neuronal cell damage in MTT assays in the differentiated SH-SY5Y cells. When the neuronal cells were treated with H2O2, levels of NO were increased; however, taurine pretreatment decreased the NO production induced by H2O2 to approximately normal levels. Interestingly, taurine treatment stimulated ERK I/II activity in the presence of AMPA or H2O2, suggesting the potential role of ERK I/II in the neuroprotection of taurine. Taken together, taurine has significant neuroprotective actions against AMPA or H2O2 induced damages in neuronal cells, possibly via activation of ERK I/II.

In vivo methylmercury exposure induced long-lasting epileptiform activity in layer II/III neurons in cortical slices from the rat

Prenatal and postnatal methylmercury (MeHg) exposure has been shown to increase neuronal excitability and seizure susceptibility. To determine if early postnatal MeHg exposure causes a similar effect, we examined changes in field potentials in layer II/III neurons in cortical slices of rat following in vivo MeHg treatment. Rats received 0 (0.9% NaCl), 0.75 mg/kg/day or 1.5 mg/kg/day MeHg subcutaneously for 15 or 30 days beginning on postnatal day 5, after which cortical slices were prepared for field potential recordings. In slices from rats treated with vehicle, single pulse stimulation of layer IV of cortical slices induced a typical field excitatory postsynaptic potential (fEPSP) with a single spike. This type of fEPSPs was also seen in slices from rats with 15 day treatment with 0.75 mg/kg/day or 1.5 mg/kg/day MeHg. However, 30-day treatment with either MeHg dose resulted in fEPSPs with multiple spikes (epileptiform activity) in 40% of animals examined. This epileptiform activity remained observable in 50–60% animals in which MeHg exposure had been terminated for 30 days. However, slices from control animals still showed fEPSPs with single spike. Thus, these data suggest that postnatal MeHg exposure in vivo altered neuronal excitability and induced a long-lasting hyperexcitability in cortical neurons.

Comparative study of quercetin and its two glycoside derivatives quercitrin and rutin against methylmercury (MeHg)-induced ROS production in rat brain slices

The hypothesis that methylmercury (MeHg) potently induces formation of reactive oxygen species (ROS) in the brain is supported by observations on the neuroprotective effects of various classes of antioxidants. Flavonoids have been reported to possess divalent metal chelating properties, antioxidant activities and to readily permeate the blood-brain barrier. They can also provide neuroprotection in a wide array of cellular and animal models of neurological diseases. Paradoxically, in vivo administration of quercetin displays unexpected synergistic neurotoxic effect with MeHg. Considering this controversy and the limited data on the interaction of MeHg with other flavonoids, the potential protective effect of quercetin and two of its glycoside analogs (i.e., rutin and quercitrin) against MeHg toxicity were evaluated in rat cortical brain slices. MeHg (100 microM) caused lipid peroxidation and ROS generation. Quercitrin (10 microg/mL) and quercetin (10 microg/mL) protected mitochondria from MeHg (5 microM)-induced changes. In contrast, rutin did not afford a significant protective effect against MeHg (100 microM)-induced lipid peroxidation and ROS production in cortical brain slices. MeHg-generated ROS in cortical slices was dependent upon an increase in intracellular Ca(2+) levels, because the over-production of MeHg-induced H(2)O(2) in mitochondria occurred with a concomitant increase in Ca(2+) transient. Here, we have extended the characterization of mechanisms associated with the neuroprotective effects of quercetin against MeHg-induced toxicity in isolated mitochondria, by performing an array of parallel studies in brain slices. We provide novel data establishing that (1) Ca(2+) plays a central role in MeHg toxicity and (2) in brain slices MeHg induces mitochondrial oxidative stress both via direct interaction with mitochondria (as previously reported in in vitro studies) as well as via mitochondria-independent (or indirect) mechanisms.