Roger Marrannes

Evidence for a role of the N-methyl-d-aspartate (NMDA) receptor in cortical spreading depression in the rat

The neurotransmitter glutamate activates the N-methyl-D-aspartate (NMDA), quisqualate and kainate receptors. It has been proposed, but also disputed, that local release of glutamate would play a pivotal role in cortical spreading depression (SD). We tested this hypothesis by investigating the influence of NMDA antagonists on SD, using the non-competitive NMDA antagonists ketamine, phencyclidine (PCP) and MK-801 and the competitive NMDA antagonist DL-2-amino-7-phosphonoheptanoate (2-APH), injected intraperitoneally in rats anesthetized with alfentanil. SD was elicited by cathodal DC-stimulation of the frontal cortex. SD propagation was followed using two ion-sensitive microelectrodes placed in the parietal and occipital cortex. The NMDA antagonists increased SD threshold, decreased the propagation velocity and decreased the duration of the accompanying extracellular DC, K+ and Ca2+ changes at the following doses: 40 mg/kg ketamine, 10 mg/kg PCP, 0.63 mg/kg MK-801, 10 and 40 mg/kg 2-APH. With each NMDA antagonist failure of SD propagation between both microelectrodes could be observed. SD elicitation (or propagation) was inhibited completely with 80 mg/kg ketamine, 3.1 mg/kg MK-801 and 160 mg/kg 2-APH. These NMDA antagonists have also anticonvulsant properties. None of these effects on SD were observed with high doses of other anticonvulsants such as 80 mg/kg phenytoin or 40 mg/kg diazepam. These experiments indicate that endogenous release of excitatory amino acids and their action on the NMDA receptor play an important role in the initiation, propagation and duration of SD.

Functional role of 5-HT2 receptors in the regulation of sleep and wakefulness in the rat

Recently developed agents specifically acting on different 5-hydroxytryptamine (5-HT) receptor populations were used to analyze the functional role of 5-HT2 receptor subtypes in the sleep-wakefulness cycle of the rat. The 5-HT2 receptor antagonist ritanserin injected intraperitoneally (IP) (0.04–2.5 mg/kg) induced an increase in deep slow wave sleep (SWS2) duration at the expense of wakefulness (W), light slow wave sleep (SWS1) and paradoxical sleep (PS). The stimulation of 5-HT2 receptors by 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) produced a dose-related increase in W and a dose-dependent decrease in both SWS2 and PS. Pretreatment with ritanserin (0.16–2.5 mg/kg) or with cinanserin (2.5–5 mg/kg), another 5-HT2 receptor antagonist, dose-dependently reversed the W enhancement and the SWS2 deficit produced by DOM, but not the PS deficit. Sleep-wakefulness alterations (increase in W and SWS1 combined with a suppression of SWS2 and PS) observed after IP injection of two putative 5-HT1 receptor agonists, 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) (2.5 mg/kg) and 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969) (0.63 mg/kg), were not modified by ritanserin pretreatment (0.16–2.5 mg/kg). These results further support the hypothesis that the serotonergic system plays an active role in the regulation of the sleep-wakefulness cycle in the rat and that 5-HT2 receptors are involved in this action. In addition it is suggested that 5-HT1 receptor subtypes are unlikely to interact with 5-HT2 receptors in the sleep-wakefulness modulation mediated through 5-HT2 receptors.

Identifying Modulators of hERG Channel Activity Using the PatchXpress® Planar Patch Clamp

The authors used the PatchXpress® 7000A system to measure compound activity at the hERG channel using procedures that mimicked the “gold-standard” conventional whole-cell patch clamp. A set of 70 compounds, including hERG antagonists with potencies spanning 3 orders of magnitude, were tested on hERG302-HEK cells using protocols aimed at either identifying compound activity at a single concentration or obtaining compound potency from a cumulative concentration dependence paradigm. After exposure to compounds and subsequent washout of the wells to determine reversibility of the block, blockade by a reference compound served as a quality control. Electrical parameters and voltage dependence were similar to those obtained using a conventional whole-cell patch clamp. Rank order of compound potency was also comparable to that determined by conventional methods. One exception was flunarizine, a particularly lipophilic compound. The PatchXpress® accurately identified the activity of 29 moderately potent antagonists, which only weakly displace radiolabeled astemizole and are false negatives in the binding assay. Finally, no false hits were observed from a collection of relatively inactive compounds. High-quality data acquisition by PatchXpress® should help accelerate secondary screening for ion channel modulators and the drug discovery process

Altered Na+-channel function as an in vitro model of the ischemic penumbra: action of lubeluzole and other neuroprotective drugs

Veratridine blocks Na(+)-channel inactivation and causes a persistant Na(+)-influx. Exposure of hippocampal slices to 10 microM veratridine led to a failure of synaptic transmission, repetitive spreading depression (SD)-like depolarizations of increasing duration, loss of Ca(+)-homeostasis, a large reduction of membrane potential, spongious edema and metabolic failure. Normalization of the amplitude of the negative DC shift evoked by high K+ ACSF 80 min after veratridine exposure was taken as the primary endpoint for neuroprotection. Compounds whose mechanisms of action includes Na(+)-channel modulation were neuroprotective (IC50-values in microM): tetrodotoxin 0.017, verapamil 1.18, riluzole 1.95, lamotrigine > or = 10, and diphenylhydantoin 16.1. Both NMDA (MK-801 and PH) and non-NMDA (NBQX) excitatory amino acid antagonists were inactive, as were NOS-synthesis inhibitor (nitro-L-arginine and L-NAME) Ca(2+)-channel blockers (cadmium, nimodipine) and a K(+)-channel blocker (TEA). Lubeluzole significantly delayed in time before the slices became epileptic, postponed the first SD-like depolarization, allowed the slices to better recover their membrane potential after a larger number of SD-like DC depolarizations, preserved Ca2+ and energy homeostasis, and prevented the neurotoxic effects of veratridine (IC50-value 0.54 microM). A concentration of lubeluzole, which was 40 x higher than its IC50-value for neuroprotection against veratridine, had no effect on repetitive Na(+)-dependent action potentials induced by depolarizing current in normal ACSF. The ability of lubeluzole to prevent the pathological consequences of excessive Na(+)-influx, without altering normal Na(+)- channel function may be of benefit in stroke.

Extracellular ions during veratridine-induced neurotoxicity in hippocampal slices: neuroprotective effects of flunarizine and tetrodotoxin

Veratridine, by blocking Na+ channel inactivation and shifting activation to more negative membrane potentials, causes Na(+)-influx and a persistent tendency for depolarization. Veratridine is neurotoxic to cultured neurones, and this neurotoxicity can be blocked by the class IV calcium antagonist, flunarizine. We were interested to know whether similar effects could be found in a functional differentiated tissue containing adult neurones and glial cells. We examined this in hippocampal slices using extracellular potential recordings and ion-selective microelectrodes sensitive to [Na+]o, [Ca2+]o and [K+]o. Veratridine blocked synaptic transmission in CA1, and induced several episodes of spreading depression (SD). This was followed by a long-lasting increase in [K+]o and a continuous decrease in [Ca+]o. Following veratridine exposure to hypoxia only revealed a small negative DC shift and small shifts in extracellular ions; indicating that the cells had lost the ability to maintain ion homeostasis before the hypoxia, and that veratridine had been neurotoxic. In hippocampal slices obtained from guinea pigs which had been pretreated with 40 mg/kg x 2 flunarizine orally the time before the first SD induced by veratridine was doubled. Although the ion shifts during the first SD were similar to controls, flunarizine reduced the time of recovery of [Ca2+]o, [K+]o and DC potential. The increase in [K+]o baseline and the massive decrease in [Ca2+]o baseline seen following the SDs in the solvent group were smaller in the flunarizine-treated slices. During the subsequent hypoxic period the negative DC shift was 8x larger in the flunarizine group, and the shifts in [K+]o, [Na+]o and [Ca2+]o were bigger. Tetrodotoxin also delayed the first SD during veratridine and increased the size of the DC shift during the subsequent hypoxic period. Both flunarizine and tetrodotoxin therefore protected adult brain tissue containing glia from the neurotoxicity of veratridine. These findings suggest that persistent Na(+)-influx and the consequent Ca2(+)-influx produce neurotoxicity, and that the ability to attenuate this neurotoxicity may be important in the mechanism of action of cerebroprotective drugs from different pharmacological classes.

NMDA Antagonists Inhibit Cortical Spreading Depression, But Accelerate the Onset of Neuronal Depolarization Induced by Asphyxia

Cortical spreading depression (SD) is a transient massive change of the local cortical microenvironment, which spreads over the cortex at a rate of approximately 3 mm/min. Asphyxia can facilitate the elicitation of SD. It has been proposed that release of glutamate may play an important role in SD1 and hypoxia. Glutamate activates the N-methyl-D-aspartate (NMDA), quisqualate and kainate receptors. We investigated whether the NMDA receptor is involved in SD during normoxia using the non-competitive NMDA antagonists ketamine, phencyclidine and MK-801 and the competitive NMDA antagonist 2-amino-7-phosphonoheptanoate (2-APH). Additionally, we tested whether NMDA antagonists affect the DC and ionic changes during asphyxia.

Analysis of the human KCNH2(HERG) gene: Identification and characterization of a novel mutation Y667X associated with long QT syndrome and a non‐pathological 9 bp insertion

Long QT (LQT) syndrome is a potentially life‐threatening disorder, characterized by a distinct cardiac arrhythmia known as torsades de pointes. Mutations within a number of genes linked to the familial form, including that coding for a cardiac potassium channel called KCNH2 (HERG), have been described based on the characterized genomic organization. A standardized method was developed to screen the entire gene for gene variants. We report a single base pair substitution, introducing a premature STOP codon at codon 667 of the gene in a healthy individual with an extended QTc interval (460 msec). In vitro expression of the codon Y667X variant in Xenopus oocyte suggests that the autosomal dominant variant does not function in a dominant/negative manner and cannot co‐assemble to form a channel, resulting in a reduction of the KCNH2 current, and an extension of the QT interval. This indicates that pathogenic LQT gene variants exist in the apparently normal population, the prognosis and clinical consequences of which remain to be determined. The assays described should facilitate future studies into this area. Hum Mutat 15:483, 2000.

Potassium translocation and spreading depression induced by electrical stimulation of the brain

Cathodal current pulses with durations from 20 ms to 80 s were applied to the surface of rat parietal neocortex, and the strength-duration properties of the threshold stimulus for initiation of spreading depression was determined. In one series, extradural stimulation was used. In a second series, a liquid electrode was used, the dura was opened, and K+-sensitive microelectrodes were used to determine the time course of extracellular K+ concentration during and after each stimulus pulse. With the dura open, the strength-duration curve was of the form It0.55 = constant. With extradural stimulation, the slope of the log-log plot relating current intensity to pulse duration changed gradually as the pulse duration increased, averaging 0.63. Theoretical analysis suggests that diffusion of K+ away from a zone of current-induced accumulation can account for these slope data. Applicability of this mechanism of K+ accumulation to observed changes in sensitivity of neurons to repeated stimulation at subcortical sites is considered.

Strength-duration properties of cathodal pulses eliciting spreading depression in rat cerebral cortex

Strength-duration curves for the threshold stimulus for initiation of spreading depression were determined for durations of 2 ms to 120 s, using cathodal surface stimulation through a well-defined area. With the dura open, the strength-duration curve was of the form I square root t = constant. With extradural stimulation, the slope of the log-log plot relating current intensity to pulse duration increased gradually as the pulse duration increased.

Effects of Flunarizine on Normal and Injured Rat Cerebral Cortex

The structure, properties, and range of therapeutic action of flunarizine are reviewed, and data from several studies are presented to support the general thesis that flunarizine in therapeutic doses acts to normalize damaged or abnormal brain tissue, but has little or no effect on normal brain tissue. When flunarizine was administered systemically in a single i.p. dose (40 mg/kg) or divided in oral doses (30 mg/kg preload, then 10 mg/kg) to 200–250 g male Wistar rats, the threshold for initiation of a spreading depression was increased by flunarizine treatment only in rats in which the cortex had been injured. When global ischemia was induced by cardiac arrest, the rise in extracellular potassium was delayed by both flunarizine and phenytoin. Flunarizine did not affect the release of potassium ion by repetitive electrical stimulation in normal cerebral cortex or the distribution of current-induced potassium translocation within normal cortex. These results support the designation of flunarizine as a “calcium overload blocker” that acts to protect damaged cells from excessive entry of calcium. Flunarizine may be a useful adjunct in the management of cerebral trauma.