Influence of lubeluzole on voltage-sensitive Ca++ channels in isolated rat neurons.

Lubeluzole is neuroprotective in a photochemical stroke model, whereas the (R)-enantiomer of the same molecule is not [De Ryck M, Keersmaekers R, Duytschaever H, Claes C, Clincke G, Janssen M and Van Reet G (1996) J Pharmacol Exp Ther 279:748-758]. We investigated the effects of lubeluzole and the (R)-enantiomer on voltage-sensitive Ca++ channels of isolated rat dorsal root ganglion cells, using whole-cell voltage-clamp, with Ba++ as the charge carrier. Both compounds blocked the low-voltage-activated Ba++ current (iLVA or T current) with an IC50 value of 1.2 microM. Lubeluzole and the (R)-enantiomer also blocked the high-voltage-activated calcium channel current (iHVA), with IC50 values of 2.6 and 3.5 microM, respectively, and accelerated the apparent inactivation of iHVA. This acceleration was more pronounced with lubeluzole than with the (R)-enantiomer at 3 and 10 microM. Both compounds produced a clear tonic block of iLVA and iHVA, even in the absence of previous stimulation. Lubeluzole and the (R)-enantiomer induced a negative shift of the inactivation curve of iLVA and showed down the recovery from inactivation. This resulted in a stronger inhibition of iLVA at more depolarized conditioning potentials and higher stimulation frequencies. The block of iHVA was voltage and frequency dependent. Lubeluzole and the (R)-enantiomer also blocked iHVA in isolated rat superior cervical ganglion cells and cerebellar Purkinje cells. The Ca++ channel-blocking properties of lubeluzole may contribute to its neuroprotective effect. However, the small difference between the two enantiomers in inhibition of Ca++ channel currents does not explain the stereospecificity of the neuroprotective properties of lubeluzole in vitro and in vivo.

Antagonism of meta-chlorophenylpiperazine-induced inhibition of exploratory activity in an emergence procedure, the open field test, in rats.

The effects of meta-chlorophenylpiperazine (mCPP) were studied on exploratory behaviour in the open field test, using a procedure designed to evaluate the emergence of rats into a novel environment. mCPP reduced the exploratory activity in a dose-related manner after subcutaneous (s.c.), intraperitoneal (i.p.) and intravenous (i.v.) administration. The inhibition was manifest in all the parameters used to quantify the exploration of the open field area. Additional neuroendocrine experiments in a parallel group of rats revealed a dose-related increase in plasma prolactin and ACTH levels after i.v. mCPP, pointing to a general state of arousal in these mCPP-treated animals. A number of 5-HT antagonists were tested for their ability to prevent or reverse the behavioural inhibition induced by an i.v. injection of 1.0 g/kg mCPP given 15 min before testing in the open field. The antagonists were injected s.c. or given orally at various time intervals before mCPP, or they were injected i.v. 10 min after mCPP. The lowest active doses for the attentuation of the mCPP-induced behavioural inhibition after s.c., oral and i.v. administration, respectively, were 0.04, 40 and 10 mg/kg for pizotifen; 0.16, 0.16 and 0.16 mg/kg for mianserin; 0.63, 0.16 and 0.16 mg/kg for methysergide, and 0.16, 2.5 and 2.5 mg/kg for ritanserin. The lowest active doses of mirtazapine after s.c. and i.v. treatment were 0.01 and 0.16 mg/kg. These data indicate that mixed 5-HT1/5-HT2 receptor antagonists such as pizotifen and methysergide, and mixed 5-HT and catecholamine antagonists such as mianserin and mirtazapine are more potent antagonists of mCPP-induced behavioural inhibition in rats than the more selective 5-HT2A/5-HT2C antagonist ritanserin.

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.

A simple, non-invasive method for the measurement of reserpine-induced tremor in rats.

A new, non-invasive method for measuring reserpine-induced tremor in rodents is described here. The test procedure is based on the piezo-electric principle and was evaluated using the tremorogenic compound reserpine and the stereotypies-inducing drug apomorphine. Whereas for reserpine an orderly and dose-related increase in activity was observed, no such effect was detected with apomorphine. In order to further evaluate the test procedure, studies on the antagonism of reserpine-induced tremor were also performed. Results from these studies indicated that the DA-agonist lisuride, but not the S2-antagonist ritanserin, were able to antagonize the reserpine-induced tremor in a dose-related manner.

Extracellular changes of taurine in the peri-infarct zone: effect of lubeluzole.

Lubeluzole is a neuroprotective compound that has been shown to stereoselectively rescue sensorimotor function and reduce infarct size in a photochemical stroke model in rats. Tissue swelling, which occurs in the peri-infarct zone, is accompanied by a compensatory taurine release. Therefore, using a microdialysis technique, we aimed at measuring changes of extracellular concentrations of taurine in the peri-infarct zone and the effects of lubeluzole and its R-isomer. Lubeluzole blocked the increase of taurine in tissue immediately surrounding a photochemically induced thrombotic neocortical infarct. By contrast, the R-isomer was completely inactive. We hypothesize that lubeluzole may reduce osmoregulatory stress in peri-infarct tissue.

Lubeluzole blocks increases in extracellular glutamate and taurine in the peri-infarct zone in rats.

A microdialysis probe was positioned inside the peri-infarct zone of a photochemically induced neocortical infarct in rats. Extracellular glutamate rose within 20 min after the start of infarct induction and continued to increase during the 5 h observation period to 5.5-fold the pre-infarct baseline value of 0.8 +/- 0.4 micromol/l. Glutamine increased only 1.4-fold. Changes in peri-infarct glutamate were preceded by steep rises in taurine (a 3.9-fold increase from the baseline value of 2.8 +/- 0.7 micromol/l), which coincided with spreading depressions during infarct induction. Post-treatment with lubeluzole ((S)-4-(2-benzothiazolylmethylamino)-alpha-[(3,4-difluoro-phenoxy) methyl]-1-piperidineethanol, 1.25 mg/kg i.v.), a new cerebroprotective drug, blocked the peri-infarct increases of glutamate and taurine, whereas the R-enantiomer was ineffective. Since lubeluzole has previously been shown to stereospecifically decrease glutamate-activated nitric oxide (NO) toxicity in vitro, the present in vivo stereospecific effect of lubeluzole may be related to modulation of the cascade of NO toxicity, thus preventing NO toxicity-mediated increases in extracellular glutamate. Blockade of the peri-infarct taurine response suggests that lubeluzole also may have reduced cellular osmotic stress in the peri-infarct zone.

Lubeluzole protects sensorimotor function and reduces infarct size in a photochemical stroke model in rats.

Posttreatment with lubeluzole, the S-isomer of a novel 3,4-difluoro benzothiazole, potently rescued tactile/proprioceptive hindlimb placing reactions contralateral to unilateral thrombotic infarcts in the hindlimb area of the parietal sensorimotor neocortex of rats. Administered at 5 min postinfarct, a single i.v. bolus of lubeluzole was three times as potent as the racemate, whereas the R-isomer was inactive. Neurological protection was near-maximal for treatment delays through 1 hr postinfarct, but declined with longer delays. However, when administered at 6 hr, 1.25 mg/kg i.v. still protected 60% of infarcted rats. An i.v. bolus followed by a 1-hr i.v. infusion produced equieffective neurologic protection at both 6- and 3-hr delays. This optimal lubeluzole regimen, started at 5 min postinfarct, reduced infarct volume by 22 to 24% at 4 hr postinfarct and by 28% at 7 days postinfarct. Again, the R-isomer was inactive. Down-regulation of the glutamate-activated nitric oxide synthase pathway leading to neurotoxicity and neuronal death may constitute a neuroprotective mechanism of action for lubeluzole.

Transgenic mouse model for the fragile X syndrome.

Transgenic fragile X knockout mice have been constructed to provide an animal model to study the physiologic function of the fragile X gene (FMR1) and to gain more insight into the clinical phenotype caused by the absence of the fragile X protein. Initial experiments suggested that the knockout mice show macroorchidism and cognitive and behavioral deficits, abnormalities comparable to those of human fragile X patients. In the present study, we have extended our experiments, and conclude that the Fmr1 knockout mouse is a reliable transgenic model to study the fragile X syndrome.

Antagonism of the discriminative stimulus properties of cocaine with the combination of a dopamine D1 and D2 antagonist.

Antagonism of the discriminative stimulus properties of 10 mg/kg cocaine was studied in rats by use of the dopamine D1 antagonist SCH 23390 and the D2 antagonist haloperidol. Whereas SCH 23390 and haloperidol were by themselves unable to antagonize the cueing properties of cocaine, the combination of both dopamine antagonists resulted in a complete blockade of the cocaine cue. In the presence of a fixed dose of 0.01 and 0.04 mg/kg haloperidol, the ED50's (it is the effective dose in 50% of the animals) of SCH 23390 for cocaine antagonism were 0.043 and 0.012 mg/kg, respectively. Similarly, the ED50's of haloperidol in combination with 0.01 and 0.04 mg/kg SCH 23390 were 0.021 and 0.024 mg/kg. The combined treatment of haloperidol and SCH 23390 resulted in strong response-rate reductions. At all combination regimens resulting in a complete blockade of the cocaine cue, response rate was reduced to less than 20% of the control values. These results indicate that the cueing properties of cocaine are both dopamine D1- and D2-mediated and that a combined antagonism of both receptor subtypes can lead to a complete antagonism of the cueing properties of cocaine which is associated with severe attenuation of response rate.

7-OHDPAT and alcohol consumption, withdrawal and discriminative stimulus properties in rats.

A series of experiments was performed, which indicated that 7-OHDPAT is only active in rats that are still in the dynamic phase of the development of a high and stable level of alcohol preference and in a drug discrimination test procedure, but not in rats with a matured preference for 3% alcohol. These data may point to a limited use of 7-OHDPAT in conditions of alcohol misuse and dependence.

Learning performance at different time periods after hypoxia in infant rats.

Infant rats were exposed to hypoxia. At different time periods after the hypoxic aggression the animals were subjected to a one-way active avoidance task consisting of 3 daily training sessions. Learning performance of hypoxic rats, expressed as the number of avoidances per 10 trials, was significantly decreased in all the sessions at posthypoxic day 35 (P < 0.001). The rate to which learning behaviour was affected by hypoxia was comparable among all 3 sessions in both groups. Histological evaluation of hippocampus and cortex at day 7 and day 35 after hypoxic exposure, did not reveal detectable structural changes. It can be concluded from this study that hypoxia, induced in 21 day old rats leads to learning performance deficits in the adult animal.

Quantification of tremor sensitivity and inhibition of exploratory behaviour during alcohol withdrawal in rats.

Rats given a 10% (v/v) alcohol liquid diet over two weeks reached high blood alcohol levels of around 200mg/dl. Discontinuation of the alcohol intake resulted within 6h in several withdrawal reactions including a tremorogenic activity and a reduction in exploratory behaviour in novel environments. The tremorogenic activity of the alcohol withdrawal could be quantified, using a piezo-film technique, in terms of a supersensitivity to both an inactive and a moderately active dose of the tremorogenic compound harmine. As compared to controls, the rats in alcohol withdrawal revealed more frequent tremor after both 5 and 10mg/kg harmine. The supersensitivity to harmine-induced tremor started within 6h after alcohol withdrawal and remained present with 10mg/kg harmine for up to 48h. The supersensitivity was independent of the length of the tremor bursts used to quantify harmine-induced tremor. Alcohol withdrawal also resulted in an inhibition of exploratory behaviour in a neutral two-chamber box. Both in terms of the number of transits into the open field as well as the time spent in the open area, rats in alcohol withdrawal were significantly less active than control animals. The reduced exploration started within 6h after withdrawal and remained present for up to 24h after the last alcohol intake. These results indicate that both alcohol withdrawal-induced sensitivity to tremorogenic agents and inhibition of exploratory behaviour can be quantified over time, allowing the pharmacological mechanisms involved to be studied.

Synaptic plasticity in rat hippocampus associated with learning.

Rats subjected to a one-way active avoidance task consisting of 3 daily training sessions, showed obvious shape changes in dendritic spines of the hippocampal supragranular molecular layer. Performance, expressed as the number of avoidances per 10 trials, significantly improved in the second and third session (P < 0.001). In trained animals, at the end of the third session, the amount of perforated concave synapses significantly increased as compared to untrained controls (P < 0.05). When compared with a group of sham-shocked rats, the increase was less pronounced. The length of the postsynaptic density in both, perforated and non-perforated synapses, significantly increased in comparison with untrained control and sham-shocked animals (perforated: P < 0.005; non-perforated: P < 0.05). The results are indicative for the existence of synaptic remodeling and turnover in rats subjected to one-way active avoidance training.

In vivo studies on the mechanism of action of the broad spectrum anticonvulsant loreclezole.

In animal models of epilepsy the anticonvulsant profile of loreclezole resembles that of barbiturates and benzodiazepines. We examined whether the increase in seizure threshold to pentylenetetrazole infusion produced by 10 mg/kg of loreclezole, pentobarbital or diazepam could be reversed by a spectrum of benzodiazepine partial inverse to full inverse agonists (FG-7142 beta-carboline carboxylate, CGS-8216, Ro-15-4513 and DMCM) or by a benzodiazepine neutral antagonist (Ro-15-1788). The doses of the benzodiazepine inverse agonists were chosen to produce a 20-40% decrease in seizure threshold. The seizure threshold increase produced by loreclezole and pentobarbital was reduced by all the benzodiazepine inverse agonists and potentiated by Ro-15-1788. Diazepam was antagonized by the benzodiazepine inverse agonists and by the neutral antagonist. The generality of this finding was examined in amygdala-kindled rats. The decrease in the duration of forepaw clonus and the reduction in behavioural stage34 produced by loreclezole, pentobarbital and diazepam was reversed by CGS-8216. Ro-15-1788, which itself showed anticonvulsant effects in this model, antagonized the effects of diazepam, but not loreclezole or pentobarbital. Thus loreclezole behaves more like a barbiturate than a benzodiazepine in these two in vivo models. This suggests a possible mechanism of action of loreclezole at a neuromodulatory site within the GABAA receptor complex, which is unlikely to be a benzodiazepine receptor.

Neocortical localization of tactile/proprioceptive limb placing reactions in the rat.

The present study was aimed at delineating the neocortical substrate of tactile/proprioceptive limb placing reactions in rats by means of behavioral tests that excluded the participation of facial stimuli in limb function. Using a photochemical technique, we made unilateral focal lesions in the frontal and parietal neocortex. Fore- and/or hindlimb placing deficits resulted from damage to a fronto-parietal region lying between the medial agranular cortex and the primary somatosensory (whisker barrel field) cortex. When the antero-posterior coordinate was varied from 4 mm anterior to 1 mm posterior to bregma, tactile/proprioceptive forelimb dysfunction was more pronounced after damage to the parietal forelimb area, but lesions confined to the frontal lateral agranular cortex also yielded clear-cut forelimb placing deficits. Damage to either area alone allowed for partial recovery of forelimb function. However, following combined, total destruction of both frontal and parietal forelimb areas, forelimb deficits did not recover. This resembled the irreversible hindlimb deficits after near-total destruction of the parietal hindlimb area. Damage to the medial agranular cortex left limb placing intact. Likewise, for as long as the medial edge of lesions to the whisker barrel field did not come closer than 3 mm to the midline, thus remaining outside the parietal hindlimb area, limb placing remained normal. This sharp medial and lateral delineation of the cortical substrate subserving tactile/proprioceptive limb placing coincides with the borders of a thick, dense subfield of large pyramidal neurons in the deeper parts of layer V. Limb placing remained intact when medial agranular cortex lesions damaged only 30% of that subfield, whereas 70% destruction of that layer following more laterally placed lesions in the parietal hindlimb area produced irreversible hindlimb dysfunction. The severity of hindlimb placing deficits was related to the amount of incursion by whisker barrel field lesions into the subfield of deep layer V large pyramidal neurons. Finally, very large lesions of the occipital cortex did not affect tactile/proprioceptive limb placing. We discuss the neocortical areal and laminar specificity of tactile/proprioceptive limb function in the context of recent neuroanatomical and electrophysiological findings, and their relevance to normal cortical function, recovery from neocortical stroke (including diaschisis), and age-related cortical dysfunction.

Effects of ritanserin and chlordiazepoxide on sleep-wakefulness alterations in rats following chronic cocaine treatment.

The effects of ritanserin, a 5-hydroxytryptamine-2 (5-HT2) receptor antagonist, and chlordiazepoxide, a benzodiazepine agonist, on sleep-wakefulness disturbances in rats after acute administration of cocaine and after discontinuation of chronic cocaine treatment were examined. Intraperitoneal (IP) injection of chlordiazepoxide (10 mg/kg) but not ritanserin (0.63 mg/kg) prevented the increase of wakefulness (W) and the reduction of light slow wave sleep (SWS1) and deep slow wave sleep (SWS2) induced by an acute injection of cocaine (20 mg/kg IP). Daily injection of cocaine (20 mg/kg for 5 days, then 30 mg/kg for 5 days IP) at the onset of the light phase elicited an increase of W and a concomitant decrease of SWS1, SWS2 and paradoxical sleep (PS) in the light phase, followed by a rebound in SWS2 and PS in the subsequent dark phase. Following cocaine discontinuation, the circadian distribution of sleep-wakefulness states remained disturbed in saline-treated rats for at least 5 days. Both ritanserin (0.63 mg/kg IP/day) and chlordiazepoxide (10 mg/kg IP/day) reduced the alteration in the distribution of W and SWS2 throughout the light-dark cycle from the first day of administration on, but failed to prevent PS alterations. The mechanisms by which both compounds exert their effect are probably quite different. For chlordiazepoxide sedative and sleep-inducing properties probably play a major role. In contrast, for ritanserin SWS2-increasing properties and its ability to reverse preference for drugs of abuse without inducing aversion might be key factors.

Sabeluzole (R58 735) increases consistent retrieval during serial learning and relearning of nonsense syllables.

Consistent retrieval during serial learning of nonsense syllables was investigated under sabeluzole (10 mg b.i.d. for 7 days) and placebo. The same material was relearned 1 week after withdrawal of the drug. During learning a twofold improvement in retrieval efficiency was seen when volunteers learned new material under steady-state levels of sabeluzole. During relearning, material originally learned under sabeluzole was significantly better retrieved than material learned under placebo. The results suggest that sabeluzole influences basic mechanisms involved in storage and retrieval of new information.

The effect of sabeluzole (R 58735) on memory retrieval functions.

In a first double-blind, placebo-controlled parallel experiment, 20 volunteers with a median age of 45 years were treated for 1 week with either sabeluzole (R 58735) or placebo. Before and after the treatment period, they were subjected to a Selective Reminding Procedure in which a 20-word list had to be learned. No differences between the two groups were seen for total recall, short-term retrieval, total long-term retrieval, random long-term retrieval and long-term storage. However, a significant improvement in consistent long-term retrieval (cLTR) was seen for the subjects treated with sabeluzole. This effect was restricted to the group of the poor performers, i.e. those with a baseline cLTR of 50% or less. In a second experiment, 12 healthy elderly volunteers with a median age of 59 years were subjected to the same test procedure. They were treated with sabeluzole in a single-blind fashion. Again the cLTR improved significantly in the group of poor performers. It was thus confirmed that sabeluzole ameliorates retrieval functions and primarily so in poor performers.