Gilbert Clincke

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.

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.

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.

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.

The effect of R 58 735 (Sabeluzole) on memory functions in healthy elderly volunteers.

The effects of chronic treatment (5 mg b.i.d. for 2 days followed by 10 mg b.i.d. for 5 days) with R 58 735 on human memory functions were studied in healthy elderly (age ≥50 years) volunteers in a double-blind placebo-controlled study. Serial learning of nonsense syllables was better under R 58 735, and relearning 1 week after termination of the treatment was superior to relearning of similar material initially learned under placebo. Free recall of nonsense syllables was significantly better when these were learned under active compound. Proactive inhibition induced by consecutive presentation of word lists was attenuated by R 58 735. Short-term memory functions, retrieval accuracy from semantic memory and unprepared reaction times were unchanged. R 58 735 ameliorated both learning and recall in conditions of age-related mild hypofunction in healthy volunteers. The compound seems to have had positive effects on encoding and consolidation of new material.

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.

Cerebral resuscitation: pathophysiology and therapy.

The interest in the possibility of cerebral resuscitation has been growing exponentially during the last decade. It became clear that pharmacotherapeutic interaction can possibly alter the outcome of cerebral hypoxia/ischemia. The present review is an attempt to provide an organizational framework for a systematic integration of studies specifically dealing with pharmacological treatment post-insult.

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.

Sabeluzole (R 58 735) 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.