mystery of yawning
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Fetal yawning assessed by 3D and 4D sonography
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Le bâillement : de l'éthologie à la médecine clinique
Le bâillement : phylogenèse, éthologie, nosogénie
 Le bâillement : un comportement universel
La parakinésie brachiale oscitante
Yawning: its cycle, its role
Warum gähnen wir ?
 
Fetal yawning assessed by 3D and 4D sonography
Le bâillement foetal
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mise à jour du
27 février 2011
Folia Biol (Praha)
2010; 56(6): 256-262
Donepezil Inhibits Diisopropylfluorophosphate-Induced Seizures
and Up-regulation of Synaptotagmin 4 mRNA
 
Saghafi MM, Pregelj P, Zivin M.
 
University of Ljubljana, Medical Faculty, Institute of Pathophysiology, Brain Research Laboratory, Slovenia

Chat-logomini

 
Abstract
 
Reversible acetylcholinesterase inhibitor donepezil displays prophylactic effects against intoxication with irreversible organophosphorous acetylcholinesterase inhibitors. We used behavioural observation of yawning and epileptic seizures, histochemical acetylcholinesterase staining, and in situ hybridization of the immediate early genes, c-fos and synaptotagmin 4 (Syt4) mRNAs in the brain, to evaluate whether donepezil could protect the brain against the effects of the organophosphate anticholinesterase, diisopropylfluorophosphate, in a rat model of intoxication.
 
Diisopropylfluorophosphatetreated animals exhibited frequent yawning, significant inhibition of acetylcholinesterase staining and upregulation of c-fos mRNA, but not the epileptic seizures or significant change of Syt4 mRNA levels. In order to reduce the threshold for the induction of cholinergic seizures, additional groups of rats were pre-treated with LiCl 24 h before the treatment with diisopropylfluorophosphate. These rats exhibited the seizures, a significant inhibition of acetylcholinesterase staining and significant upregulation of c-fos and Syt4 mRNA levels. All the above-mentioned effects of diisopropylfluorophosphate were inhibited by donepezil pre-treatment. Donepezil pre-treatment by itself induced only a comparatively weaker inhibition of acetylcholinesterase staining and infrequent yawning.
 
We conclude that donepezil protects the brain against diisopropylfluorophosphate-induced effects and that Syt4 mRNA upregulation may serve as a novel marker for organophosphate-induced seizures.

Introduction
 
Irreversible organophosphorous (UP) inhibitors of acetyicholinesterase (AChE) were developed as chemical warfare agents (e.g., soman) and agricultural insecticides (e.g., diisopropyl fluorophosphate - DFP). These compounds exert their toxic effects by (pseudo)irreversible inhibition of AChE, resulting in prolonged acetylcholine (ACh) activity, with subsequent excessive stimulation of both muscarinic and nicotinic ACh receptors (Taylor, 1996). Due to the localization of cholinergic synapses in the central nervous system (CNS), autonomic ganglia, and neuromuscular junction, this leads to a cholinergic toxic syndrome, characterized by numerous symptoms of central and peripheral origin. Since UP anticholinesterases are highly soluble in lipid membranes, they easily cross the blood-brain-barrier (BBB). The spectrum of acute CNS effects includes confusion, ataxia, slurred speech, loss of reflexes, Cheyene-Stokes respiration, coma, and central respiratory paralysis (Watson et al., 2009). Cholinergic mechanisms can also trigger the onset of seizures (Turski et al., 1983), although the propagation and maintenance of status epilepticus occurs primarily via activation of excessive glutamatergic transmission (Smolders et al., 1997) and subsequent excitotoxic brain damage (Solberg and Belkin, 1997). Conventional treatment of organophosphate poisoning includes combined administration of a cholinesterase reactivator (an oxime), a muscarinic cholinergic receptor antagonist (atropine) and a benzodiazepine anticonvulsant (diazepam) (Watson et al., 2009).
 
One of the strategies for prevention of irreversible inhibition of AChE by UP is the use of reversible inhibitors, such as carbamates (Lallement et al., 2001). AChE inhibited by carbamates is resistant against irreversible inhibition by UP nerve agents and the reversibly inhibited enzyme recovers spontaneously, without the use of AChE reactivators. The peripherally acting carbamate AChE inhibitor, pyridostigmine bromide (PB), has been advocated as a prophylaxis against potential threat with OP warfare agents (Keeler et al., 1991). However, PB does not cross the BBB and thus may not counteract CNS symptoms. Indeed, the CNS effects of OP agents may actually be potentiated by pre-treatment with PB (Amourette et al., 2009). In this regard, centrally acting reversible anticholinesterases that have been used for the treatment of Alzheimer's disease (AD) may offer a better prophylactic choice against OP intoxication than PB.
 
Donepezil (DON) is a reversible, primarily non-competitive, selective inhibitor of AChE used for the treatment of AD. Acute pre-treatment with oral DON, with and without scopolamine, decreased the hypothermic, hypokinetic, and diarrhoea-inducing effects of DFP (Janowsky et al., 2004, 2005). A combined pre-treatment with DON and procyclidine, a muscarinic receptor antagonist, protected against soman-induced seizures (Haug et al., 2007).
 
Here, we further explored the prophylactic potential of DON against the central effects of DFP in the brain, such as yawning (Ogura et al., 2001) and seizures (Zivin et al., 1999). In addition, we performed AChE histochemical staining and in situ hybridization of immediate early genes (lEGs) c-fos and synaptotagmin 4 (Syt4) mRNA for this purpose. c-fos induction in the brain is a well-established marker of neuronal hyperactivity induced by cholinergic seizures (Zivin et al., 1999), while there is rising evidence for the pathophysiological and adaptive changes of membrane trafficking in the brain induced by seizures, thus implicating the potential role of the plasticity of Syt4 expression in these processes (Glavan et al., 2009).
 
Discussion
 
The main findings of our experiments confirmed that DON, which is the dominant drug in the treatment of Alzheimer's disease, could also display prophylactic activity against central signs of intoxication with OP nerve agents.
 
AChE staining showed that DFP almost fully inhibited striatal, cortical and hippocampal AChE. Although to a lesser extent, DON also inhibited AChE in these regions. However, it should be remembered that DFP induces irreversible inhibition of AChE, while the inhibition of AChE by DON is mostly reversible. The only physiological mechanism for restoring DFP-inhibited AChE activity thus depends on the relatively slow process of de novo synthesis of AChE. It may be speculated that the pre-treatment with DON has protected AChE against the irreversible inhibition by DFP, and that the relatively high AChE activity observed in our experiments 1-4 h after the treatment with DFP reflects spontaneously recovered activity of AChE.
 
In rats without seizures, we did not find cortical and hippocampal c-fos mRNA up-regulation after DON, as compared to the clear up-regulation in DFP-treated animals. DON in fact completely prevented the DFP-induced up-regulation of c-fos mRNA levels. lEGs such as c-fos respond to various stimuli and their protein products transactivate other genes, resulting in longterm changes in the nervous system. Various types of noxious stimulation (e.g., thermal, mechanical, and chemical), including DFP, have been shown to induce c-fos in the brain and spinal cord of various species (Zivin et al., 1999; Gupta et al., 2000). Since the massive induction of c-fos mRNA is indicative of pathologic processes, such as excitotoxic and neurodegenerative changes (Gupta et al., 2000), the prevention of DFP-induced upregulation of c-fos mRNA indicates that prophylaxis with DON may prevent neurodegenerative changes induced by DFR Syt4 is also considered to be an lEG, since its induction does not depend on previous protein synthesis (Vician et al., 1995). However, Syt4 does not seem to be involved in the regulation of delayed gene response. Syt4 has rather been recognized to be implicated in phenotypic changes involving membrane trafficking (Glavan et al., 2009). Syt4 mRNA levels were not significantly affected by either DON or DFP. This suggests that in rats without seizures, the membrane trafficking may not be significantly affected by the cholinergic stimulation and that the increase of c-fos mRNA levels is a more rapid and sensitive marker for increased brain cholinergic activity.
 
So far, no data exist in the literature in regard to the prophylactic effects of DON against DFP-induced seizures. Syt4 mRNA is known to be strongly up-regulated in the brains of animals with seizures induced with glutamate analogue kainic acid (Glisovic et al., 2007). There is rising evidence for the pathophysiological and adaptive changes of membrane trafficking in neurodegeneration in neuronal plasticity and in glial activation induced by seizures, thus implicating the potential role of the plasticity of Syt4 expression in these processes.
 
So far, Syt4 mRNA levels (Tocco et al., 1996) and protein (Glisovic et al., 2007) were found to be up-regulated in the brain regions associated with the propagation of temporal lobe epilepsy only following kainic acid-induced seizures, and in hemi-seizures in rats after unilateral striatal injection of glutamate analogue and excitotoxin quinolinic acid (Glavan et al., 2009). Numerous studies have demonstrated that excitatory amino acid glutamate also plays a prominent role in the maintenance of organophosphate-induced seizures and in the subsequent neuropathology especially through over-activation of the N-methyl-D-aspartate (NMDA) receptor subtype (Lallement et al., 1999). We have shown previously that prevention of hemi-seizures in rats after unilateral striatal injection of glutamatergic NMDA agonist quinolinic acid by pre-treatment with NMDA antagonist dizolcipine (MK-801) prevented up-regulation of c-fos and Syt4 mRNAs (Glavan et al., 2009). In the present experiment, we also found that the prerequisite for the up-regulation of Syt4 mRNA was the glutamatergic hyperactivity due to the induction of seizures, and not just cholinergic hyperactivity by itself. The increase of cholinergic activity in DON-treated rats without seizures or in rats in which LiCIIDFP-induced seizures were prevented by DON namely did not result in increased Syt4 mRNA levels. It is noteworthy in this regard that DON has been shown to protect cortical neurons against glutamate-induced neurotoxicity and apoptotic death via nicotinic ACh receptors (Takada et al., 2003). It remains to be determined whether this may be an additional pharmacological mechanism by which DON could prevent the up-regulation of Syt4 mRNA in animals with seizures.
 
We conclude that the pre-treatment with DON protects the brain against the effects of central cholinergic and glutamatergic over-activity induced by DFP. This study has also revealed that up-regulation of Syt4 mRNA may be considered as a novel marker for increased membrane trafficking in animals with OP-induced seizures.
 
-Ogura H, Kosasa T, Kuriya Y, Yamanishi Y Central and peripheral activity of cholinesterase inhibitors as revealed by yawning and fasciculation in rats. Eur J Pharmacol. 2001;415(2-3):157-64