Absence of tolerance to the anticonvulsant and neuroprotective effects of imidazenil against DFP-induced seizure and neuronal damage Bashkim Kadriu a , James Gocel a , John Larson a , Alessandro Guidotti a , John M. Davis a , Madhusoodana P. Nambiar b , James Auta a, * a The Psychiatric Institute, Department of Psychiatry, College of Medicine, University of Illinois at Chicago,1601 W. Taylor St., Chicago, IL 60612, USA b Closed-Head Injury Branch, Center for Military Psychiatry and Neuroscience, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA article info Article history: Received 26 April 2011 Received in revised form 15 August 2011 Accepted 24 August 2011 Keywords: Imidazenil Diazepam Benzodiazepine Organophosphates Neuropathology Seizure Tolerance abstract The clinical use of diazepam or midazolam to control organophosphate (OP) nerve agent-induced seizure activity is limited by their unwanted effects including sedation, amnesia, withdrawal, and anticonvulsant tolerance. Imidazenil is an imidazo-benzodiazepine derivative with high intrinsic efficacy and selectivity for a2-, a3-, and a5- but low intrinsic efficacy for a1-containing GABA A receptors. We have previously shown that imidazenil is more efficacious than diazepam at protecting rats and mice from diisopropyl fluorophosphate (DFP)-induced seizures and neuronal damage without producing sedation. In the present study, we compared the tolerance liability of imidazenil and diazepam to attenuate the seizure activity and neurotoxic effects of DFP. Rats received protracted (14 days) oral treatment with increasing doses of imidazenil (1e4 mg/kg), diazepam (5e20 mg/kg), or vehicle. Eighteen hours after the last dose of the protracted treatment schedule, rats were tested for anticonvulsant tolerance after a 30 min pretreatment with a single test dose of imidazenil (0.5 mg/kg) or diazepam (5 mg/kg) prior to a DFP challenge (1.5 mg/kg). The anticonvulsant (modified Racine score scale) and neuroprotective (fluoro-jade B staining) effects of diazepam were significantly reduced in protracted diazepam-treated animals whereas the effects of imidazenil were not altered in protracted imidazenil-treated animals. The present findings indicate that protracted imidazenil treatment does not produce tolerance to its protective action against the neurotoxic effects of OP exposure. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction In an era of increased global risk from terrorist attacks, chemical warfare nerve agent (CWNA) exposure is of great concern (Holstege et al., 1997). Examples of the use of these agents in attacks targeting civilians and military in recent decades include the Japan subway attacks of 1995 (Tochigi et al., 2002; Okumura et al., 2003; Miyaki et al., 2005), the IraqieIranian war of 1998 (Brown and Brix, 1998), and the possible low-level exposure of the US military during the Gulf War in 1991 (Haley et al., 1997, 2000; Couzin, 2004). Moreover, our military combat personnel are confronted on a daily basis with the threat of CWNA exposure during active military operations. The deliberate use of nerve agents in these examples clearly explains why defense and health departments take the threat of CWNA attack or exposure very seriously (Brown and Brix, 1998; Lee, 2003). A major concern with CWNA exposure is the rapid development of self-sustaining status epilepticus which once established, is difficult to treat and is often refractory to all thera- pies (Martin et al., 1985; Mazarati et al., 1998; McDonough et al., 2010). The current medical countermeasures against CWNA, including organophosphate (OP) nerve agent poisons, consist of a pre- exposure treatment (when an attack is anticipated) with a revers- ible AChE inhibitor (usually pyridostigmine bromide) and post- exposure treatment with a muscarinic receptor antagonist [atro- pine sulfate (AT)] to counteract the acute cholinergic crisis. These treatments are usually accompanied by administration of an oxime [pyridine-2-aldoxime methochloride (2-PAM)] to reactivate CWNA-inhibited acetylcholinesterases. However, these treatments do not protect victims from intense CWNA-induced seizures. Hence, when a CWNA attack is anticipated, preventive treatment with an anticonvulsant benzodiazepine (BZ) [primarily diazepam (DZ) or midazolam (MDZ)] would be desirable to prevent the Abbreviations: GABA, g-aminobutyric acid; DFP, diisopropyl fluorophosphate; OP, organophosphates; CWNA, chemical warfare nerve agent; AT, atropine sulfate; 2-PAM, pyridine-2-aldoxime methochloride; BZ, benzodiazepine; IMD, imidazenil; DZ, diazepam; MDZ, midazolam; FJB, fluoro-jade B. * Corresponding author. The Psychiatric Institute, Department of Psychiatry, College of Medicine, University of Illinois,1601 W. Taylor St., Room 283, Chicago, IL 60612, USA. Tel.: þ1 312 355 5940; fax: þ1 312 413 4569. E-mail address: jauta@psych.uic.edu (J. Auta). Contents lists available at SciVerse ScienceDirect Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm 0028-3908/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2011.08.043 Neuropharmacology 61 (2011) 1463e1469