title: Combined treatment with diazepam and allopregnanolone reverses tetramethylenedisulfotetramine (TETS)-induced calcium dysregulation in cultured neurons and protects TETS-intoxicated mice against lethal seizures.
abstract: Tetramethylenedisulfotetramine (TETS) is a potent convulsant GABAA receptor blocker. Mice receiving a lethal dose of TETS (0.15 mg/kg i.p.) are rescued from death by a high dose of diazepam (5 mg/kg i.p.) administered shortly after the second clonic seizure (∼20 min post-TETS). However, this high dose of diazepam significantly impairs blood pressure and mobility, and does not prevent TETS-induced neuroinflammation in the brain. We previously demonstrated that TETS alters synchronous Ca(2+) oscillations in primary mouse hippocampal neuronal cell cultures and that pretreatment with the combination of diazepam and allopregnanolone at concentrations having negligible effects individually prevents TETS effects on intracellular Ca(2+) dynamics. Here, we show that treatment with diazepam and allopregnanolone (0.1 μM) 20 min after TETS challenge normalizes synchronous Ca(2+) oscillations when added in combination but not when added singly. Similarly, doses (0.03-0.1 mg/kg i.p.) of diazepam and allopregnanolone that provide minimal protection when administered singly to TETS intoxicated mice increase survival from 10% to 90% when given in combination either 10 min prior to TETS or following the second clonic seizure. This therapeutic combination has negligible effects on blood pressure or mobility. Combined treatment with diazepam and allopregnanolone also decreases TETS-induced microglial activation. Diazepam and allopregnanolone have distinct actions as positive allosteric modulators of GABAA receptors that in combination enhance survival and mitigate neuropathology following TETS intoxication without the adverse side effects associated with high dose benzodiazepines. Combination therapy with a benzodiazepine and neurosteroid represents a novel neurotherapeutic strategy with potentially broad application.
authors.affiliation: Department of Molecular Biosciences, School of Veterinary Medicine, Davis, CA 95616, USA.
authors.forename: Donald A
authors.initials: DA
authors.lastname: Bruun
authors.affiliation: Department of Molecular Biosciences, School of Veterinary Medicine, Davis, CA 95616, USA.
authors.forename: Zhengyu
authors.initials: Z
authors.lastname: Cao
authors.affiliation: Department of Entomology, College of Agricultural and Environmental Sciences, University of California, Davis, Davis, CA 95616, USA.
authors.forename: Bora
authors.initials: B
authors.lastname: Inceoglu
authors.affiliation: Department of Entomology, College of Agricultural and Environmental Sciences, University of California, Davis, Davis, CA 95616, USA.
authors.forename: Stephen T
authors.initials: ST
authors.lastname: Vito
authors.affiliation: Department of Pediatrics, Sacramento, CA 95817, USA.
authors.forename: Adam T
authors.initials: AT
authors.lastname: Austin
authors.affiliation: Department of Molecular Biosciences, School of Veterinary Medicine, Davis, CA 95616, USA.
authors.forename: Susan
authors.initials: S
authors.lastname: Hulsizer
authors.affiliation: Department of Entomology, College of Agricultural and Environmental Sciences, University of California, Davis, Davis, CA 95616, USA; Comprehensive Cancer Center, Sacramento, CA 95817, USA.
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Hammock
authors.affiliation: Department of Pediatrics, Sacramento, CA 95817, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Tancredi
authors.affiliation: Department of Neurology, School of Medicine, University of California, Davis, Sacramento, CA 95817, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Rogawski
authors.affiliation: Department of Molecular Biosciences, School of Veterinary Medicine, Davis, CA 95616, USA.
authors.forename: Isaac N
authors.initials: IN
authors.lastname: Pessah
authors.affiliation: Department of Molecular Biosciences, School of Veterinary Medicine, Davis, CA 95616, USA. Electronic address: pjlein@ucdavis.edu.
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: Lein
mesh_terms: D000818:Animals; D000927:Anticonvulsants / Q000494:pharmacology*; D001952:Bridged-Ring Compounds; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003975:Diazepam / Q000494:pharmacology*; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004359:Drug Therapy, Combination; D006624:Hippocampus / Q000187:drug effects / Q000473:pathology / Q000503:physiopathology; D008297:Male; D051379:Mice; D015213:Neuroimmunomodulation / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000187:drug effects / Q000473:pathology / Q000502:physiology; D011280:Pregnanolone / Q000494:pharmacology*; D012640:Seizures / Q000188:drug therapy* / Q000473:pathology / Q000503:physiopathology
chemical_list: D000927:Anticonvulsants; D001952:Bridged-Ring Compounds; D011280:Pregnanolone; C010349:tetramethylenedisulfotetramine; D003975:Diazepam; D002118:Calcium
keywords: Allopregnanolone; Allopregnanolone (PubChem CID: 262961); Combination therapy; Diazepam; Diazepam (PubChem CID: 3016); Neurosteroid; Seizures; Tetramethylenedisulfotetramine; Tetramethylenedisulfotetramine (PubChem CID: 64148)