The recovery of acetylcholinesterase activity and the progression of neuropathological and pathophysiological alterations in the rat basolateral amygdala after soman-induced status epilepticus: Relation to anxiety-like behavior
Introduction
Nerve agents are organophosphorus compounds that exert their toxic effects by rapidly and irreversibly inhibiting acetylcholinesterase (AChE) activity (Bajgar, 1997, Bajgar et al., 2008, Hajek et al., 2004, Shih et al., 2005, Sirin et al., 2012). In the brain, the resulting increase of acetylcholine and overstimulation of cholinergic receptors raises the excitation level to the point of seizure generation, and gives way to excessive activation of the glutamatergic system, which sustains and intensifies seizure activity (status epilepticus, SE), causing profound brain damage (McDonough and Shih, 1997, Petras, 1994). If death is prevented, long-term behavioral impairments may ensue due to brain pathologies (Coubard et al., 2008, Filliat et al., 2007).
Five years after the attacks with the nerve agent sarin in Matsumoto and Tokyo, individuals exposed to sarin reported persistent increases in symptoms that characterize anxiety disorders, including irritability and restlessness, avoidance of places that triggered recollection of the trauma, tension, and insomnia (Ohtani et al., 2004, Yanagisawa et al., 2006). Electrographic abnormalities indicative of epileptic activity were also present in exposed individuals (Murata et al., 1997, Nishiwaki et al., 2001, Yanagisawa et al., 2006). In animal models, exposure to nerve agents also results in long-term increases in anxiety and fear-like behaviors (Coubard et al., 2008, Filliat et al., 2007, Langston et al., 2012, Mamczarz et al., 2010, Moffett et al., 2011), as well as in the appearance of spontaneous recurrent seizures (de Araujo Furtado et al., 2010). The progression of pathological and pathophysiological alterations leading to these persistent behavioral and neurological abnormalities has not yet been elucidated. A better understanding of these alterations and the time course of their occurrence may allow for the development of treatment interventions that will prevent or minimize long-term neurological and behavioral deficits.
The amygdala is well recognized for its central role in emotional behavior (Phelps and LeDoux, 2005), and the basolateral nucleus of the amygdala (BLA), in particular, is closely associated with the generation and expression of anxiety and fear (Davis et al., 1994, Etkin and Wager, 2007, LeDoux, 2003); a common feature of anxiety disorders is hyperexcitability in the BLA (Rauch et al., 2006, Villarreal and King, 2001). In addition, evidence points to the BLA as a key brain region for seizure initiation and propagation after nerve agent poisoning (McDonough et al., 1987), and we recently found that after exposure to the nerve agent soman, SE is induced only when AChE activity is sufficiently inhibited in the BLA (Prager et al., 2013). The BLA is also one of the most severely damaged regions after nerve agent-induced SE (Apland et al., 2010, Aroniadou-Anderjaska et al., 2009, Baille et al., 2005, Carpentier et al., 2000, Figueiredo et al., 2011b, Shih et al., 2003).
Since inhibition of AChE is the primary mechanism of nerve agent poisoning, and sustained cholinergic dysregulation of the BLA may alter its excitability contributing to behavioral abnormalities, in the present study we examined the time course of recovery of AChE activity in the BLA; for comparison with other brain regions that may play an important role in seizure generation and propagation after nerve agent exposure (Myhrer, 2007), we also measured AChE activity in the piriform cortex, hippocampus, and prelimbic cortex. In addition, we investigated the progression of neuronal loss and degeneration in the BLA, during a 30-day period after soman exposure. To gain insight into the impact of the neuropathology on emotional behavior, we tested for the presence of increased anxiety at 14 and 30 days after soman-induced SE, and correlated the behavioral observations with synaptic alterations in the BLA, at the same time points.
Section snippets
Animal model
Experiments were performed using 6-week old (150–200 g) male, Sprague-Dawley rats (Taconic Farms, Derwood, MD). Animals were individually housed in an environmentally controlled room (20–23 °C, ∼44% humidity, 12-h light/12-h dark cycle [350–400 lux], lights on at 6:00 am), with food (Harlan Teklad Global Diet 2018, 18% protein rodent diet; Harlan Laboratories; Indianapolis, IN) and water available ad libitum. Cages were cleaned weekly and animal handling was minimized to reduce animal stress (
Results
Soman was administered to 161 rats. The average latency to Stage 3 seizure onset (SE onset) was 8.7 ± 1.2 min. Of the surviving 104 rats that developed prolonged SE (see Apland et al., 2013; Figueiredo et al., 2011a, Figueiredo et al., 2011b), 51 rats were used to measure AChE activity, and 31 rats were used for electrophysiological experiments. From the remaining 22 soman-exposed rats, 5 rats were used for evaluation of neuropathology at 24-h after soman exposure, 6 rats were used for
Discussion
After exposure of rats to lethal doses of soman, we found that more than 90% of AChE is inhibited at the onset of SE in brain regions that play a key role in seizure generation, and it takes up to two weeks for AChE activity to recover to normal levels. Significant neuronal loss and neurodegeneration was present in the BLA throughout the time period examined, from 24 h to 30 days after soman exposure. Interestingly, at the 24-h time point, there was no significant loss of GABAergic
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This work was supported by the CounterACT Program, National Institutes of Health, Office of the Director and the National Institute of Neurologic Disorders and Stroke [Grant Number 5U01NS058162-07], and the Defense Threat Reduction Agency-Joint Science and Technology Office, Medical S&T Division [Grant Numbers CBM.NEURO.01.10.US.18 and CBM.NEURO.01.10.US.15].
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