Preventing Long-Term Brain Damage by Nerve Agent-Induced Status Epilepticus in Rat Models Applicable to Infants: Significant Neuroprotection by Tezampanel Combined with Caramiphen but Not by Midazolam Treatment.

Acute exposure to nerve agents induces a peripheral cholinergic crisis and prolonged status epilepticus (SE), causing death or long-term brain damage. To provide preclinical data pertinent to the protection of infants and newborns, we compared the antiseizure and neuroprotective effects of treating soman-induced SE with midazolam (MDZ) versus tezampanel (LY293558) in combination with caramiphen (CRM) in 12- and 7-day-old rats. The anticonvulsants were administered 1 hour after soman exposure; neuropathology data were collected up to 6 months postexposure. In both ages, the total duration of SE within 24 hours after soman exposure was significantly shorter in the LY293558 plus CRM groups compared with the MDZ groups. Neuronal degeneration was substantial in the MDZ-treated groups but absent or minimal in the groups treated with LY293558 plus CRM. Loss of neurons and interneurons in the basolateral amygdala and CA1 hippocampal area was significant in the MDZ-treated groups but virtually absent in the LY293558 plus CRM groups. Atrophy of the amygdala and hippocampus occurred only in MDZ-treated groups. Neuronal/interneuronal loss and atrophy of the amygdala and hippocampus deteriorated over time. Reduction of inhibitory activity in the basolateral amygdala and increased anxiety were found only in MDZ groups. Spontaneous recurrent seizures developed in the MDZ groups, deteriorating over time; a small percentage of rats from the LY293558 plus CRM groups also developed seizures. These results suggest that brain damage can be long lasting or permanent if nerve agent-induced SE in infant victims is treated with midazolam at a delayed timepoint after SE onset, whereas antiglutamatergic treatment with tezampanel and caramiphen provides significant neuroprotection. SIGNIFICANCE STATEMENT: To protect the brain and the lives of infants in a mass exposure to nerve agents, an anticonvulsant treatment must be administered that will effectively stop seizures and prevent neuropathology, even if offered with a relative delay after seizure onset. The present study shows that midazolam, which was recently approved by the Food and Drug Administration for the treatment of nerve agent-induced status epilepticus, is not an effective neuroprotectant, whereas brain damage can be prevented by targeting glutamate receptors.

Delayed tezampanel and caramiphen treatment but not midazolam protects against long-term neuropathology after soman exposure.

Prolonged status epilepticus (SE) can cause brain damage; therefore, treatment must be administered promptly after seizure onset to limit SE duration and prevent neuropathology. Timely treatment of SE is not always feasible; this would be particularly true in a mass exposure to an SE-inducing agent such as a nerve agent. Therefore, the availability of anticonvulsant treatments that have neuroprotective efficacy even if administered with a delay after SE onset is an imperative. Here, we compared the long-term neuropathology resulting from acutely exposing 21-day-old male and female rats to the nerve agent soman, and treating them with midazolam (3 mg/kg) or co-administration of tezampanel (10 mg/kg) and caramiphen (50 mg/kg), at 1 h postexposure (~50 min after SE onset). Midazolam-treated rats had significant neuronal degeneration in limbic structures, mainly at one month postexposure, followed by neuronal loss in the basolateral amygdala and the CA1 hippocampal area. Neuronal loss resulted in significant amygdala and hippocampal atrophy, deteriorating from one to six months postexposure. Rats treated with tezampanel-caramiphen had no evidence of neuropathology, except for neuronal loss in the basolateral amygdala at the six-month timepoint. Anxiety was increased only in the midazolam-treated rats, at one, three, and six months postexposure. Spontaneous recurrent seizures appeared only in midazolam-treated rats, at three and six months postexposure in males and only at six months in females. These findings suggest that delayed treatment of nerve agent-induced SE with midazolam may result in long-lasting or permanent brain damage, while antiglutamatergic anticonvulsant treatment consisting of tezampanel and caramiphen may provide full neuroprotection.

Antiseizure and Neuroprotective Efficacy of Midazolam in Comparison with Tezampanel (LY293558) against Soman-Induced Status Epilepticus.

Acute exposure to nerve agents induces status epilepticus (SE), which can cause death or long-term brain damage. Diazepam is approved by the FDA for the treatment of nerve agent-induced SE, and midazolam (MDZ) is currently under consideration to replace diazepam. However, animal studies have raised questions about the neuroprotective efficacy of benzodiazepines. Here, we compared the antiseizure and neuroprotective efficacy of MDZ (5 mg/kg) with that of tezampanel (LY293558; 10 mg/kg), an AMPA/GluK1 receptor antagonist, administered 1 h after injection of the nerve agent, soman (1.2 × LD50), in adult male rats. Both of the anticonvulsants promptly stopped SE, with MDZ having a more rapid effect. However, SE reoccurred to a greater extent in the MDZ-treated group, resulting in a significantly longer total duration of SE within 24 h post-exposure compared with the LY293558-treated group. The neuroprotective efficacy of the two drugs was studied in the basolateral amygdala, 30 days post-exposure. Significant neuronal and inter-neuronal loss, reduced ratio of interneurons to the total number of neurons, and reduction in spontaneous inhibitory postsynaptic currents accompanied by increased anxiety were found in the MDZ-treated group. The rats treated with LY293558 did not differ from the control rats (not exposed to soman) in any of these measurements. Thus, LY293558 has significantly greater efficacy than midazolam in protecting against prolonged seizures and brain damage caused by acute nerve agent exposure.

Acetylcholinesterase inhibitors (nerve agents) as weapons of mass destruction: History, mechanisms of action, and medical countermeasures.

Nerve agents are organophosphorus acetylcholinesterase inhibitors. Acute exposure to nerve agents can cause rapid death. In this review, we summarize the history of nerve agent development and use in warfare, the mechanisms by which these agents cause death or long-term brain damage, and the treatments for preventing death or long-term morbidity. The G-series nerve agents, tabun, sarin, soman, ethyl sarin, and cyclosarin, were developed by the Nazis. VX, the best-known of the V-series agents, was synthesized in the 1950's by a British scientist. Little is known about the development of the novichoks (the "A-series") by the former Soviet Union. Nerve agents were used for the first time in the battlefield by the Iraqi government in the Iran-Iraq War, in the 1980s. The Chemical Weapons Convention, in 1993, banned all chemical weapons production and use, yet, sarin was subsequently used in terrorist attacks in Japan and, recently, in the war in Syria. Pyridostigmine has been used as a prophylactic treatment, and bioscavengers are presently investigated as a better alternative. Atropine, along with an oxime, can prevent rapid death from the nerve agent-induced peripheral cholinergic crisis. Treatment with diazepam or midazolam for the cessation of nerve agent-induced status epilepticus cannot protect against brain damage, and, therefore, these benzodiazepines should be replaced by novel anticonvulsants and neuroprotectants. The AMPA/GluK1 receptor antagonist LY293558 (tezampanel) has shown superior antiseizure and neuroprotective efficacy against soman, particularly when administered in combination with caramiphen, an antagonist of muscarinic and NMDA receptors. This article is part of the special issue entitled 'Acetylcholinesterase Inhibitors: From Bench to Bedside to Battlefield'.

Functional basis for dose-dependent antagonism of rat and rabbit neuromuscular transmission by the bis-pyridinium oxime MMB4.

Organophosphorus (OP) compounds inhibit central and peripheral acetylcholinesterase (AChE) activity, overstimulating cholinergic receptors and causing autonomic dysfunction (e.g., bronchoconstriction, excess secretions), respiratory impairment, seizure and death at high doses. Current treatment for OP poisoning in the United States includes reactivation of OP-inhibited AChE by the pyridinium oxime 2-pyridine aldoxime (2-PAM). However, 2-PAM has a narrow therapeutic index and its efficacy is confined to a limited number of OP agents. The bis-pyridinium oxime MMB4, which is a more potent reactivator than 2-PAM with improved pharmaceutical properties and therapeutic range, is under consideration as a potential replacement for 2-PAM. Similar to other pyridinium oximes, high doses of MMB4 lead to off-target effects culminating in respiratory depression and death. To understand the toxic mechanisms contributing to respiratory depression, we evaluated the effects of MMB4 (0.25-16 mM) on functional and neurophysiological parameters of diaphragm and limb muscle function in rabbits and rats. In both species, MMB4 depressed nerve-elicited muscle contraction by blocking muscle endplate nicotinic receptor currents while simultaneously prolonging endplate potentials by inhibiting AChE. MMB4 increased quantal content, endplate potential rundown and tetanic fade during high frequency stimulation in rat but not rabbit muscles, suggesting species-specific effects on feedback mechanisms involved in sustaining neurotransmission. These data reveal multifactorial effects of MMB4 on cholinergic neurotransmission, with the primary toxic modality being reduced muscle nicotinic endplate currents. Evidence of species-specific effects on neuromuscular function illustrates the importance of comparative toxicology when studying pyridinium oximes and, by inference, other quaternary ammonium compounds.

Electroencephalographic analysis in soman-exposed 21-day-old rats and the effects of midazolam or LY293558 with caramiphen.

Acute nerve agent exposure induces status epilepticus (SE), which can cause brain damage or death. Research aiming at developing effective therapies for controlling nerve agent-induced SE is commonly performed in adult rats. The characteristics of nerve agent-induced SE in young rats are less clear; relevant knowledge is necessary for developing effective pediatric therapies. Here, we have used electroencephalographic (EEG) recordings and analysis to study seizures in postnatal day 21 rats exposed to 1.2 × LD50 of soman, and compared the antiseizure efficacy of midazolam (MDZ)-currently considered by the Food and Drug Administration to replace diazepam for treating SE in victims of nerve agent exposure-with that of LY293558, an AMPA/GluK1 receptor antagonist, administered in combination with caramiphen, an antimuscarinic with N-methyl-d-aspartate receptor antagonistic properties. Prolonged SE developed in 80% of the rats and was reflected in behavioral seizures/convulsions. Both MDZ and LY293558 + caramiphen stopped the SE induced by soman, but there was a significant recurrence of seizures within 24 h postexposure only in the MDZ-treated group, as revealed in the raw EEG data and their representation in the frequency domain using a fast Fourier transform and in spectral analysis over 24 hours. In contrast to the high efficacy of LY293558 + caramiphen, MDZ is not an effective treatment for SE induced by soman in young animals.

Targeting the glutamatergic system to counteract organophosphate poisoning: A novel therapeutic strategy.

One of the devastating effects of acute exposure to organophosphates, like nerve agents, is the induction of severe and prolonged status epilepticus (SE), which can cause death, or brain damage if death is prevented. Seizures after exposure are initiated by muscarinic receptor hyperstimulation-after inhibition of acetylcholinesterase by the organophosphorus agent and subsequent elevation of acetylcholine-but they are reinforced and sustained by glutamatergic hyperexcitation, which is the primary cause of brain damage. Diazepam is the FDA-approved anticonvulsant for the treatment of nerve agent-induced SE, and its replacement by midazolam is currently under consideration. However, clinical data derived from the treatment of SE of any etiology, as well as studies on the control of nerve agent-induced SE in animal models, have indicated that diazepam and midazolam control seizures only temporarily, their antiseizure efficacy is reduced as the latency of treatment from the onset of SE increases, and their neuroprotective efficacy is limited or absent. Here, we review data on the discovery of a novel anticonvulsant and neuroprotectant, LY293558, an AMPA/GluK1 receptor antagonist. Treatment of soman-exposed immature, young-adult, and aged rats with LY293558, terminates SE with limited recurrence of seizures, significantly protects from brain damage, and prevents long-term behavioral deficits, even when LY293558 is administered 1 h post-exposure. More beneficial effects and complete neuroprotection is obtained when LY293558 administration is combined with caramiphen, which antagonizes NMDA receptors. Further efficacy studies may bring the LY293558 + caramiphen combination therapy on the pathway to approval for human use.

Acute and long-term consequences of exposure to organophosphate nerve agents in humans.

Nerve agents are organophosphate (OP) compounds and among the most powerful poisons known to man. A terrorist attack on civilian or military populations causing mass casualties is a real threat. The OP nerve agents include soman, sarin, cyclosarin, tabun, and VX. The major mechanism of acute toxicity is the irreversible inhibition of acetylcholinesterase. Acetylcholinesterase inhibition results in the accumulation of excessive acetylcholine levels in synapses, leading to progression of toxic signs including hypersecretions, tremors, status epilepticus, respiratory distress, and death. Miosis and rhinorrhea are the most common clinical findings in those individuals acutely exposed to OP nerve agents. Prolonged seizures are responsible for the neuropathology. The brain region that shows the most severe damage is the amygdala, followed by the piriform cortex, hippocampus, cortex, thalamus, and caudate/putamen. Current medical countermeasures are only modestly effective in attenuating the seizures and neuropathology. Anticonvulsants such as benzodiazepines decrease seizure activity and improve outcome, but their efficacy depends upon the administration time after exposure to the nerve agent. Administration of benzodiazepines may increase the risk for seizure recurrence. Recent studies document long-term neurologic and behavior deficits, and technological advances demonstrate structural brain changes on magnetic resonance imaging.

Full Protection Against Soman-Induced Seizures and Brain Damage by LY293558 and Caramiphen Combination Treatment in Adult Rats.

Acute exposure to nerve agents induces status epilepticus (SE), which causes brain damage or death. LY293558, an antagonist of AMPA and GluK1 kainate receptors is a very effective anticonvulsant and neuroprotectant against soman; however, some neuronal damage is still present after treatment of soman-exposed rats with LY293558. Here, we have tested whether combining LY293558 with an NMDA receptor antagonist can eliminate the residual damage. For this purpose, we chose caramiphen (CRM), an antimuscarinic compound with NMDA receptor antagonistic properties. Adult male rats were exposed to 1.2 × LD50 soman, and at 20 min after soman exposure, were injected with atropine + HI-6, or atropine + HI-6 + LY293558 (15 mg/kg), or atropine + HI-6 + LY293558 + CRM (50 mg/kg). We found that (1) the LY293558 + CRM treatment terminated SE significantly faster than LY293558 alone; (2) after cessation of the initial SE, seizures did not return in the LY293558 + CRM-treated group, during 72 h of monitoring; (3) power spectrum analysis of continuous EEG recordings for 7 days post-exposure showed increased delta and decreased gamma power that lasted beyond 24 h post-exposure only in the rats who did not receive anticonvulsant treatment; (4) spontaneous recurrent seizures appeared on day 7 only in the group that did not receive anticonvulsant treatment; (5) significant neuroprotection was achieved by LY293558 administration, while the rats who received LY293558 + CRM displayed no neurodegeneration; (6) body weight loss and recovery in the LY293558 + CRM-treated rats did not differ from those in control rats who were not exposed to soman. The data show that treatment with LY293558 + CRM provides full antiseizure and neuroprotective efficacy against soman.

Comparing the Antiseizure and Neuroprotective Efficacy of LY293558, Diazepam, Caramiphen, and LY293558-Caramiphen Combination against Soman in a Rat Model Relevant to the Pediatric Population.

The currently Food and Drug Administration-approved anticonvulsant for the treatment of status epilepticus (SE) induced by nerve agents is the benzodiazepine diazepam; however, diazepam does not appear to offer neuroprotective benefits. This is of particular concern with respect to the protection of children because, in the developing brain, synaptic transmission mediated via GABAA receptors, the target of diazepam, is weak. In the present study, we exposed 21-day-old male rats to 1.2 × LD50 soman and compared the antiseizure, antilethality, and neuroprotective efficacy of diazepam (10 mg/kg), LY293558 (an AMPA/GluK1 receptor antagonist; 15 mg/kg), caramiphen (CRM, an antimuscarinic with NMDA receptor-antagonistic properties; 50 mg/kg), and LY293558 (15 mg/kg) + CRM (50 mg/kg), administered 1 hour after exposure. Diazepam, LY293558, and LY293558 + CRM, but not CRM alone, terminated SE; LY293558 + CRM treatment acted significantly faster and produced a survival rate greater than 85%. Thirty days after soman exposure, neurodegeneration in limbic regions was most severe in the CRM-treated group, minimal to severe-depending on the region-in the diazepam group, absent to moderate in the LY293558-treated group, and totally absent in the LY293558 + CRM group. Amygdala and hippocampal atrophy, a severe reduction in spontaneous inhibitory activity in the basolateral amygdala, and increased anxiety-like behavior in the open-field and acoustic startle response tests were present in the diazepam and CRM groups, whereas the LY293558 and LY293558 + CRM groups did not differ from controls. The combined administration of LY293558 and CRM, by blocking mainly AMPA, GluK1, and NMDA receptors, is a very effective anticonvulsant and neuroprotective therapy against soman in young rats.

Susceptibility to Soman Toxicity and Efficacy of LY293558 Against Soman-Induced Seizures and Neuropathology in 10-Month-Old Male Rats.

Acute nerve agent exposure causes prolonged status epilepticus (SE), leading to death or long-term brain damage. We have previously demonstrated that LY293558, an AMPA/GluK1 kainate receptor antagonist, terminates SE induced by the nerve agent soman and protects from long-term brain damage, in immature rats and young-adult rats, even if administered with a relatively long latency from the time of exposure. However, susceptibility to the lethal consequences of SE increases with age, and mortality by SE induced by soman is substantially greater in older animals. Therefore, in the present study, we compared the susceptibility to soman toxicity of 10-month-old male rats with that of young-adult male rats (42 to 50 days old) and examined the protective efficacy of LY293558 in the older group. A lower percentage of the 10-month-old rats developed SE after injection of 1.2 × LD50 soman, compared to the young adults, the latency to seizure onset was longer in the older rats, and seizure intensity did not differ between the two age groups. However, mortality rate in the older rats who developed SE was higher than in the young adults. Acetylcholinesterase activity in the amygdala, hippocampus, and piriform cortex did not differ between the two age groups. Administration of LY293558 at 20 or 60 min post-exposure suppressed SE, increased 24-h survival rate, decreased the long-term risk of death, reduced neuronal degeneration in the amygdala, hippocampus, piriform, and entorhinal cortices, and facilitated recovery from body weight loss. Thus, LY293558 is an effective countermeasure against soman toxicity also in older animals.

The M1 Muscarinic Receptor Antagonist VU0255035 Delays the Development of Status Epilepticus after Organophosphate Exposure and Prevents Hyperexcitability in the Basolateral Amygdala.

Exposure to organophosphorus toxins induces seizures that progress to status epilepticus (SE), which can cause brain damage or death. Seizures are generated by hyperstimulation of muscarinic receptors, subsequent to inhibition of acetylcholinesterase; this is followed by glutamatergic hyperactivity, which sustains and reinforces seizure activity. It has been unclear which muscarinic receptor subtypes are involved in seizure initiation and the development of SE in the early phases after exposure. Here, we show that pretreatment of rats with the selective M1 receptor antagonist, VU0255035 [N-(3-oxo-3-(4-(pyridine-4-yl)piperazin-1-yl)propyl)-benzo[c][1,2,5]thiadiazole-4 sulfonamide], significantly suppressed seizure severity and prevented the development of SE for about 40 minutes after exposure to paraoxon or soman, suggesting an important role of the M1 receptor in the early phases of seizure generation. In addition, in in vitro brain slices of the basolateral amygdala (a brain region that plays a key role in seizure initiation after nerve agent exposure), VU0255035 blocked the effects produced by bath application of paraoxon-namely, a brief barrage of spontaneous inhibitory postsynaptic currents, followed by a significant increase in the ratio of the total charge transferred by spontaneous excitatory postsynaptic currents over that of the inhibitory postsynaptic currents. Furthermore, paraoxon enhanced the hyperpolarization-activated cation current Ih in basolateral amygdala principal cells, which could be one of the mechanisms underlying the increased glutamatergic activity, an effect that was also blocked in the presence of VU0255035. Thus, selective M1 antagonists may be an efficacious pretreatment in contexts in which there is risk for exposure to organophosphates, as these antagonists will delay the development of SE long enough for medical assistance to arrive.

Long-term neuropathological and behavioral impairments after exposure to nerve agents.

One of the deleterious effects of acute nerve agent exposure is the induction of status epilepticus (SE). If SE is not controlled effectively, it causes extensive brain damage. Here, we review the neuropathology observed after nerve agent-induced SE, as well as the ensuing pathophysiological, neurological, and behavioral alterations, with an emphasis on their time course and longevity. Limbic structures are particularly vulnerable to damage by nerve agent exposure. The basolateral amygdala (BLA), which appears to be a key site for seizure initiation upon exposure, suffers severe neuronal loss; however, GABAergic BLA interneurons display a delayed death, perhaps providing a window of opportunity for rescuing intervention. The end result is a long-term reduction of GABAergic activity in the BLA, with a concomitant increase in spontaneous excitatory activity; such pathophysiological alterations are not observed in the CA1 hippocampal area, despite the extensive neuronal loss. Hyperexcitability in the BLA may be at least in part responsible for the development of recurrent seizures and increased anxiety, while hippocampal damage may underlie the long-term memory impairments. Effective control of SE after nerve agent exposure, such that brain damage is also minimized, is paramount for preventing lasting neurological and behavioral deficits.

Alpha-Linolenic Acid-Induced Increase in Neurogenesis is a Key Factor in the Improvement in the Passive Avoidance Task After Soman Exposure.

Exposure to organophosphorous (OP) nerve agents such as soman inhibits the critical enzyme acetylcholinesterase (AChE) leading to excessive acetylcholine accumulation in synapses, resulting in cholinergic crisis, status epilepticus and brain damage in survivors. The hippocampus is profoundly damaged after soman exposure leading to long-term memory deficits. We have previously shown that treatment with three sequential doses of alpha-linolenic acid, an essential omega-3 polyunsaturated fatty acid, increases brain plasticity in naïve animals. However, the effects of this dosing schedule administered after a brain insult and the underlying molecular mechanisms in the hippocampus are unknown. We now show that injection of three sequential doses of alpha-linolenic acid after soman exposure increases the endogenous expression of mature BDNF, activates Akt and the mammalian target of rapamycin complex 1 (mTORC1), increases neurogenesis in the subgranular zone of the dentate gyrus, increases retention latency in the passive avoidance task and increases animal survival. In sharp contrast, while soman exposure also increases mature BDNF, this increase did not activate downstream signaling pathways or neurogenesis. Administration of the inhibitor of mTORC1, rapamycin, blocked the alpha-linolenic acid-induced neurogenesis and the enhanced retention latency but did not affect animal survival. Our results suggest that alpha-linolenic acid induces a long-lasting neurorestorative effect that involves activation of mTORC1 possibly via a BDNF-TrkB-mediated mechanism.

A rat model of nerve agent exposure applicable to the pediatric population: The anticonvulsant efficacies of atropine and GluK1 antagonists.

Inhibition of acetylcholinesterase (AChE) after nerve agent exposure induces status epilepticus (SE), which causes brain damage or death. The development of countermeasures appropriate for the pediatric population requires testing of anticonvulsant treatments in immature animals. In the present study, exposure of 21-day-old (P21) rats to different doses of soman, followed by probit analysis, produced an LD50 of 62µg/kg. The onset of behaviorally-observed SE was accompanied by a dramatic decrease in brain AChE activity; rats who did not develop SE had significantly less reduction of AChE activity in the basolateral amygdala than rats who developed SE. Atropine sulfate (ATS) at 2mg/kg, administered 20 min after soman exposure (1.2×LD50), terminated seizures. ATS at 0.5mg/kg, given along with an oxime within 1 min after exposure, allowed testing of anticonvulsants at delayed time-points. The AMPA/GluK1 receptor antagonist LY293558, or the specific GluK1 antagonist UBP302, administered 1h post-exposure, terminated SE. There were no degenerating neurons in soman-exposed P21 rats, but both the amygdala and the hippocampus were smaller than in control rats at 30 and 90days post-exposure; this pathology was not present in rats treated with LY293558. Behavioral deficits present at 30 days post-exposure, were also prevented by LY293558 treatment. Thus, in immature animals, a single injection of atropine is sufficient to halt nerve agent-induced seizures, if administered timely. Testing anticonvulsants at delayed time-points requires early administration of ATS at a low dose, sufficient to counteract only peripheral toxicity. LY293558 administered 1h post-exposure, prevents brain pathology and behavioral deficits.

LY293558 prevents soman-induced pathophysiological alterations in the basolateral amygdala and the development of anxiety.

Exposure to nerve agents can cause brain damage due to prolonged seizure activity, producing long-term behavioral deficits. We have previously shown that LY293558, a GluK1/AMPA receptor antagonist, is a very effective anticonvulsant and neuroprotectant against nerve agent exposure. In the present study, we examined whether the protection against nerve agent-induced seizures and neuropathology conferred by LY293558 translates into protection against pathophysiological alterations in the basolateral amygdala (BLA) and the development of anxiety, which is the most prevalent behavioral deficit resulting from exposure. LY293558 (15 mg/kg) was administered to rats, along with atropine and HI-6, at 20 min after exposure to soman (1.2 × LD50). At 24 h, 7 days, and 30 days after exposure, soman-exposed rats who did not receive LY293558 had reduced but prolonged evoked field potentials in the BLA, as well as increased paired-pulse ratio, suggesting neuronal damage and impaired synaptic inhibition; rats who received LY293558 did not differ from controls in these parameters. Long-term potentiation of synaptic transmission was impaired at 7 days after exposure in the soman-exposed rats who did not receive anticonvulsant treatment, but not in the LY293558-treated rats. Anxiety-like behavior assessed by the open field and acoustic startle response tests was increased in the soman-exposed rats at 30 and 90 days after exposure, while rats treated with LY293558 did not differ from controls. Along with our previous findings, the present data demonstrate the remarkable efficacy of LY293558 in counteracting nerve agent-induced seizures, neuropathology, pathophysiological alterations in the BLA, and anxiety-related behavioral deficits.

Pathophysiological mechanisms underlying increased anxiety after soman exposure: reduced GABAergic inhibition in the basolateral amygdala.

The recent sarin attack in Syria killed 1429 people, including 426 children, and left countless more to deal with the health consequences of the exposure. Prior to the Syrian chemical assault, nerve agent attacks in Japan left many victims suffering from neuropsychiatric illnesses, particularly anxiety disorders, more than a decade later. Uncovering the neuro-pathophysiological mechanisms underlying the development of anxiety after nerve agent exposure is necessary for successful treatment. Anxiety is associated with hyperexcitability of the basolateral amygdala (BLA). The present study sought to determine the nature of the nerve agent-induced alterations in the BLA, which could explain the development of anxiety. Rats were exposed to soman, at a dose that induced prolonged status epilepticus. Twenty-four hours and 14-days after exposure, neurons from the BLA were recorded using whole-cell patch-clamp techniques. At both the 24h and 14-day post-exposure time-points, the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) in the BLA were reduced, along with reduction in the frequency but not amplitude of miniature IPSCs. In addition, activation of α7-nicotinic acetylcholine receptors, a cholinergic receptor that participates in the regulation of BLA excitability and is involved in anxiety, increased spontaneous excitatory postsynaptic currents (sEPSCs) in both soman-exposed rats and controls, but was less effective in increasing sIPSCs in soman-exposed rats. Despite the loss of both interneurons and principal cells after soman-induced status epilepticus, the frequency of sEPSCs was increased in the soman-exposed rats. Impaired function and cholinergic modulation of GABAergic inhibition in the BLA may underlie anxiety disorders that develop after nerve agent exposure.

The limitations of diazepam as a treatment for nerve agent-induced seizures and neuropathology in rats: comparison with UBP302.

Exposure to nerve agents induces prolonged status epilepticus (SE), causing brain damage or death. Diazepam (DZP) is the current US Food and Drug Administration-approved drug for the cessation of nerve agent-induced SE. Here, we compared the efficacy of DZP with that of UBP302 [(S)-3-(2-carboxybenzyl)willardiine; an antagonist of the kainate receptors that contain the GluK1 subunit] against seizures, neuropathology, and behavioral deficits induced by soman in rats. DZP, administered 1 hour or 2 hours postexposure, terminated the SE, but seizures returned; thus, the total duration of SE within 24 hours after soman exposure was similar to (DZP at 1 hour) or longer than (DZP at 2 hours) that in the soman-exposed rats that did not receive the anticonvulsant. Compared with DZP, UBP302 stopped SE with a slower time course, but dramatically reduced the total duration of SE within 24 hours. Neuropathology and behavior were assessed in the groups that received anticonvulsant treatment 1 hour after exposure. UBP302, but not DZP, reduced neuronal degeneration in a number of brain regions, as well as neuronal loss in the basolateral amygdala and the CA1 hippocampal area, and prevented interneuronal loss in the basolateral amygdala. Anxiety-like behavior was assessed in the open field and by the acoustic startle response 30 days after soman exposure. The results showed that anxiety-like behavior was increased in the DZP-treated group and in the group that did not receive anticonvulsant treatment, but not in the UBP302-treated group. The results argue against the use of DZP for the treatment of nerve agent-induced seizures and brain damage and suggest that targeting GluK1-containing receptors is a more effective approach.

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.

Organophosphorus nerve agents are powerful neurotoxins that irreversibly inhibit acetylcholinesterase (AChE) activity. One of the consequences of AChE inhibition is the generation of seizures and status epilepticus (SE), which cause brain damage, resulting in long-term neurological and behavioral deficits. Increased anxiety is the most common behavioral abnormality after nerve agent exposure. This is not surprising considering that the amygdala, and the basolateral nucleus of the amygdala (BLA) in particular, plays a central role in anxiety, and this structure suffers severe damage by nerve agent-induced seizures. In the present study, we exposed male rats to the nerve agent soman, at a dose that induce SE, and determined the time course of recovery of AChE activity, along with the progression of neuropathological and pathophysiological alterations in the BLA, during a 30-day period after exposure. Measurements were taken at 24 h, 7 days, 14 days, and 30 days after exposure, and at 14 and 30 days, anxiety-like behavior was also evaluated. We found that more than 90% of AChE is inhibited at the onset of SE, and AChE inhibition remains at this level 24 h later, in the BLA, as well as in the hippocampus, piriform cortex, and prelimbic cortex, which we analyzed for comparison. AChE activity recovered by day 7 in the BLA and day 14 in the other three regions. Significant neuronal loss and neurodegeneration were present in the BLA at 24 h and throughout the 30-day period. There was no significant loss of GABAergic interneurons in the BLA at 24 h post-exposure. However, by day 7, the number of GABAergic interneurons in the BLA was reduced, and at 14 and 30 days after soman, the ratio of GABAergic interneurons to the total number of neurons was lower compared to controls. Anxiety-like behavior in the open-field and the acoustic startle response tests was increased at 14 and 30 days post-exposure. Accompanying pathophysiological alterations in the BLA - studied in in vitro brain slices - included a reduction in the amplitude of field potentials evoked by stimulation of the external capsule, along with prolongation of their time course and an increase in the paired-pulse ratio. Long-term potentiation was impaired at 24 h, 7 days, and 14 days post-exposure. The loss of GABAergic interneurons in the BLA and the decreased interneuron to total number of neurons ratio may be the primary cause of the development of anxiety after nerve agent exposure.

Acetylcholinesterase inhibition in the basolateral amygdala plays a key role in the induction of status epilepticus after soman exposure.

Exposure to nerve agents induces intense seizures (status epilepticus, SE), which cause brain damage or death. Identification of the brain regions that are critical for seizure initiation after nerve agent exposure, along with knowledge of the physiology of these regions, can facilitate the development of pretreatments and treatments that will successfully prevent or limit the development of seizures and brain damage. It is well-established that seizure initiation is due to excessive cholinergic activity triggered by the nerve agent-induced irreversible inhibition of acetylcholinesterase (AChE). Therefore, the reason that when animals are exposed to lethal doses of a nerve agent, a small proportion of these animals do not develop seizures, may have to do with failure of the nerve agent to inhibit AChE in brain areas that play a key role in seizure initiation and propagation. In the present study, we compared AChE activity in the basolateral amygdala (BLA), hippocampus, and piriform cortex of rats that developed SE (SE rats) after administration of the nerve agent soman (154µg/kg) to AChE activity in these brain regions of rats that received the same dose of soman but did not develop SE (no-SE rats). The levels of AChE activity were measured at the onset of SE in SE rats, 30min after soman administration in no-SE rats, as well as in controls which received saline in place of soman. In the control group, AChE activity was significantly higher in the BLA compared to the hippocampus and piriform cortex. Compared to controls, AChE activity was dramatically lower in the hippocampus and the piriform cortex of both the SE rats and the no-SE rats, but AChE activity in the BLA was reduced only in the SE rats. Consistent with the notion that soman-induced neuropathology is due to intense seizures, rather than due to a direct neurotoxic effect of soman, no-SE rats did not present any neuronal loss or degeneration, 7 days after exposure. The results suggest that inhibition of AChE activity in the BLA is necessary for the generation of seizures after nerve agent exposure, and provide strong support to the view that the amygdala is a key brain region for the induction of seizures by nerve agents.