Mitigation of adverse behavioral impact from predator exposure by the nociceptin/orphanin FQ peptide antagonist J-113397 in rats.

The nociceptin/orphanin FQ peptide (NOP) receptor is believed to have an integral modulatory function in the stress response system. We evaluated the highly selective NOP antagonist J-113397 (7.5 and 20.0 mg/kg), using a predator exposure in which rats were exposed to predator cats as a stressor. A single dose of J-113397 or vehicle was administered (intraperitoneally) shortly before exposure to the predators or a sham exposure. Behavioral impact was measured using elevated plus maze (EPM), open field activity (OFA), and an olfactory discrimination (OD). The predator exposure produced a relatively long-lasting deficit (decreased time in open arms, decreased basic activity) on the EPM while having little effect on performance on the OFA or OD. J-113397 mitigated the performance deficits on the EPM in a dose-dependent manner while having little effect on performance on the OFA or OD. The largest dose of J-113397, administered with a sham exposure, was essentially devoid of effects on the EPM, OFA, and OD. These results demonstrate that J-113397 can significantly and selectively mitigate the effects of a stressor typically used in a preclinical model of post-traumatic stress disorder. Furthermore, these results are consistent with and extend previous results showing that the NOP receptor has an important role in the response to stress and that NOP antagonism may, potentially, have therapeutic benefit in stress disorders.

Adulthood stress responses in rats are variably altered as a factor of adolescent stress exposure.

Stress exposure during development may influence adulthood stress response severity. The present study investigates persisting effects of two adolescent stressors upon adulthood response to predator exposure (PE). Rats were exposed to underwater trauma (UWT) or PE during adolescence, then to PE after reaching adulthood. Rats were then exposed to predator odor (PO) to test responses to predator cues alone. Behavioral and neuroendocrine assessments were conducted to determine acute effects of each stress experience. Adolescent stress altered behavioral response to adulthood PE. Acoustic startle response was blunted. Bidirectional changes in plus maze exploration were revealed as a factor of adolescent stress type. Neuroendocrine response magnitude did not predict severity of adolescent or adult stress response, suggesting that different adolescent stress events may differentially alter developmental outcomes regardless of acute behavioral or neuroendocrine response. We report that exposure to two different stressors in adolescence may differentially affect stress response outcomes in adulthood. Acute response to an adolescent stressor may not be consistent across all stressors or all dependent measures, and may not predict alterations in developmental outcomes pertaining to adulthood stress exposure. Further studies are needed to characterize factors underlying long-term effects of a developmental stressor.

Ketamine administration diminishes operant responding but does not impair conditioned fear.

While not well understood, the NMDA (N-methyl-D-aspartate) antagonist ketamine, a dissociative anesthetic, has been reported to be efficacious in depression and related psychological disorders. Conditioned fear is a normal emotional conditioning process that is known to become dysfunctional in individuals suffering from Post-Traumatic Stress Disorder (PTSD) and related stress disorders. We examined the effects of ketamine to determine the potential modulation of the acquisition and extinction of a conditioned fear using a conditioned suppression procedure. Rats were trained on a variable interval (VI), food maintained, operant conditioning task to establish a general measure of performance. Rats were exposed to inescapable shock (IES, unconditioned stimulus) paired (×20) with an audio/visual conditioned stimulus (CS) to establish conditioning. Conditioning was quantified by measuring response suppression following CS presentation during subsequent extinction trials where the CS alone was presented. Ketamine or vehicle was administered either after initial conditioning or after each of the subsequent extinction trials. For each regimen, a series of four injections were administered 60 min apart (100, 50, 50, 50 mg/kg, respectively) in order to sustain a ketamine effect for a minimum of 4 h. Ketamine produced a general decrease in responding on the VI, relative to baseline, as response rates were slower on the operant task when tested 24 h later and longer. Ketamine did not affect the acquisition of the conditioned fear when the regimen was administered shortly after the initial pairings of IES and CS. Ketamine did not alter extinction to the conditioned fear when the regimen was administered following each CS only presentation following initial conditioning. Our conclusion from these findings is that while ketamine alters behavior on an appetitively motivated operant task it does not, however, appear to directly modulate learning and memory processes associated with conditioned fear.

Multiple presentations reduce the behavioral impact of protected predator exposure in rats.

Exposure of rats to a predator species, such as a cat, or stimuli associated with a predator species has been used to model the effects of traumatic stress. We further investigated this procedure to determine if the behavioral effects from such exposure could be increased by multiple exposures. In rats (n=8 for each treatment group), we evaluated single (1×) and multiple (1×/day for 3 consecutive days [3×] and 2×/day for 3 consecutive days [6×]) exposures using cats and soiled cat litter. All exposures were 15min in duration and the rats were directly exposed to the cats but in a protected fashion that did not allow the predator to physically injure the rat. Sham exposures were conducted using similar conditions without the presence of the predator or litter. The effects of the exposures were evaluated using an elevated plus maze (EPM). Sessions on the EPM were conducted before the exposures and at various times after the exposure. Difference scores (post-pre) were calculated for dependent measures from the EPM, and statistical analyses compared the slopes and intercept values derived from regression functions from these scores over the post-exposure sessions. During the first 30 days after exposure, a significant reduction in activity on the EPM was observed for the 1× treatment and a smaller reduction was observed for the 3× treatment, but no reduction was observed for the 6× and sham control treatments. Thus, increasing the number of exposures did not increase the magnitude of the effect but, instead, resulted in a decrease. These results show that adaptation to the effects of the predator exposure occurred with repeated sessions.

Adolescent traumatic stress experience results in less robust conditioned fear and post-extinction fear cue responses in adult rats.

Early exposure to a traumatic event may produce lasting effects throughout the lifespan. Traumatic stress during adolescence may deliver a distinct developmental insult compared with more-often studied neonatal or juvenile traumatic stress paradigms. The present study describes the lasting effects of adolescent traumatic stress upon adulthood fear conditioning. Adolescent rats were exposed to a traumatic stressor (underwater trauma, UWT), then underwent fear conditioning during adulthood. Fear extinction was tested over five conditioned suppression extinction sessions three weeks later. The efficacies of two potential extinction-enhancing compounds, endocannabinoid reuptake inhibitor AM404 (10mg/kg) and M1 muscarinic positive allosteric modulator BQCA (10mg/kg), were also assessed. Finally, post-extinction fear responses were examined using a fear cue (light) as a prepulse stimulus. Rats traumatically stressed during adolescence showed blunted conditioned suppression on day 1 of extinction training, and AM404 reversed this effect. Post-extinction startle testing showed that fear conditioning eliminates prepulse inhibition to the light cue. Startle potentiation was observed only in rats without adolescent UWT exposure. AM404 and BQCA both ameliorated this startle potentiation, while BQCA increased startle in the UWT group. These results suggest that exposure to a traumatic stressor during adolescence alters developmental outcomes related to stress response and fear extinction compared to rats without adolescent traumatic stress exposure, blunting the adulthood fear response and reducing residual post-extinction fear expression. Efficacy of pharmacological interventions may also vary as a factor of developmental traumatic stress exposure.

Differential severity of anxiogenic effects resulting from a brief swim or underwater trauma in adolescent male rats.

Clinical studies have shown a link between early-life adversity and severity of adulthood responses to a traumatic stress event (post-traumatic stress disorder, PTSD). Despite a need for basic research, few rodent models are available to test the lasting impacts of early-life traumatic stressors. Underwater trauma (UWT) has been used previously to model traumatic stress; however, effects of this procedure have only been characterized in adulthood. Susceptibility of younger animals to physiological or psychological damage from a forced submersion procedure is unknown. A procedure involving swimming may be a stressful stimulus outside of the underwater component of the experience, as well. The acute effects of a 1-minute sham exposure (empty water tank), swim-only, and UWT (40s swim followed by 20s underwater) were compared in adolescent rats at postnatal day 37. No effects on blood oxygenation or lung tissue were observed. Stepwise decreases in open arm behavior were observed on the elevated plus maze (EPM) in swim-only rats, while UWT rats showed an immediate, lasting decrease in open arm behavior. UWT rats showed a significant decrease in basal corticosterone one week after trauma. These results show that while water immersion is a stressor, UWT causes a distinct syndrome of traumatic stress response in adolescent rats.

Determination of threshold adverse effect doses of percutaneous VX exposure in African green monkeys.

Percutaneous exposure to the chemical warfare nerve agent VX was evaluated in African green monkeys (n=9). Doses of VX (7.5-100 µg/kg) were applied to the skin for 60 min and residual agent was quantified (before decontamination) to estimate the absorbed dose. Monkeys were evaluated for the presence or absence of clinical signs of toxicity and blood was sampled periodically (30 min--12 weeks) following exposure to measure the degree of circulating acetylcholinesterase (AChE) inhibition. Monkeys were also evaluated for behavioral changes from VX exposure using a serial probe recognition (SPR) task. The lowest observable adverse effect level (LOAEL) for the production of major clinical signs was determined to be 42.22 µg/kg (absorbed dose estimate=17.36 µg/kg) and the LOAEL for AChE inhibition was 13.33 µg/kg (absorbed dose estimate=6.53 µg/kg). Behavioral performance was unaffected at doses that, while producing substantial AChE inhibition, did not produce clinical signs. VX represents a substantial threat as a contact hazard and these results complement previous studies using the percutaneous route of exposure with VX and extend the findings to a non-human primate species.

Evaluation of miosis, behavior and cholinesterase inhibition from low-level, whole-body vapor exposure to soman in African green monkeys (Chlorocebus sabeus).

BACKGROUND: Relatively little is known about the effects of very low-level exposures to nerve agents where few signs or symptoms are present. METHODS: African green monkeys (Chlorocebus sabeus) (n = 8) were exposed for 10 min, whole-body, to a single concentration of soman (0.028-0.891 mg/m³). RESULTS: EC50 values for miosis were determined to be 0.055 mg/m³ and 0.132 mg/m³ when defined as a 50 percent reduction in pupil area and diameter, respectively. In general, performance on a serial probe recognition task remained unchanged at lower concentrations, but responding was suppressed at the largest concentration tested. Soman produced concentration-dependent inhibition of acetylcholinesterase activity and, to a lesser extent, butyrylcholinesterase activity. CONCLUSIONS: These results characterize threshold soman exposure concentrations that produce miosis in the absence of other overt signs of toxicity and extend previous studies indicating that miosis is a valuable early indicator for the detection of soman vapor exposure.

Safety of administration of human butyrylcholinesterase and its conjugates with soman or VX in rats.

We evaluated the effects of conjugated enzyme-nerve agent product resulting from the inhibition of bioscavenger human serum butyrylcholinesterase (Hu BChE) by nerve agents soman or VX. Rats were trained on a multiple Fixed-Ratio 32, Extinction 30 sec. (FR32, Ext30) schedule of food reinforcement and then injected (i.m.) with Hu BChE (30 mg/kg), equivalent amounts of Hu BChE-soman conjugate (GDC), Hu BChE-VX conjugate, oxotremorine (OXO) (0.316 mg/kg) or vehicle (n = 8, each group). On the day of injection and on 10 subsequent daily sessions, performance was evaluated on the FR32, Ext30 schedule. Neither conjugates nor Hu BChE produced a performance deficit under the schedule. OXO produced a substantial decrease in responding on the day of administration, with complete recovery observed on subsequent sessions. None of the treatments affected circulating acetylcholinesterase (AChE) activity when evaluated 24-72 hr after injection. The dose of Hu BChE produced a 20,000-fold increase above baseline in circulating BChE activity. Pathological evaluation of organ systems approximately 2 weeks following administration of conjugates or Hu BChE alone did not show toxicity. Taken together, these results suggest that Hu BChE - nerve agent conjugates produced following bioscavenger protection against nerve agents soman and VX do not appear to be particularly toxic. These results add to the safety assessment of Hu BChE as a bioscavenger countermeasure against nerve agent exposure.

Assessment of low level whole-body soman vapor exposure in rats.

We evaluated biochemical and behavioral effects of single, low-level exposures to the chemical warfare nerve agent soman (GD). Male Sprague-Dawley rats were trained on a variable-interval, 56-sec schedule of food reinforcement (VI56). The schedule specifies that a single lever press, following an average interval of 56 s, produces food reinforcement (i.e., a single food pellet). After training, rats received a single 60 min exposure to soman vapor at concentrations of 1.0-7.0 mg/m(3), or air control (n=8 for each treatment condition). Blood was sampled before and after the exposure. Following exposures, performance on the VI56 was evaluated for approximately 11 weeks. Additionally, the acquisition and maintenance of a radial-arm maze (RAM) spatial memory task were evaluated in the same subjects during the same 11-week period. Soman exposures produced miosis in all subjects but were otherwise essentially asymptomatic. That is, no convulsions or major signs of toxicity were observed in any subjects, a result consistent with a low-level concentration. Soman exposures produced significant and concentration-dependent decreases in circulating acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity. Soman exposures also produced concentration-dependent levels of regenerated soman in plasma and red blood cell fractions that served to verify the systemic exposure and estimate the total body burden. Soman exposure did not disrupt performance on the VI56 schedule as responding was maintained at pre-exposure levels throughout the 11-week period in all treatment groups. All subjects acquired, and maintained, performance on the RAM task and no significant differences were observed as a result of soman exposure. That is, soman-exposed rats learned the RAM task at the same general rate and to the same general level of accuracy as air-control rats. No delayed effects from exposures were observed. These results demonstrate that, in rats, single exposures to soman vapors at levels that produce substantial AChE and BChE inhibition, but below those producing convulsions and other severe clinical signs of toxicity, may not produce observable effects on the performance of a previously learned task or the acquisition of a new task.

Behavioral evaluation of rats following low-level inhalation exposure to sarin.

We evaluated the effects, in rats, of single and multiple low-level inhalation exposures to sarin. Rats were trained on a variable-interval, 56 s (VI56) schedule of food reinforcement and then exposed to sarin vapor (1.7-4.0 mg/m(3) x 60 min) or air control. The exposures did not produce clinical signs of toxicity other than miosis. Subsequently, performance on the VI56 and acquisition of a radial-arm maze spatial memory task was evaluated over approximately 11 weeks. Single exposures did not affect performance on the VI56 and had little effect on acquisition of the radial-arm maze task. Multiple exposures (4.0 mg/m(3) x 60 min/day x 3) disrupted performance on the VI56 schedule during the initial post-exposure sessions. The disruption, however, resolved after several days. Multiple exposures also produced a deficit on the radial-arm maze task in that sarin-exposed rats tended to take it longer to complete the maze and to make more errors. The deficit, however, resolved during the first three weeks of acquisition. These results demonstrate that in rats, inhalation exposure to sarin at levels below those causing overt signs of clinical toxicity can produce cognitive and performance deficits. Furthermore, the observed deficits do not appear to be persistent.

Determination of miosis threshold from whole-body vapor exposure to sarin in African green monkeys.

We determined the threshold concentration of sarin vapor exposure producing miosis in African green monkeys (Chlorocebus aethiops). Monkeys (n=8) were exposed to a single concentration of sarin (0.069-0.701mg/m3) for 10min. Changes in pupil size were measured from photographs taken before and after the exposure. Sarin EC50 values for miosis were determined to be 0.166mg/m3 when miosis was defined as a 50% reduction in pupil area and 0.469mg/m3 when miosis was defined as a 50% reduction in pupil diameter. Monkeys were also evaluated for behavioral changes from sarin exposure using a serial probe recognition test and performance remained essentially unchanged for all monkeys. None of the concentrations of sarin produced specific clinical signs of toxicity other than miosis. Sarin was regenerated from blood sampled following exposure in a concentration-dependent fashion. Consistent with a predominant inhibition of acetylcholinesterase (AChE), more sarin was consistently found in RBC fractions than in plasma fractions. Further, elimination of regenerated sarin from RBC fractions was slower than from plasma fractions. Blood samples following exposure also showed concentration-dependent inhibition of AChE activity and, to a lesser extent, butyrylcholinesterase activity. At the largest exposure concentration, AChE inhibition was substantial, reducing activity to approximately 40% of baseline. The results characterize sarin exposure concentrations that produce miosis in a large primate species in the absence of other overt signs of toxicity. Further, these results extend previous studies indicating that miosis is a valid early indicator for the detection of sarin vapor exposure.

Understanding artemisinin-induced brainstem neurotoxicity.

Artemisinins are fast-acting and highly efficacious antimalarials. There has been a rapid increase in their use in response to increasing drug resistance and further increases in their use are anticipated as they continue to replace existing therapies. In laboratory studies, artemisinins can produce relatively specific brainstem neurotoxicity. Select nuclei in the medulla, pons and mesencephalon are usually found to be most vulnerable. Species-specific differences in the vulnerability of nuclei may also exist. While not yet completely understood, occurrence of the lesion seems to be dependent upon a sustained, rather than peak, level of circulating drug or metabolite. With daily administrations, the onset of signs of brainstem neurotoxicity frequently develops abruptly and sometimes is observable only at the end of, or after, a regimen of administration. Behavioral correlates of brainstem neurotoxicity in laboratory animals include ataxic symptoms such as tremor, gait impairment and balance disturbance. Symptoms may also include auditory impairment. Screening and diagnostic procedures to guard against artemisinin-induced brainstem neurotoxicity in humans need to be based on the available, albeit limited, data from laboratory studies. Substantial and fundamental gaps in our understanding of artemisinin brainstem neurotoxicity exist including the mode of action of neurotoxicity and the specific conditions under which it occurs. Further, the possibility of increased vulnerability from age-related factors, drug interactions and cumulative administration regimens has not yet been investigated. Substantial progress addressing these issues is needed to maintain appropriate pharmacovigilance as the use of these powerful and life-saving antimalarials increases.

Behavioral and biochemical evaluation of sub-lethal inhalation exposure to VX in rats.

We evaluated the effects of low-level inhalation exposures (whole body, 60min duration) to the chemical warfare nerve agent VX (0.016, 0.15, 0.30 or 0.45mg/m(3)) in rats. The range of concentrations was approximately equivalent to 0.02-0.62 times 1.0 LC50. Biochemical effects were assessed by evaluating blood acetylcholinesterase (AChE) activity and by a regeneration assay that quantified the amount of VX (as the G analog) present in blood. Behavioral effects were assessed using a variable-interval 56-s schedule of reinforcement (VI56), in which rats were trained to press a lever to receive a food reward. VI56 training was established before exposure and evaluations continued after exposure. Additionally, after exposure, acquisition and maintenance of an eight-arm radial maze (RAM) task was evaluated in which rats learned to locate the four arms of the maze that presented a single food pellet reward. Behavioral assessments were conducted over approximately 3 months following exposure. Transient miosis was observed following exposure to all concentrations of VX and exposures to the 0.45mg/m(3) concentration also produced mild and temporary signs of toxicity (i.e., slight tremor and ataxia) in some subjects. All concentrations of VX also inhibited circulating AChE and the highest concentration inhibited AChE activity to less than 10% of pre-exposure values. Regenerated VX-G was found in red blood cell (RBC) and plasma blood fractions. In this respect, more VX-G was seen in plasma than RBC. Only small disruptions were observed on the VI56 or RAM following some VX exposures. In general, however, behavioral effects were minor and not clearly systematic. Taken together these results demonstrate that largely asymptomatic exposures to VX vapors in rats can produce substantial biochemical effects while having only minor performance effects on a previously learned behavioral task and on the acquisition of a new behavioral task.

Evaluation of cognitive and biochemical effects of low-level exposure to sarin in rhesus and African green monkeys.

We investigated the potential of low-level exposures to the chemical warfare nerve agent, sarin, to produce adverse effects. Rhesus (Macaca mulatta) and African green monkeys (Chlorocebus acthiops) were trained on a serial probe recognition (SPR) task before IM administration of a low-level concentration (5.87 microg/kg or 2.93 microg/kg) of sarin. Blood was sampled before agent administration and at various times following administration. Sarin administration did not disrupt performance on the SPR task in either species. Major dependent measures characterizing performance (accuracy, number of completed trials per session, average choice response time) were largely unaffected on the day sarin was administered as well as on subsequent testing sessions occurring over several weeks following administration. Analyses of red blood cell (RBC) and plasma samples revealed that sarin administration produced a substantial degree of inhibition of circulating acetylcholinesterase (AChE) in RBC fractions and butyrylcholinesterase (BChE) in plasma fractions, which only slowly recovered. In this regard, AChE activity was inhibited to a greater extent than BChE activity. Blood samples were also evaluated for regenerated sarin, which was found in RBC and plasma fractions in both species and showed orderly elimination functions. More sarin was regenerated from RBC fractions than from plasma fractions. Elimination of regenerated sarin was much slower in RBC than plasma and exceeded the expected time of AChE aging, suggesting the presence of additional sarin binding sites. In general, effects were similar in both species. Taken together, our results show that while the concentrations of sarin administered were clearly biochemically active, they were below those that are required to produce a disruption of behavioral performance.

Effects of low-level inhalation exposure to cyclosarin on learned behaviors in Sprague-Dawley rats.

Behavioral and biochemical effects of low-level whole-body inhalation exposure to the chemical warfare nerve agent cyclosarin (GF) were evaluated. Sprague-Dawley rats were first trained on a variable-interval, 56-s (VI56) schedule of food reinforcement. The VI56 schedule specifies that a single lever press, following an average interval of 56 s, produces food reinforcement (i.e., a single food pellet). Subjects were then exposed to GF vapor at concentrations of 1.6-5.2 mg/m3, or air control, for 60 min. Following exposures, performance on the VI56 and acquisition and maintenance of a radial-arm maze (RAM) spatial memory task were evaluated during 55 test sessions over approximately 11 wk. GF exposures produced miosis in all subjects and other mild clinical signs of toxicity at the highest concentration. Convulsions were not observed in any subjects. GF exposures produced concentration-dependent decreases in acetylcholinesterase and butyrylcholinesterase activity. Additionally, blood assays revealed concentration-dependent levels of regenerated GF, thus verifying systemic exposure. The largest concentration of GF disrupted performance on the VI56 task. The deficit, however, resolved by the third postexposure test session. All subjects acquired, and maintained, performance on the RAM task, and no significant differences were seen as a result of GF exposure. No delayed effects from exposures were observed. These results demonstrate that, in rats, inhalation exposure to GF at levels below those producing convulsions and other severe clinical signs of toxicity may produce performance deficits on learned behaviors, but the deficits appear to not be persistent.