Cholinesterase inhibitors and stress: effects on brain muscarinic receptor density in mice.

Exposure to the reversible cholinesterase inhibitor, pyridostigmine bromide (PB), in conjunction with stress, has been suggested as a possible cause of Gulf War Syndrome. This work explores the hypothesis that PB exposure coupled with stress will alter cholinergic receptor density based on the rationale that prolonged exposure to PB and stress will lead to increased stimulation of cholinergic receptors due to the reduced capacity to degrade acetylcholine, leading to changes in receptor levels. Male C57Bl6 mice were exposed to PB (3 or 10 mg/kg/day) or physostigmine (2.88 mg/kg/day) for 7 days via ALZET mini-osmotic pumps implanted subcutaneously. The mice were stressed by shaking at random intervals (avg of 2 min/30 min) for 1 week, which was sufficient to increase blood cortisol levels. Brain tissue for autoradiographic analysis was collected on day 7 of treatment. While we examined many brain regions, analysis revealed that most of the significant changes (p<0.05) were seen in cholinergic nuclei. Stress typically increased muscarinic receptor density, while PB and PHY generally decreased muscarinic receptor density.

A murine model for sarin exposure using the carboxylesterase inhibitor CBDP.

Sub-lethal exposure to sarin (GB), a potent chemical warfare agent, produces long-term neurological deficits in both humans and rodents. However, rodents express much higher levels of carboxylesterase (CaE) than humans and require a much higher dose of GB in rodents to produce neurotoxicity. In mice, the combination of the carboxylesterase inhibitor 2-(o-cresyl)-4H-1:3:2-benzodioxaphosphorin-2-oxide (CBDP) with the organophosphorus (OP) nerve agent GB renders mice more sensitive to OP poisoning. After the reduction in CaE, GB inhibits acetylcholinesterase at doses similar to those in human toxicity. A dose-response curve for GB was determined in male C57BL/6 mice after 1.5mg/kg CBDP. A functional observational battery (FOB) for behavior was used to determine the dose needed to elicit seizure activity but maintain a mortality of less than 50%. Neuronal cell death was evaluated at 4, 7, 10 and 14 days post-GB exposure. Multiple brain areas were examined using cresyl violet: CA1 and the dentate gyrus of the hippocampus, amygdala and piriform cortex. GFAP staining was then measured as an index of cell death in the dentate gyrus of the hippocampus. The dentate gyrus and CA1 exhibited significant neuronal death indicated by both cresyl violet and GFAP staining. The treated animals also had a significant decrease in tissue and blood acetylcholinesterase, in addition to decreases in plasma CaE. CBDP renders mice more sensitive to the effects of GB exposure and mirrors a human symptomatic exposure dose.

Delayed behavioral and endocrine effects of sarin and stress exposure in mice.

The organophosphorus agent sarin is a potent inhibitor of acetylcholinesterase. Experiments tested the influence of exposure to low doses of sarin along with psychological stress on delayed behavioral and endocrine changes in mice. Motor activity, acoustic startle response (ASR), pre-pulse inhibition (PPI) of ASR, activity of cholinesterase in blood and catecholamine levels in adrenals were evaluated after low dose sarin exposure (3 x 0.4 LD50 subcutaneously) combined with chronic intermittent stress in C57BL/6J mice. While sarin alone produced depression of motor activity, no interaction of the stress with sarin exposure was observed. Cholinesterase activity was significantly reduced 24 h after exposure to sarin; however, the basal activity was re-established 3 weeks later. The combination of low dose sarin exposure and stress produced delayed behavioral change manifested as excessive grooming together with endocrine alterations in adrenals 7 weeks after exposure. The size of the adrenals in the combined exposure group was increased and the concentration of catecholamines was significantly decreased. In conclusion, these findings indicate that sarin in low doses is more dangerous when combined with shaker stress inducing delayed behavioral and endocrine changes.

Behavioral changes after acetylcholinesterase inhibition with physostigmine in mice.

The effect of the central and peripheral acetylcholinesterase (AChE) inhibitor, physostigmine (PHY), was examined on spatial memory using a water maze, motor activity as well as acoustic startle response (ASR) and prepulse inhibition (PPI) in C57BL/6J mice. PHY was administered intraperitoneally (IP) at doses of 0.0, 0.01, 0.03, 0.1 and 0.3 mg/kg and the mice were tested 30 min after injection. Administration of PHY reduced motor activity in the open field in a dose-dependent fashion, with notable decreases in activity observed at 0.1 and 0.3 mg/kg. The results also showed that animals receiving 0.1 mg/kg spent more total time in the peripheral zone than in the central zone. The water maze data showed impairment of acquisition and performance of the task, accompanied by a reduced swimming time and enhanced thigmotaxis at a dose of 0.1 mg/kg. We also found that the ASR was significantly decreased after 0.03 and 0.1 mg/kg with no change in PPI. These results indicate that central plus peripheral cholinesterase inhibition (ChEI) decreased ASR, which is contrary to our previous experiments with the peripheral ChEI pyridostigmine bromide (PB), suggesting different involvement of cholinergic systems in modulating ASR in mice.

Effect of chronic pyridostigmine bromide treatment on cardiovascular and behavioral parameters in mice.

Experiments were performed to determine the effect of chronic low-dose pyridostigmine bromide (PB) treatment on blood acetylcholinesterase (AChE), cardiovascular (CV) function, and behavior in C57BL/6J male mice. Chronic carotid arterial catheters were used for long-term CV measurements and for collection of blood samples. Separate groups of mice were used for behavioral open field tests. PB was administered subcutaneously using osmotic minipumps at 1 and 3 mg/kg/day for 7 days. Blood pressure and heart rate (HR) were measured continuously for 24 h before treatment and on Days 3 and 7 after minipump insertion. Blood samples were collected on the same days. Mean arterial pressure (MAP) of the control group was 108+/-2 and 104+/-2 mm Hg during the dark and light periods, respectively. HR was 510+/-18 and 493+/-19 beats/min during the dark and light periods, respectively. PB treatment had no effect on MAP or HR in either dark or light period. Basal AChE activity was 0.42+/-0.1 micromol/min/ml, with no changes observed with PB at 1 mg/kg/day. The higher PB dose (3 mg/kg/day) decreased blood AChE activity by 85% on Day 7. Despite the reduction in blood AChE activity, there were no alterations in open field behaviors (locomotor activity, rearing, distance traveled, rest time, number of entries, and pokes). In conclusion, chronic low-dose PB exposure decreased blood AChE activity but had no effect on CV function or behavior in mice.

Intracellular magnesium and magnesium buffering.

The development of new techniques for measuring intracellular free Mg2+ during the 1980s has provided investigators with the tools needed to produce new insights into the regulation of cellular magnesium. Within the limits of this technology, it appears that all mammalian cells maintain free cytosolic Mg2+ levels within the fairly narrow range of 0.25-1 mM. While transport mechanisms and sequestration within cellular organelles will contribute to this regulation, it is binding of Mg2+ to an as yet poorly defined system of buffers that is largely responsible for determining the set point of this regulation. The lack of an adequately Mg2+-selective ionophore remains an impediment to progress in this area.