Robert D. Grubbs

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.

Determination of cytosolic Mg2+ activity and buffering in BC3H-1 cells with mag-fura-2

The magnesium buffer coefficient (BMg) was calculated for BC3H-1 cells from the rise in cytosolic Mg2+ activity observed when magnesium was released from ATP after iodoacetate (IAA) and NaCN treatment. The basal cytosolic Mg2+ activity (0.54±0.1 mM) measured with mag-fura-2 doubled when 4.54 mM magnesium was liberated from ATP:BMg was 12.9 indicating that a 1 mM increase in Mg2+ activity requires an addition of about 13 mM magnesium. The accuracy of this value depends on these assumptions: (a) all of the magnesium released from ATP stayed in the cells; (b) the rise in Mg2+ was not secondary to pH-induced changes inBMg; (c) mag-fura-2 measured Mg2+ and not Ca2+; and (d) the accuracy of the mag-fura-2 calibration. Total magnesium did not change in response to IAA/CN treatment, thus the change in Mg2+ activity reflected a redistribution of cell magnesium. pH changes induced by NH4Cl pulse and removal had little effect on Mg2+ activity and the changes were slower than and opposite to pH-induced changes in Ca2+ activity measured by fura-2. Ca2+ responses were temporally uncopled from Mg2+ responses when the cells were treated with IAA only and in no cases did Ca2+ levels rise above 1 μM, showing that the mag-fura-2 is responding to Mg2+. Additional studies demonstrated that ∼90% of the mag-fura-2 signal was cytosolic in origin. The remaining non-diffusible mag-fura-2 either was bound to cytosolic membranes or sequestered in organelles with the fluorescence characteristics of the Mg2+-complexed form, even when cytosolic free Mg2+ activity was approximately 0.5 mM. This bound mag-fura-2 would appear to increase the Kd and thus clearly limits the accuracy of our estimmate forBMg. Despite this limitation, we demonstrate that Mg2+ is tightly regulated in face of large changes in extracellular Mg2+, and that the interplay observed between pH, Ca2+ and Mg2+ activities strongly supports the hypothesis that these factors interact through a shared buffer capacity of the cell.

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.

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.

Phenotypic stability of chick cardiomyocytes in serum-free media: Preservation of muscarinic receptor expression

Chick cardiomyocytes cultured in fetal bovine serum (FBS)-supplemented media are phenotypically unstable, becoming noncontractile and unresponsive to stimuli after several days. We report a culturing protocol that preserves the differentiated cardiomyocyte phenotype for at least 9 days in culture. Cardiomyocytes isolated from 11-day chicken embryos, and cultured in either Dulbeccos Modified Earles Medium (DMEM)/Hams F12 medium with N-2 supplement or Medium 199 (M199) with 10% FBS continued to beat spontaneously for 4-5 days; only cells cultured in N-2-supplemented medium exhibited spontaneous beating beyond 5 days. Immunostaining for alpha-actinin after 9 days in culture revealed that myofibrils persisted in N-2-supplemented cells, while no myofibrils were observed in the FBS-supplemented cells. For cells in FBS-supplemented media, [3H]thymidine incorporation rates were 7.5 and 3 times greater than that of cells in N-2-supplemented media at Days 4 and 9 in culture, respectively. The effect of growth media on the binding parameters of the muscarinic antagonist, [3H]N-methyl-scopolamine (NMS), was also compared. While B(max) decreased 34% between Days 4 and 9 for cells maintained in N-2-supplemented media, a 77% decrease was observed for cells cultured in FBS-supplemented media. The phenotypic stability of this preparation makes it feasible for the first time to use these cells in experiments that require more than 4 days to complete.

Chemical Messenger Systems

Neurotransmitters, neurohormones, and neuromodulators are the chemical messengers that allow one cell to communicate with another cell or with itself.