Methanesulfonyl fluoride (MSF) blocks scopolamine-induced amnesia in rats

Neurobiology t~['Aging,Vol. 14, pp. 93-96, 1993

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Methanesulfonyl Fluoride (MSF) Blocks Scopolamine-Induced Amnesia in Rats R. L. PALACIOS-ESQUIVEL I, G. P A C H E C O A N D D. E. M O S S Psychobiochemistry Laboratory, Department of Psychology, University of Texas at El Paso, El Paso, TX, 79968 R e c e i v e d 2 J a n u a r y 1992; A c c e p t e d 8 J u l y 1992 PALACIOS-ESQUIVEL, R. L., G. PACHECO, AND D. E. MOSS. Methanesul/bnylfluoride (MSF) blocks scopolamineinduced amnesia in rats. NEUROBIOL AGING 14(1)93-96, 1993.--Cholinesterase inhibitors, such as physostigmine and tetrahydroaminoacridine, have been found to alleviate some of the memory deficits characteristic of senile dementia of the Alzheimer's type (SDAT). Many toxic side effects, however, have been associated with the use of these compounds. Recently, a cholinesterase inhibitor, methanesulfonyl fluoride (MSF), was discovered to have low toxicity, central nervous system (CNS) selectivity, and a long therapeutic duration. The purpose of this research was to determine if MSF ( 1.5 mg/kg) would be effective in reducing or blocking amnesia induced by various doses of scopolamine (0.2, 0.6, and 2.0 mg/kg). One hundred and twentytwo female Sprague-Dawley albino rats were trained and retention tested in a Y-maze brightness discrimination task. MSF was highly effective in reducing scopolamine-induced amnesia. Cholinergic hypothesis

Cholinesterase inhibitors

CNS selectivity

M E M O R Y deficits in senile dementia of the Alzheimer's type (SDAT) may result from insufficient cholinergic function within the central nervous system (6,8,25). Facilitation ofcholinergic activity in the CNS through the use of cholinesterase inhibitors, therefore, has become a treatment strategy for SDAT. Cholinesterase inhibitors such as physostigmine and T H A have been shown to have marginal effects on memory in SDAT patients in various studies (3,5,9,23,24). However, these drugs produce toxic side effects such as nausea, diarrhea, emesis, excessive micturition, and diaphoresis at doses at or very near those required for a therapeutic effect. A drug with CNS selectivity would minimize or avoid peripheral toxicity relatively (7,19). A second problem that arises is due to the short half-lives of physostigmine and THA. A drug with a long halflife would be more appropriate in a long-term treatment program such as SDAT patients would require. Methanesulfonyl fluoride (MSF) is a very long acting (irreversible) cholinesterase inhibitor that shows high relative selectivity for the CNS. Small doses of MSF administered repeatedly to rats can produce up to 90% inhibition within the CNS with less than 35% inhibition in the peripheral tissues (heart, smooth muscle, and skeletal muscle). MSF, therefore, has greatly reduced toxicity from peripheral cholinesterase poisoning compared with other cholinesterase inhibitors (16,19). In fact, 86% inhibition o f C S F cholinesterase can be produced by a single injection of 1.5 mg/kg (IM) MSF in monkeys without a deleterious effect (16). Also, monkeys were treated with doses of MSF increasing to 1.5 mg/kg (IM) and then maintained on

MSF

SDAT

Scopolamine

this dose for twelve injections given over approximately 5 weeks. These monkeys showed no loss of body weight, no loss of appetite, no general behavioral indications of toxicity (e.g., no lethargy or unusual behaviors), and no changes in clinical blood chemistry. These MSF-treated monkeys showed 80% inhibition of cholinesterase in cortex biopsies compared to controls. These data show the remarkably low general toxicity produced by sulfonyl fluorides even in the presence of extreme CNS enzyme inhibition. Besides producing prolonged decreases in AChE activity, as expected, acute treatment with MSF (1.5 mg/kg SC) also increases forebrain acetylcholine content in free (extraterminal) and labile bound (intraplasmic) but not in stable bound (vesicular) fractions of mouse brain. After three daily treatments, MSF increased levels of free acetylcholine for more than 5 days (20). Interestingly, however, QNB binding and high affinity choline uptake were decreased by either the single or repeated doses oforganophosphates or MSF (20). Treatment with MSF did not produce signs of cholinesterase toxicity observed with similar treatment with an organophosphate inhibitor (16,18,20,21). The special characteristics of MSF reviewed above suggest that it might be well suited as a treatment for dementia in Alzheimer's disease or other disorders caused by declining cholinergic function. A recent study (15) has shown that pretreatment with 0.5 mg/kg MSF (IP), causing 50.4% inhibition of brain ACHE, improved retention in terms of speed and errors in 18-month-old rats in a one trial discriminative reward learn-

~Requests for reprints should be addressed to D. E. Moss, D e p a r t m e n t o f Psychology, University of Texas at E1 Paso, El Paso, T X 79968.

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ing task. Not only did the MSF-treated 18-month-old rats perform better than the untreated controls, these rats performed as well as young rats (2- to 3-months old). This experiment indicates that MSF may be useful in treating age-related memory impairment and perhaps, Alzheimer's disease. However, the degree to which memory impairment in normal older rats is caused specifically by insufficient cholinergic function is not known. Although it is clear that Alzheimer's disease causes many changes that may be involved in memory impairment, major losses in cholinergic function are involved (6,8,25). The purpose of the present experiment was to determine the degree to which MSF could reduce the amnestic effect of scopolamine. Scopolamine HBr is a muscarinic antagonist that is highly effective in producing memory impairment (4,10,12,14,17) through direct cholinergic involvement (1). In addition to acting as a muscarinic antagonist, scopolamine reduces acetylcholine levels (14,22). In view of these characteristics, scopolamine is widely used to induce cognitive dysfunction by reducing cholinergic function. Scopolamine was used to induce memory deficits in a rat model of cholinergic insufficiency. Methanesulfonyl fluoride was found to be highly effective in reducing the amnestic effects of scopolamine. METHOD

Subjects The subjects were 122 female Sprague-Dawley albino rats, reared and maintained with ad lib food and water in a 12L: 12D cycle (on at 1800 h) in the animal colony at the University of Texas at El Paso. The age of the rats ranged from 3 to 6 months. The animals were reared in a closed colony to insure that the animals were not exposed to cholinesterase inhibiting pesticides.

Drug Treatment In a factorial design, three different dosages of scopolamine HBr (SCOP) were administered to produce different levels of cholinergic disruption (0.2, 0.6, and 2.0 mg/kg) compared to controls. A group of rats was treated with MSF (1.5 mg/kg) alone to determine the effects of this drug on normal memory. Other groups of rats were treated with both SCOP and MSF to determine the degree to which MSF would reduce SCOP-induced amnesia (0.2, 0.6, and 2.0 mg/kg). Twenty three, 14, 10, and 14 rats were in each group (no-MSF and MSF-pretreated) receiving no SCOP, 0.2 mg/kg SCOP, 0.6 mg/kg SCOP, and 2.0 mg/kg SCOP. The relatively large number of controls (both no-MSF and MSF-pretreated) resulted from the practice of running these controls concurrently in every experimental session. MSF (Aldrich Chemical Co., Milwaukee, WI) or peanut oil vehicle was administered once (IP), 2 h prior to t ~ i n g , One injection was sufficient for this 24-hour experiment because of the long acting irreversible effects of MSF (11 days) (19). SCOP HBr (Sigma Chemical, St. Louis, MO) was prepared in deionized water and was administered (IP) 30 rain prior to training. It was also given approximately 8 h after training and then again at 30 rain prior to retention testing (approximately 24 h after training). Repeated doses of SCOP were required to simulate a state of cholinergic dysfunction throughout the experiment.

Apparatus The rats were trained on a Y-maze similar to that used by Deutsch (10). Each arm of the maze was 50 ~< 12.5 cm with walls 15 cm high and at the end of each arm was a piece of 3 mm thick translucent white Plexiglas behind which was a 60 W light bulb that was supplied with 65 volts.

Procedure The rats were trained to escape 1.0 mA toot shock by entering the lighted arm of the Y-maze. Training was concluded when the rat met the learning criterion of 10 successive correct trials or when a maximum of 80 trials were completed. The animals were then retention tested on the same Y-maze brightness discrimination task 24 h later as a measure of memory. performance.

Cholinesterase Assays A sample of 8 MSF-treated and a sample of 8 control rats were sacrificed by decapitation and their brains were assayed for cholinesterase activity in triplicate according to the method of Ellman et al. (13) as modified by Moss et al. (18,19). Although the results reflect the enzyme inhibition at a time after retention testing (28 h after injection), it has been shown that MSF-induced inhibition is at a steady state level between 3 and 72 h (19). Therefore, the results of these assays give an indication of the level of inhibition present both during training and retention testing. RESULTS

A two-way analysis of variance (ANOVA) (MSF × SCOP) was computed for training scores ( 11 ). SCOP affected training performance as each dose increased the number of trials needed to meet learning criterion, F(3, 114) = 8.65, p < 0.00 ! (Fig. 1). Pretreatment with MSF had no effect on training scores and did not significantly improve scores of rats treated with SCOP. A two-way ANOVA (MSF × SCOP) was also computed for retention scores (11). SCOP-induced amnesia was shown by the increased number of trials required for animals to meet the relearning retention criterion of 10 out of 10 responses, F(3, 114) = 12.58, p _<_0.001. Rats pretreated with MSF showed reduced SCOP-induced amnesia as shown by the reduced number of trials it took to meet the retention criterion. F( 1. 114) = 18.84.p < 0.001 (Fig 2). A Bonferroni analysis of the source of differences in retention scores was also computed (11). The significance levels shown represent the overall Bonferroni-adjusted significance levels. This comparison of individual groups showed the group receiving no MSF and no SCOP was different from the no MSF/0.2 SCOP, t~8 = 3.08, p ~ 0.05, no MSF/0.6 SCOP. t~z = 4.84, p ~ 0.01, and no MSF/2.0 SCOP. t~8 = 4.53, p _< 0.01, groups. Within each dose of SCOP, however, individual comparisons of MSF-pretreated groups with SCOP-only groups did not show any differences except at 0.6 rng/kg SCOP where the MSF-pretreated group showed better retention, t~: = 3.00, p ~ 0.05. Brains removed from vehicle-treated control rats hydrolyzed 6.96 (+0.28) X 10 -6 moles of substratelgram/minute. Brains removed from MSF-pretreated rats hydrolyzed 1.82 ( + 0.17) >( 10-6 moles of substrate/gram/min, indicating that pretreatment with 1.5 mg/kg MSF produced approximately 74% inhibition of brain ACHE.

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MSF BLOCKS SCOPOLAMINE

RETENTION

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FIG. 1. The effects of scopolamine HBr (SCOP)and methanesulfonyl fluoride (MSF) on training. The solid black bars represent the performance of control rats (no MSF) whereas the hatched bars represent the performance of rats treated with MSF (1.5 mg/kg). The greater the number of trials to criterion, the worse the training performance.

DISCUSSION Individual group comparisons showed that SCOP treatment produced increasing retention scores (amnesia). As predicted from the powerful inhibition of CNS cholinesterase, MSF produced significant improvements in retention scores of animals with SCOP-induced amnesia• Individual group comparisons showed that groups treated with both MSF and SCOP were not different from the no MSF/no SCOP control group. A high level of AChE inhibition was intentionally induced to counteract the strong effect of high doses of SCOP. It might be expected that MSF pretreatment sufficient to produce 74% CNS inhibition would interfere with learning or memory performance in the group that did not receive SCOP. However, prior research has shown that rats 5, 12, and 24 months of age maintained eleven weeks on high doses of phenyimethanesulfonyl fluoride (PMSF) (76% inhibition in brain) showed no effect on total responses and only a small decrement in number of consecutive correct responses in a radial arm maze (19). This PMSF treatment also did not have any effect on motor behaviors as measured by a string test (19). In addition, the sulfonyl fluorides (both PMSF and MSF) had no effect on methylphenidate-induced stereotypy even at 64% inhibition of CNS

2.0

(mg/kg)

FIG. 2. The effects of scopolamine HBr (SCOP)and methanesulfonyl fluoride (MSF) on retention. The legend is the same as shown for Fig. 1.

enzyme (18). At this level of inhibition, physostigmine produced complete suppression (18). The sulfonyl fluorides do not appear to interfere with behavior like other cholinesterase inhibitors, even at relatively high doses. It is unclear how much interference with behavior from cholinesterase inhibitors is caused from cognitive or motor effects. In summary, as a possible treatment for dementia in AD, MSF has many favorable characteristics that other cholinesterase inhibitors do not possess: (a) low toxicity, (b) long therapeutic duration, and (c) CNS selectivity. All of these MSF characteristics are advantageous for SDAT patients who are encountering toxic side effects and/or the discomfort of numerous drug administrations. If the experimental data on MSF in this SCOP-model of the memory defect in SDAT is any evidence of the effects this drug will have on humans, then MSF has the potential to become an efficacious treatment for cholinergic insufficiency in SDAT. ACKNOWLEDGEMENTS This research was supported in part by the E1 Paso Chapter of the Alzheimer's Association, MIRDP MH47167, and private donors. The technical assistance of Twila Harris-OIson is appreciated.

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