Rescue of the acetylcholinesterase knockout mouse by feeding a liquid diet; phenotype of the adult acetylcholinesterase deficient mouse

Abstract

Acetylcholinesterase (AChE, EC3.1.1.7) functions in nerve impulse transmission, and possibly as a cell adhesion factor during neurite outgrowth. These functions predicted that a mouse with zero AChE activity would be unable to live. It was a surprise to find that AChE −/− mice were born alive and survived an average of 14 days. The emaciated appearance of AChE −/− mice suggested an inability to obtain sufficient nutrition and experiments were undertaken to increase caloric intake. Pregnant and lactating dams (+/−) were fed 11% high fat chow supplemented with liquid Ensure^®. AChE −/− pups were weaned early, on day 15, and fed liquid Ensure. Although nullizygous animals showed slow but steady weight gain with survival over 1 year (average 100 days), they remained small at all ages compared to littermates. They demonstrated delays in temperature regulation (day 22 vs. 15), eye opening (day 13 vs. 12), righting reflex (day 18 vs. 12), descent of testes (week 7–8 vs. 4), and estrous (week 15–16 vs. 6–7). Significant physical findings in adult AChE −/− mice included body tremors, abnormal gait and posture, absent grip strength, inability to eat solid food, pinpoint pupils, decreased pain response, vocalization, and early death caused by seizures or gastrointestinal tract ileus. Behavioral deficits included urination and defecation in the nest, lack of aggression, reduced pain perception, and sexual dysfunction. These findings support the classical role for AChE in nerve impulse conduction and further suggest that AChE is essential for timely physical development and higher brain function.

Introduction

Acetylcholinesterase has a central role in neurotransmission at cholinergic synapses. AChE hydrolyzes acetylcholine, thus preventing overstimulation of nicotinic and muscarinic receptors. Overstimulation of these receptors may lead to respiratory failure and death. AChE has been proposed to have a second function independent of its catalytic activity [1], [2], [3], [4], [9], [11], [13], [14], [15], [16], [23], [25], [26], [28], [30], [31], [32], possibly serving as a cell adhesion factor in morphogenesis of neurites. These important roles for AChE predicted that the absence of AChE activity in mutant mice would be lethal. It was a surprise, therefore, to find that mice with no AChE enzyme activity and no AChE protein were born alive, and that they were capable of breathing and moving [35]. They were not normal, however. Homozygous mutant mice were smaller than heterozygous littermates, they gained body weight more slowly, their eyes never opened, they had no righting reflex, the external ear did not mature, body tremor was persistent, they circled when walking, and they died at an early age. About 50% of the nullizygotes died by postnatal day 14 and 100% died by day 21 (n=63). This phenotype could be due to deficiency in cholinergic neurotransmission caused by absence of AChE enzyme activity, or to developmental problems caused by the absence of the AChE protein, or to a combination of both.

In the present work our goal was to prolong the life of AChE −/− mice. Injection of purified AChE or of atropine had no beneficial effect, so we tried a different approach. Their emaciated appearance and absence of body fat suggested that the cause of death might be starvation. Therefore, efforts were made to increase their caloric intake. This report describes the success of feeding dams a high fat diet supplemented with liquid Ensure^® to enrich their milk during the nursing period. After weaning, the AChE −/− mice subsist on liquid Ensure. This diet increased the lifespan of AChE −/− mice to an average of 100 days. Several AChE −/− mice have lived up to 15 months.

Having succeeded in producing an abundant supply of AChE −/− mice, we were able to investigate which characteristics were due to deficiency of AChE catalytic activity. Since pinpoint pupils, body tremor, and muscle weakness are present in AChE −/− mice and are also diagnostic for poisoning by AChE inhibitors, we concluded that these abnormalities were due to lack of AChE catalytic function. However, other abnormalities, including postnatal developmental delay, low body weight, lack of housekeeping behavior, and sexual dysfunction have no obvious link to absence of AChE catalytic function and could be due to a combination of effects.