Acetylcholinesterase Dynamics at the Neuromuscular Junction of Live Animals

Abstract

At cholinergic synapses, acetylcholinesterase (AChE) is critical for ensuring normal synaptic transmission. However, little is known about how this enzyme is maintained and regulated in vivo. In this work, we demonstrate that the dissociation of fluorescently-tagged fasciculin 2 (a specific and selective peptide inhibitor of AChE) from AChE is extremely slow. This fluorescent probe was used to study the removal and insertion of AChE at individual synapses of living adult mice. After a one-time blockade of AChEs with fluorescent fasciculin 2, AChEs are removed from synapses initially at a faster rate (t1 ⁄ 2 of ϳ3 days) and later at a slower rate (t1 ⁄ 2 of ϳ12 days). Most of the removed AChEs are replaced by newly inserted AChEs over time. However, when AChEs are continuously blocked with fasciculin 2, the removal rate increases substantially (t1 ⁄ 2 of ϳ12 h), and most of the lost AChEs are not replaced by newly inserted AChE. Furthermore, complete one-time inactivation of AChE activity significantly increases the removal of postsynaptic nicotinic acetylcholine receptors (AChRs). Finally, time lapse imaging reveals that synaptic AChEs and AChRs that are removed from synapses are co-localized in the same pool after being internalized. These results demonstrate a remarkable AChE dynamism and argue for a potential link between AChE function and postsynaptic receptor lifetime.

Assembly and regulation of acetylcholinesterase at the vertebrate neuromuscular junction

“…The half-life of Fas2 dissociation is approximately 36 h whereas the dissociation half-life of α-Btx is closer to 8-10 days, as previously reported. Thus the rate of Fas2 dissociation from previously frozen skeletal muscle sections is essentially the same as reported for the recycling if AChE at the living NMJ of adult mice [22,23].…”

Neurally Mediated Airway Constriction in Human and Other Species: A Comparative Study Using Precision-Cut Lung Slices (PCLS)

“…Additionally, the experiments with magnesium exclude direct electric activation of ASM. Possible explanations are differences in neurotransmitter release or degrading enzymes such as the acetylcholine esterase that may vary in amount or activity [37]–[39]. However, we believe that the type of innervation, i.e., cholinergic, adrenergic, eNANC or iNANC, is the most relevant reason for the differences between species, as for instance the repetitive reversible contraction in rat is cholinergic [21], whereas the steady contraction without relaxation in guinea pigs (figure 1) suggests an additional mechanism other than a cholinergic one in which the neurotransmitter is not metabolized.…”

Distinct localization of collagen Q and PRiMA forms of acetylcholinesterase at the neuromuscular junction

“…In brief, after incubation overnight in a solution containing 0.8 µM biotinylated fasciculin2 (Krejci et al, 2006) or 30nM biotinylated α-BTX (Molecular Probes), biotin was detected using streptavidin coupled to gold particles (1.4 nm in diameter; Nanoprobes, NY, USA, 1:100 in PBS/BSA). Intensification of gold particles, fixation, embedding and observations were processed as described above.…”