Functional repair of motor endplates after botulinum neurotoxin type A poisoning: biphasic switch of synaptic activity between nerve sprouts and their parent terminals

Abstract

Blockade of acetylcholine release by botulinum neurotoxin type A at the neuromuscular junction induces the formation of an extensive network of nerve-terminal sprouts. By repeated in vivo imaging of N-(3-triethyl ammonium propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide uptake into identified nerve endings of the mouse sternomastoid muscle after a single intramuscular injection of the toxin, inhibition of stimulated uptake of the dye at the terminals was detected within a few days, together with an increase in staining of the newly formed sprouts. After 28 days, when nerve stimulation again elicited muscle contraction, regulated vesicle recycling occurred only in the sprouts [shown to contain certain soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAREs) and to abut acetylcholine receptors] and not at the parent terminals. Therefore, only these sprouts could be responsible for nerve-muscle transmission at this time. However, a second, distinct phase of the rehabilitation process followed with a return of vesicle turnover to the original terminals, accompanied by an elimination of the by then superfluous sprouts. This extension and later removal of "functional" sprouts indicate their fundamental importance in the repair of paralyzed endplates, a finding with ramifications for the vital process of nerve regeneration.

Figures

Figure 1

Repeated in vivo visualization of FM1-43 uptake into nerve endings on the mouse sternomastoid muscle (taken before and after BoTx/A-poisoning) indicated a transient switch in the locus of stimulation-dependent vesicular uptake and release between the nerve terminals and their toxin-induced sprouts. (a, b, e, and f) At d0, before BoTx/A injection, FM1-43 (red) uptake was seen in the nerve terminals also stained with 4-di-2-ASP (green), and their overlap appears as yellow. (a, d3) Imaging at d3 indicated a dramatic diminution in depolarization-induced FM1-43 uptake. Although staining of the original terminals (b, open arrows) remained suppressed, FM1-43 endocytosis became apparent in the sprouts (b, closed arrows), after their appearance and extension (b; d14 and d28). In a separate experiment, FM1-43-loaded sprouts (c) at d28 were destained visibly on depolarization (d), establishing that they also can mediate stimulated exocytosis. On further extension (e; up to d42) the sprouts continued to be labeled with FM1-43, but imaging at d63 and thereafter indicated that uptake recovered in the parent terminals, while such staining in the sprouts began to decline preceding their retraction (e and f; d63). (f; d91) By d91, FM1-43 uptake at the original endplate closely resembled that recorded immediately before injection. Luminance levels were quantified for both 4-di-2-ASP and FM1-43 staining in the original endplates and sprouts, as illustrated, for example, at d14 (b); filled arrows in that image pinpoint two of the four sprouts present. (g) The average pixel-intensity values calculated from bands of lines (depicted schematically for sprout 1) are plotted for red (filled curve) and green (open curve) channels for these two processes and the original terminals. (h) The integrated red and green intensity values for all sprouts (closed circles) and their parent terminals (open circles) obtained in this manner were averaged ±SEM (from 11–16 endplates in three or four mice at each time point), and their ratios were plotted for the entire time course. a, b, e and f show repeated visualizations of the same endplates with each series having been obtained from different animals. (Bars = 20 μm.)

Figure 2

Control and BoTx/A-treated endplates dual-labeled with rhodamine-conjugated α-bungarotoxin and FITC-conjugated secondary IgGs reactive with antibodies bound to synaptobrevin, SNAP-25, or neurofilament. On control sternomastoid muscle fibers, synaptobrevin (a) and SNAP-25 (d) staining was colocalized within the areas occupied by the nAChRs; unlike synaptobrevin, SNAP-25 was also detected in the axons. At d9, both synaptobrevin (b) and SNAP-25 (e) were detected in the sprouts extending beyond the area containing nAChRs (arrows). By d28, sprouts containing the v-SNARE (c) and t-SNARE (f) had extended to form a complicated network. (c and f, arrowheads) At this time, clusters of nAChRs were detected abutting these sprouts. (g) This clustering of nAChRs was also seen in mouse LAL injected with BoTx/A 18 days earlier and dual-stained with antineurofilament IgG. (Bars = 20 μm.)

Figure 3

Quantitation of activity-dependent uptake of FM1-43 in excised living motor-nerve terminals from control and mouse LAL injected with BoTx/A. The first two bars represent measured fluorescence levels in stimulated (S) and nonstimulated (NS) terminals of control muscle. The next five bars show the progressive increase of depolarization-dependent uptake of FM1-43 into the terminals and sprouts in previously paralyzed tissue. The control level is reached only at d97. Values presented represent the mean index of FM1-43 uptake ±SEM of 100–200 measurements performed on two to six neuromuscular preparations. ∗, P < 0.01; Student’s t test.