Recovery of mouse neuromuscular junctions from single and repeated injections of botulinum neurotoxin A

Abstract

Botulinum neurotoxin type A (BoNT/A) paralyses muscles by blocking acetylcholine (ACh) release from motor nerve terminals. Although highly toxic, it is used clinically to weaken muscles whose contraction is undesirable, as in dystonias. The effects of an injection of BoNT/A wear off after 3-4 months so repeated injections are often used. Recovery of neuromuscular transmission is accompanied by the formation of motor axon sprouts, some of which form new synaptic contacts. However, the functional importance of these new contacts is unknown. Using intracellular and focal extracellular recording we show that in the mouse epitrochleoanconeus (ETA), quantal release from the region of the original neuromuscular junction (NMJ) can be detected as soon as from new synaptic contacts, and generally accounts for > 80% of total release. During recovery the synaptic delay and the rise and decay times of endplate potentials (EPPs) become prolonged approximately 3-fold, but return to normal after 2-3 months. When studied after 3-4 months, the response to repetitive stimulation at frequencies up to 100 Hz is normal. When two or three injections of BoNT/A are given at intervals of 3-4 months, quantal release returns to normal values more slowly than after a single injection (11 and 15 weeks to reach 50% of control values versus 6 weeks after a single injection). In addition, branching of the intramuscular muscular motor axons, the distribution of the NMJs and the structure of many individual NMJs remain abnormal. These findings highlight the plasticity of the mammalian NMJ but also suggest important limits to it.

Figure 1. Recovery of CMAP after exposure of mouse ETA to BoNT/A

A, recordings of CMAPs at representative times during the course of recovery. B, changes in amplitude of negative peak of CMAP as a function of time after exposure to BoNT/A. Each point represents a single muscle. Open symbol on ordinate shows mean value for 10 control muscles ± s.d.

Figure 2. Recovery of evoked quantal release after exposure of mouse ETA to BoNT/A

Quantal content of the EPP_in, calculated from mean amplitudes of mEPP_ins and EPP_ins (see Methods). Each point represents the mean of values from approximately 10 NMJs in a single muscle. Open symbol on ordinate shows mean value for 10 control muscles ± s.d.

Figure 3. Axonal sprouting induced by exposure of mouse ETA muscle to BoNT/A

A, images of terminal motor innervation at representative times during recovery. Nerves labelled with antibodies to neurofilament protein and synaptophysin (green), AChRs in muscle labelled with R-BgTx (red). The appearances illustrated in each panel are typical of those seen at the indicated times after injection of BoNT/A. B, time course of events in the response of NMJs to exposure to BoNT/A, expressed as the fraction of NMJs (%) having the features shown. Sprouts form within the first week or so and AChR clusters appear a few days later. The increase in quantal release is indicated by curves showing the fraction of NMJs with QC > 5, representing an unambiguous increase from the blocked level, and of NMJs with QC > 25, a value likely to cause muscle fibre excitation and contraction. Each point represents the mean of values from 1–5 muscles.

Figure 4. Spatial discrimination of focal extracellular recording

A, a fluorescence image of an ETA NMJ after labelling with 4-Di-2-ASP merged with a bright field image showing muscle fibre and electrodes. Two extracellular electrodes (‘Ex 1’ and ‘Ex 2’) are positioned on different parts of the nerve terminal, 8.9 μm apart. The position of the two intracellular electrodes (‘V’ and ‘I’), both slightly out of focus in this image, are indicated by arrows. Electrode ‘V’ is used to record membrane potential and electrode ‘I’ is used to pass current to maintain membrane potential near −75 mV. B, the bottom trace shows mEPPs recorded with the intracellular electrode (‘V’) and the other two traces show recordings by the two extracellular electrodes. The difference in the recordings from these two electrodes gives an indication of the spatial resolution of this approach.

Figure 5. Localization of evoked quantal release

A, image of nerve terminal, labelled with 4-Di-2-ASP, from mouse ETA 21 days after exposure to BoNT/A. Two extracellular recording positions are indicated, one over part of the original terminal and one over a newly formed synaptic spot about 10 μm away. The limits of what is considered the original terminal are shown by a continuous white line. B, recordings from the two sites depicted in A. For each site the upper traces are from the extracellular electrode, with downward deflections indicating evoked synaptic currents. The lower traces are from an intracellular electrode. Six pairs of traces, which were selected from a series of 100 to show the range of values observed, are shown for each site. One pair from each site has been shown in grey. At each site most stimuli failed to evoke any localized extracellular response even though an EPP_in was present. At the ‘new’ site the trace in grey was the only clear response seen in 100 trials. Further details in text.

Figure 6. Comparison of the intensity of evoked quantal release at new and original synaptic sites during recovery of mouse ETA NMJs from exposure to BoNT/A

The local quantal contents (QC_local) of the extracellular responses to nerve stimulation, calculated from the fraction of ‘failures’ using the Poisson distribution (see text for further details), are shown as a function of time after exposure to BoNT/A. Each point represents the mean of values from 1 to 27 (mean 18) fibres in 2–8 (mean 5) muscles at times < 40 days, and 1–2 muscles for times > 40 days, pooled into 5-day intervals after exposure to BoNT/A.

Figure 7. Extent of AChR clusters at new synaptic spots that form during recovery of mouse ETA muscles after exposure to BoNT/A

A, images of AChRs, labelled with R-BgTx, at representative times during recovery. Arrows point to new synaptic spots. At 17 days these are fainter and less well-defined than at 24 days. B, total area, per muscle fibre, of AChR clusters at original NMJs and at new synaptic sites as a function of time after exposure to BoNT/A. At all times the area of new sites is substantially less than that of the original NMJs. C, mean number of separate regions of high AChR density on individual muscle fibres as a function of time after exposure to BoNT/A. Each point represents the mean of data from 1 to 4 muscles pooled into 5-day intervals after exposure to BoNT/A.

Figure 8. Estimated contribution of quantal release from new synaptic spots to neuromuscular transmission during recovery from exposure of mouse ETA to BoNT/A

Open symbols, total quantal content recorded with an intracellular electrode; filled symbols, estimated quantal release from all new synaptic spots on individual muscle fibres (see text). Points represent mean values from 1 to 8 (mean 3.4) muscles, pooled into 5-day intervals after exposure to BoNT/A. Typically 2–3 fibres were studied in detail in each muscle. Note that at all times, the estimated contribution from new synaptic sites is a small fraction of the total.

Figure 9. Temporal changes in onset of evoked responses during recovery from BoNT/A

A, records showing averaged EPP_exs recorded from a control NMJ, from new synaptic contacts 16 days after exposure to BoNT/A and from the site of an original NMJs 21 days after exposure to BoNT/A. B, changes in EPP_ex latency (time from stimulus artefact to 20% of peak amplitude of EPP). C, changes in synaptic delay (time from negative peak of nerve action potential to 20% of peak amplitude of EPP_ex). Note that the time course with which both latency and synaptic delay approach normal values is similar at the new contacts and at the original NMJs. Each data point represents averaged data from one recording site.

Figure 10. Changes in kinetic properties of EPP_ins during recovery from exposure of mouse ETA muscles to BoNT/A

All recordings show EPP_ins selected from trains of 100 stimuli (1 Hz). A, typical EPPs (14 superimposed) from uninjected muscles. Note minimal variation of amplitude or time course. B, superimposed EPP_ins from ETA injected 21 days previously. Note great variation of both amplitude and time course, and much smaller amplitude than in control. C and D, changes in mean value of EPP_in rise time (C, ‘RT’) and decay time (D, ‘DT’) during recovery from BoNT/A. The total quantal content is shown in each graph for comparison. Note that the kinetic properties of the EPP_in return to normal values when the total quantal content has only reached approximately one-third of its normal value. E, variable time courses of EPP_ins recorded from the same muscle fibre, 15 days after exposure to BoNT/A. EPP_ins of two quite distinct time courses could be recorded from this fibre. Examples of these are shown in F (fast EPP_ins), G (mixed time-course EPP_ins) and H (slow EPP_ins). A single response showing both fast and slow components is highlighted in G.

Figure 11. Response to repetitive stimulation of uninjected control NMJs and NMJs 4 months after recovery from BoNT/A

In each case trains of 100 stimuli were given at frequencies from 1 to 100 Hz and the peak amplitudes of the resulting EPP_ins are plotted. For both control (A) and recovered (B and C) NMJs, stimulation at 50 and 100 Hz caused initial facilitation followed by substantial depression. At 1–20 Hz, there was depression but no facilitation. The graphs show averaged pooled data from 75 NMJs from 5 uninjected control mice (A), from 45 NMJs from 3 mice 4 months after a single injection (B), and from 45 NMJs from 3 mice after a 2nd injection, given 4 months after the first (C).

Figure 12. Recovery of quantal content at mouse ETA NMJs after one, two or three injections of BoNT/A

Injections were given at intervals of 3–4 months. Time is shown after the final injection. Note that recovery after 2 and 3 injections is slower than after a single injection. Each point indicates averaged values of pooled data from a number of fibres, typically 10, in one mouse. Regression lines have been drawn through the data for each number of injections: dotted, 1 injection; dashed, 2 injections; continuous, 3 injections. Statistical analysis is described in the text.

Figure 13. Abnormal structural aspects of motor innervation after three injections of BoNT/A

Nerves (green) immunolabelled with anti-NFP and anti-synaptophysin, AChR (red) labelled with R-BgTx). A and C, low magnification (scale bar in A also applies to C) views of normal (saline injected) ETA and an ETA from a mouse of approximately the same age (476 days) which had received 3 injections of BoNT/A at approximately 3-monthly intervals. The band containing NMJs is about twice as wide as normal indicating persisting abnormal distribution of NMJs. B and D–G, higher magnification views (scale bar in B applies to all 5 panels). B is from an uninjected muscle showing NMJ structure typical of mice of this age, consisting of a compact cluster of spot-like regions. D–G show a selection of NMJs from injected muscles, 124 days after the 3rd injection. While the NMJ in E has a structure within the normal range, the others show synaptic spots arising from ultraterminal sprouts (D) or short lateral axonal branches (F and G) that are very rarely seen in normal muscles. H–K, ETA muscle 223 days after 3rd injection of BoNT/A. The lower magnification views (H and I, same magnification) show axons coursing along the muscle fibres while the higher magnification views (J and K, same magnification) show abnormal distributions of AChR clusters.