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NEUROSCIENCE |
1 Kazan State Medical Academy, Kazan, Russia 420012
2
Institute of Neuroscience, Newcastle University, UK
3
Kazan Institute of Biochemistry and Biophysics, Kazan, Russia 420111
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Abstract |
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(Received 5 March 2008;
accepted after revision 7 May 2008;
first published online 8 May 2008)
Corresponding author C. R. Slater: Institute of Neuroscience, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, UK. Email: c.r.slater{at}ncl.ac.uk
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Introduction |
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BoNT/A is a metalloendoprotease. After binding to the synaptic vesicle protein SV2 at the surface of the motor nerve terminal (Dong et al. 2006), it is internalized and activated. It then specifically cleaves SNAP-25, an essential protein in exocytosis, thus preventing the ‘quantal’ release of ACh in response to nerve impulses (Schiavo et al. 2000). During the period of recovery from BoNT/A, sprouts grow out from the motor axon and may make contact with the muscle fibre (Duchen, 1970; Angaut-Petit et al. 1990; de Paiva et al. 1999; Santafe et al. 2000). Key synaptic proteins are subsequently expressed at many of these sites (Duchen, 1970; Yee & Pestronk, 1987; Juzans et al. 1996b; de Paiva et al. 1999). Although it is generally assumed that synaptic transmission occurs at these new contacts and mediates the initial functional recovery from BoNT/A (e.g. Molgo et al. 1990; Angaut-Petit et al. 1990) we are unaware that this view has ever been directly tested.
Some evidence concerning the role of new contacts in the recovery from BoNT/A comes from a study in which changes in the ability of identified NMJs to take up the membrane soluble fluorescent dye FM1-43 (Cochilla et al. 1999) were studied in vivo at various times after administering BoNT/A (de Paiva et al. 1999). Exposure to BoNT/A blocked activity-dependent FM1-43 labelling of the original nerve terminals within a few days. Newly formed axonal sprouts were seen to take up FM1-43 at a stage of recovery when uptake by the original terminal was still undetectable. However, 2–3 months after intoxication, the original terminal began to recover its ability to take up the dye and the sprouts were then withdrawn. These results were interpreted to mean that during the initial stage of recovery the sprouts, which appeared to be the only possible source of quantal ACh release, were exclusively responsible for the initial recovery of neuromuscular transmission.
The observations just described do not specifically demonstrate ACh release. Indeed, it is uncertain that the sensitivity of FM1-43 uptake experiments is adequate to detect the low levels of quantal release that may be able to excite muscle fibres that have been functionally denervated for some time. Using focal extracellular recording, capable of detecting the effects of a single ACh quantum with a spatial resolution of less than 10 µm (del Castillo & Katz, 1954), we have found that in the mouse ETA muscle (Bradley et al. 1989), although quantal release from the newly formed synaptic contacts does occur, substantially more quanta are released in the region of the original NMJ at all stages of recovery.
The action of BoNT/A is known to result in changes in the kinetics of quantal release at some synapses (Sakaba et al. 2005). In addition, there is evidence that types of voltage-gated calcium channel that are not normally expressed at mammalian NMJs are active during recovery from BoNT/A (Santafe et al. 2000). Although a number of previous studies have noted an increase in the incidence of ‘slow’ or ‘giant’ spontaneous mEPPs after exposure to BoNT/A (Thesleff et al. 1983; Kim et al. 1984), little has been reported about the properties of the evoked quantal release that account for functional recovery of the NMJ. We have therefore investigated changes in the temporal aspects of evoked ACh release and found that evoked responses are also significantly slower during the recovery from the effects of BoNT/A.
Although multiple injections of BoNT/A are widely used clinically to maintain muscle weakness, very little is known about the ability of the NMJ to recover from such repeated exposure. We have investigated the effects on NMJs in the mouse ETA of up to three injections, given at successive intervals of 3–4 months. Although there is substantial functional recovery even after three injections, this recovery is significantly slower than after a single injection and is associated with persisting structural abnormalities of the terminal and preterminal motor axon.
Preliminary accounts of some of our observations have been presented previously (Pang et al. 2002; Slater & Rogozhin, 2006).
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Methods |
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All studies were authorized by Project Licence PPL 60/3024 issued by the UK Home Office and approved by the Newcastle University Ethics Committee. A total of 180 mice were used in the study.
All experiments were made on the epitrochleoanconeus muscle (ETA), located superficially on the medial aspect of the proximal forelimb (Bradley et al. 1989). To expose the muscle to BoNT/A, the mice were anaesthetized (3% isoflurane) and a small incision (1 mm) made in the skin in the right axilla. Ten microlitres of a solution containing 0.5 U of BoNT/A (BOTOX®, Allergan) in sterile saline was injected under the skin over the ETA using a 10 µl Hamilton syringe fitted with a 26 gauge needle. The skin was closed with a single suture and 0.2 ml Buprenorphine (1: 100, subcutaneous) given to control pain. At the time of the acute experiment, the mice were killed by cervical dislocation.
Nerve–muscle preparations
In the adult mice used (female, ca 30 g) the ETA contains about 400 muscle fibres, each about 9 mm long with a mean diameter of about 35 µm. It is usually innervated by five to seven motor neurons contained in a small branch of the radial nerve. More than 90% of the muscle fibres are ‘fast’ as defined by myosin heavy chain immunolabelling.
The results from mice injected with BoNT/A were compared to those from uninjected mice, from mice injected with 10 µl of sterile saline (using the same injection procedure outlined above), or from the uninjected muscles contralateral to the experimental muscles.
The experiments reported here were made on mice of the strains CD1, MF1 or TO. No differences in the response to BoNT/A between the strains was noted.
Electrophysiological studies
Animals were killed at various times after BoNT/A injection by stunning and cervical dislocation. ETA nerve–muscle preparations were then dissected out and maintained at room temperature (20–23°C) in Liley's solution containing (mM): 137.8 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 1 KH2PO4, 12 NaHCO3, 11 glucose, gassed with 95% O2–5% CO2 (Liley, 1956). The preparations were pinned out in a recording chamber (Warner RC 27, Warner Instruments) with a volume of 2.5 ml, lined with Sylgard (Dow Corning). A continuous flow of gassed bathing fluid was maintained at 1 ml min–1 by a peristaltic pump. The muscle was viewed with a Leica DLFSA fluorescence microscope (Leica Microsystems) fitted with water immersion objectives (10–40x).
The nerve was drawn up into a suction electrode and routinely stimulated supramaximally with pulses of 0.1 ms duration. Intracellular recordings were made with glass electrodes (ca 10 M
resistance when filled with 3 M KCl), amplified by an Axoclamp 2B amplifier (Axon Instruments/Molecular Devices), further amplified and processed (filtered at 3 kHz) by a CyberAmp 380 (Axon Instruments), and digitized at a sampling rate of 41.6 kHz by a Digidata 1322A A/D converter (Axon Instruments) before being displayed and saved using pCLAMP 9 software (Axon Instruments). Extracellular signals were recorded with glass electrodes with an internal diameter of 2 µm, filled with bathing fluid, and were amplified by an AI402 head stage (Axon Instruments) feeding into the CyberAmp (filtered at 10 kHz) and digitized as above. Spontaneous events (mEPPs) were recorded in continuous samples of 1–2 min duration.
The preparations were initially viewed in bright field transmitted light with a 2.5x dry objective with a long working distance. Compound muscle action potentials (CMAPs) were recorded during stimulation of the nerve at 1 Hz to provide an indication of the overall state of functional innervation of the muscles before exposure to labelling and blocking reagents (see below) (Fig. 1). For this, an extracellular electrode (see above) was positioned approximately 100 µm above the surface of the muscle and moved parallel to the muscle fibres until a response was observed starting with an abrupt negative deflection, indicating proximity to the innervation zone. The electrode position was then further optimized by moving it laterally to the position where the largest response was observed.
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Muscle action potentials were then blocked by exposing the muscles to µ-conotoxin GIIIB (µCTX, 2 µM; Peptide Institute, Osaka, Japan) for 15 min while the flow was stopped (Plomp et al. 1992). The toxin solution was then sucked out, the preparation returned to fresh bathing fluid and the flow resumed. This procedure blocked muscle contraction for 45–60 min, at which time the preparation was re-exposed to the toxin.
After blocking contraction with µCTX, one or two intracellular and two extracellular electrodes were positioned approximately and the objective changed to 40x water immersion (2 mm working distance) to search for NMJs located on the surface of the muscle. To record synaptic potentials, the intracellular electrodes were inserted into the muscle fibre 50–100 µm away from the NMJ, to minimize damage to the nerve terminal. When two intracellular electrodes were used, current was passed through one to maintain the membrane potential close to –75 mV. This ensured that measurements of the amplitude and time course of synaptic events, which are both influenced by the value of the membrane potential, were all made at nearly the same membrane potential. EPPs were routinely evoked by trains of 100 stimuli delivered at a frequency of 1 Hz. In some cases, described in the text, higher frequency trains were used. Spontaneous mEPPs were recorded during continuous records of 1 or 2 min duration. The EPPs and mEPPs recorded in this way are referred to below as EPPin and mEPPin to distinguish them from events recorded with extracellular electrodes.
To make focal recordings of synaptic events (del Castillo & Katz, 1954) an extracellular electrode was positioned close to a site of interest along the axon terminal or a sprout. Gentle pressure was then applied, under visual control, to bring the electrode into contact with the axon without causing damage (signalled by an abrupt increase in mEPP frequency).
The response to nerve stimulation recorded from a single extracellular electrode positioned near the innervation zone in a muscle blocked with µCTX is the sum of a diffuse field potential from the summed EPPs at a large number of NMJs, analogous to the CMAP, and a much more localized response from the site of interest, which we refer to here as EPPex. The field response varied little with movements of the electrode of even 100 s of µm, but the local response was very sensitive to movements of a few micrometres. To obtain the local response without ‘contamination’ by the field response, a second extracellular electrode was frequently positioned 5–10 µm away from the site of focal recording and the signal from this reference electrode was subtracted from the signal from the focal electrode (Katz & Miledi, 1965). Successful placement of the focal electrode on the nerve was indicated by the presence of a nerve action potential in the average of a set of 25–100 responses (Angaut-Petit et al. 1990).
In muscles that had been treated with BoNT/A, the structure of the original NMJs was sometimes so altered that they could not be identified with certainty. However, fibres with clearly identifiable original NMJs were used when possible, to allow reliable comparison of events associated with the new sprouts with those arising in the region of the original NMJs. In these muscles, most recordings from sprouts were made within 50 µm of the original NMJs.
Analysis of electrophysiological data
All electrophysiological recordings were analysed with the Clampfit package in pCLAMP 9 (Axon Instruments). Spontaneous events were extracted using the ‘template search’ facility.
Kinetics of synaptic events. EPPex latency was defined as the time from the onset of the stimulus artefact to the time when the potential reached 20% of its peak value. The rise time and decay time of synaptic events was defined as the time taken to go from 10 to 90%, or 90–10%, respectively, of the peak amplitude.
Quantal content. The quantal content of the EPPin was estimated from the ratio of EPPin amplitude to mEPPin amplitude, after suitable correction of the signals for membrane potential and nonlinear summation, assuming a reversal potential of 0 mV (Martin, 1955; Slater et al. 1992). No additional adjustment was made for the effect of membrane capacity on quantal content (McLachlan & Martin, 1981) (equivalent to setting their f = 1).
Immunolabelling
Whole ETA muscles were incubated in rhodamine–
-bungarotoxin (R-BgTx, 10–6
M; Molecular Probes/Invitrogen) in Liley's solution for 1 h and then washed thoroughly. They were then fixed in 0.5–1% paraformaldehyde in 0.1 M phosphate buffer for 30 min. After washing in buffer, muscles were permeabilized in alcohols (ethanol for 10 min followed by methanol for 10 min, both at –20°C) followed by incubation in Triton X-100 (0.1%, 10 min, room temperature). They were then incubated overnight at 4°C in a mixture of rabbit anti-neurofilament protein (NFP; AB1982, Chemicon) and rabbit anti-synaptophysin (RB1461, Neomarkers, Stratech), each at a dilution of 1/100, in PBS containing 3% BSA (Sigma-Aldrich) and 0.1 M lysine (Sigma-Aldrich). The next day, after washing in PBS, they were incubated for 3 h at room temperature in FITC–swine anti-rabbit (1/100) (DAKO) which had been preincubated in half the volume of neat rat serum to reduce non-specific binding. After washing overnight in PBS, the muscles were mounted on slides in Vectashield (Vector Laboratories).
Labelling of single teased muscle fibres
To assess the distribution of differentiated postsynaptic sites, muscles were labelled with R-BgTx (10–6 M, 1 h) and then fixed in 1% paraformaldehyde 1 h, 0.1 M phosphate buffer. After fixation junctional regions, several millimetres long, of 30–50 individual fibres from each muscle studied were dissected out and mounted in Vectashield for subsequent analysis.
Microscopy
Image recording. Fixed preparations and teased fibres were viewed with a Leica DMRA fluorescence microscope (Leica Microsystems) using filter blocks N21 (TMRITC) and L5 (FITC, selective). Images were captured with a SPOT-II camera (Diagnostics Instruments) under control by MetaMorph. When appropriate, Z-series were recorded at a spacing of 1 or 2 µm using a 50x oil immersion objective.
Image analysis. The area and number of regions of postsynaptic differentiation were determined from teased fibres. For each fibre, each of what appeared to be separate regions of high R-BgTx labelling was outlined by hand. The regions of high R-BgTx labelling at what appeared to be the original endplate, i.e. to consist of a closely packed cluster of highly intense spots, were also outlined. The areas of all regions were calculated using MetaMorph.
Statistical analysis and data presentation
Averaged data are presented with the S.D. and relevant sample size. The significance of differences between means was determined using Student's two-sided paired t test, accounting for small samples where appropriate. The relationship between number of BoNT/A injections and rate of functional recovery, illustrated in Fig. 12, was investigated using analysis of covariance (http://udel.edu/~mcdonanld/statancova.html).
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Results |
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Time course of recovery from a single injection of BoNT/A
Neuromuscular transmission. Nerve-evoked contraction of the ETA was fully blocked within 1–2 days of BoNT/A injection. The first visible signs of recovery occurred 2–3 weeks later, when weak contractions in response to nerve stimulation could be seen. During the next 3–4 weeks the contractions grew in strength and became hard to distinguish from normal by eye. During the period of impaired transmission, many muscles displayed spontaneous fibrillation, in a manner typical of denervated muscles. This was most prominent 1–4 weeks after BoNT/A treatment. Occasionally spontaneous contractions were seen up to 4 months after BoNT/A treatment but not after that time.
To provide a more quantitative measure of the overall recovery of function, compound muscle action potentials (CMAPs) were recorded (Fig. 1A). In control ETA muscles these have a mean amplitude of 1.92 mV (S.D. 0.48 mV, n = 10). They were completely abolished within 1–2 days after BoNT/A injection (Fig. 1B). During recovery, CMAPs were first detected at 2–3 weeks, and some were within the normal range of amplitudes 8–10 weeks after BoNT/A injection. Nonetheless, the CMAP amplitude was usually less than normal even 10–12 weeks after BoNT/A.
To assess the changes in quantal release of ACh from the whole motor nerve terminal we used intracellular electrodes to record EPPins and mEPPins. From their amplitudes we calculated the quantal content (see Methods). These values are shown, as a function of time after BoNT/A injection, in Fig. 2. At NMJs in normal ETA muscles, the quantal content in our conditions was 57.9 (S.D. 26.8, n = 10). The quantal content fell to < 0.1 within 1–2 days after BoNT/A. The first NMJs where the QC had increased to > 1 were seen 12 days after injecting BoNT/A. By 6–7 weeks after BoNT/A the QC was about 60% (33.2, S.D. 13.1, n = 3) of normal and after 10–14 weeks was indistinguishable from normal (61.6, S.D. 15.2, n = 5). The mean EPPin amplitude was significantly correlated with the CMAP amplitude in the same muscle throughout the recovery period (r = 0.92, n = 53, P < 0.001). This implies that the experimental treatments used to record EPPs (µCTX, 4-Di-2-ASP) did not have a disproportionate effect on, for example, quantal release early in recovery. Mice of several strains were used in the course of this project (see Methods). No differences between the strains were observed in either the timing or extent of recovery.
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Axonal sprouts could be seen extending from the motor axon terminals within a week or less of BoNT/A injection (Fig. 3A, ‘1–2 weeks’). Over the next few weeks, these sprouts grew further, primarily along the muscle fibres. From about 2 weeks after BoNT/A injection, lateral branches began to emerge from the main longitudinal sprouts and then gradually increased in length. From an early stage, many of the growing ends of the sprouts ended in roughly circular varicosities, typically about 3 µm in diameter.
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We quantified the incidence of sprouts and AChR clusters after BoNT/A intoxication in order to relate their appearance to the onset of evoked quantal release (Fig. 3B). At a population level, the AChR clusters form about 5 days after the formation of sprouts. A clear increase in quantal content, represented by the fraction of NMJs where the quantal content has increased to > 5 (normal value 58), is present at 50% of NMJs by about a week after AChR clusters form. To trigger a muscle action potential and contraction probably requires the release of about 25 quanta (based on data from rat soleus muscles; Wood & Slater, 1997). As shown in Fig. 3B, this level of release occurs at 50% of NMJs by about 40 days after BoNT/A.
In summary, our observations of the ETA confirm earlier observations (de Paiva et al. 1999) that after an injection of BoNT/A, well formed axonal sprouts whose terminals are associated with AChR clusters are already present for several days before quantal release of ACh becomes readily detectable. However, the entire recovery process occurs more rapidly in ETA than in the mouse soleus, extensor digitorum longus or levator auris (LAL) (Duchen, 1970; Tonge, 1974; Santafe et al. 2000). We have confirmed the slower recovery in soleus (L. Gurney, K. K. Pang & C. R. Slater, unpublished observations).
How much do new synaptic contacts contribute to functional recovery?
To estimate the contribution of new synaptic sites to the initial recovery after exposure to BoNT/A, we first located individual new synaptic sites and then estimated the intensity of release from them. We then determined the extent and number of such new synaptic contacts. Finally we used these data to estimate the summed contribution of release from all the new sites on a muscle fibre to the total quantal content.
Location of release sites. The spatial resolution of intracellular recording does not allow quanta released from new synaptic sites to be easily distinguished from those released from the site of the original NMJ. To study release from new sprouts, we therefore used focal extracellular recording (del Castillo & Katz, 1954), which allows the sites of synaptic current generation to be defined with a resolution of 5–10 µm.
The nerve–muscle preparations were routinely labelled with the mitochondrial dye 4-Di-2-ASP to allow the nerve and its sprouts to be visualized during electrophysiological recording (Magrassi et al. 1987). Normal nerve terminals labelled with this dye generally appear as a compact cluster of discrete varicosities, each typically 2–3 µm in diameter (Fig. 4). Extracellular electrodes placed near such labelled spots frequently record both spontaneous and evoked quantal events which we refer to as EPPex. Comparison of records from two electrodes placed at different spots at the same normal NMJ confirms that this mode of recording can differentiate between quanta released from sites less than 10 µm apart (Fig. 4).
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An important additional indication that the recording electrode was placed over a sprout was the ability to record an extracellular nerve action potential (Brigant & Mallart, 1982; Mallart & Brigant, 1982; Angaut-Petit et al. 1990). The detection of these signals was improved if the electrode was pressed against the sprout. However, this risked causing mechanical damage. We therefore generally looked for the nerve action potentials in averages of 25–100 traces. Only when both 4-Di-2-ASP labelling and an extracellular nerve action potential were present was the absence of detectable synaptic events taken as a significant negative finding.
Assessment of release. Early in recovery only occasional stimuli evoked detectable responses at any extracellular recording site (Fig. 5B). For example, at the NMJ illustrated in Fig. 5, 21 days after BoNT/A, responses from the original NMJ were seen in 28/100 trials and in only 1/100 trials at the ‘new’ site. In contrast, EPPins were recorded in 82/100 and 84/100 in these two series of trials.
The very low frequency of spontaneous mEPPexs observed made it impractical to estimate directly the mean number of quanta released by a single nerve impulse from new synaptic spots. Instead, we made the assumption that during the early stages of recovery, as in many other situations where quantal content is low, the release process obeyed Poisson statistics (Katz, 1969). Analysis of the distribution of amplitudes of EPPexs suggested that this was a valid assumption. For example, at the NMJ illustrated in Fig. 5, the series of 100 trials recorded from the ‘original’ site included 72 ‘failures’, i.e. stimuli that failed to evoke a detectable response, 24 responses whose amplitude was intermediate in amplitude (mean 0.15 ± 0.024 mV) and four responses whose amplitude was clearly greater (mean 0.27 ± 0.030 mV). The Poisson distribution predicts that for a failure rate of 72%, 24% of the responses should consist of 1 quantum and 4% of 2. In this case, the observed distribution of EPPex amplitudes agreed well with that predicted by the Poisson distribution for a mean quantal content of 0.33.
We therefore used the fraction of response ‘failures’ to estimate the mean number of quanta released in the pickup area of the electrode. We defined this value as the ‘local’ quantal content (QClocal), i.e. the mean number of quanta detected by the extracellular electrode at a given recording site in response to a single stimulus. An inherent limitation of this method for estimating QClocal is that it can only be used when the fraction of failures is greater than about 5%. This was generally the case up to 6–8 weeks after exposure to BoNT/A.
Comparison of the QClocal at original and new synaptic sites shows that at the earliest stages of functional recovery, 2–3 weeks after exposure to BoNT/A, more quanta were released by a single nerve action potential at recording sites at the original NMJ than at the new synaptic spots (Fig. 6). This situation was maintained for at least 2 months, by which time overall recovery of quantal release was more than 50% complete. During the period 2–12 weeks after exposure to BoNT/A, the mean QClocal at ‘new’ synaptic spots was 17% (S.D. 19%, n = 10 muscles) of that from sites in the original junctional region.
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Contribution of new synaptic contacts to functional recovery. We estimated the quantal content from new contacts by assuming that our extracellular recordings detected quanta from a single region of new synaptic contact. This seemed justified because the average area of these regions (13.6 µm2) is well within the pickup area of the extracellular electrodes (100–300 µm2), and there was generally a distance of at least 10 µm between these regions (cf. Fig. 7A; 17 and 24 days). On this basis, the total quantal content is given by the product of the number of regions per muscle fibre and the local quantal content. When new AChR clusters were closer together than 10 µm, this procedure would tend to overestimate release from new spots. The resulting values are shown in Fig. 8, in which data have been pooled into bins 5 days wide.
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Temporal features of evoked synaptic events during recovery
In the course of our studies it became clear that there were significant changes in both the latency and time course of the evoked synaptic responses at NMJs during the process of recovery from BoNT/A. Since it is known that cleavage of SNAP-25 by BoNT/A influences the dynamics of quantal release at other synapses (Sakaba et al. 2005), we attempted to define these changes in more detail.
Latency and synaptic delay of evoked synaptic events during recovery from BoNT/A. In a previous study, made on the LAL muscle of mice, it was found that the latency of EPPs increased during recovery from BoNT/A (Santafe et al. 2000). The latency of the EPP has two components: the conduction time in the nerve and the synaptic delay between the arrival of the nerve impulse and detectable quantal release. To distinguish between these factors as determinants of the increased latency, we studied EPPexs. During the first 30 days after BoNT/A, the average latency from stimulus to onset of EPPex was nearly twice as long as normal (control, 1.1 ± 0.13 ms, n = 5 versus BoNT/A, 1.9 ± 0.33 ms, n = 23, P < 0.0001) (Fig. 9A and B).
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In contrast, we found clear evidence for an increase in the second component of EPP latency, the synaptic delay. We defined this as the time from the negative peak of the nerve action potential to the foot of the EPPex (Fig. 9C). At those NMJs where both a clear nerve action potential and EPPex were recorded, the synaptic delay increased from a normal value of 0.32 ± 0.034 ms (n = 8) to 0.73 ± 0.16 ms (n = 26 NMJs studied up to 30 days after exposure to BoNT/A, P < 0.0001). There was no significant difference between the delays observed at new and original sites (0.70 ± 0.14 ms, n = 6 versus 0.74 ± 0.17 ms, n = 20). Thus, during recovery from BoNT/A, the events coupling nerve terminal depolarization to quantal release occur more slowly than normal.
Kinetics of synaptic potentials during recovery. During the early stages of recovery of transmission, evoked synaptic potentials with an unusually slow time course were sometimes seen (Fig. 10). Some of these recalled the ‘giant’ or ‘slow’ spontaneous miniature potentials previously described by others (Thesleff & Molgo, 1983; Thesleff et al. 1983; Kim et al. 1984). In those earlier studies, changes in the time course of evoked events were not reported.
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Of particular interest were a small number of fibres where more than one population of EPPins, differing in time course were present (Fig. 10E–H). Figure 10E shows most of the 71 events evoked during a series of 100 trials from one of these fibres. Of the 100 responses, 29 were ‘failures’, indicating that the quantal content of the EPPin was 1.24. Of the events that were evoked, 52 were ‘pure fast’ (e.g. Fig. 10F), 7 were ‘pure slow’ (Fig. 10H) and 12 were ‘mixed’ (Fig. 10G), apparently the sum of a fast and a slow response. Thus 64 responses included a fast response and 19 a slow response. If the responses of these two classes arose independently, the probability that both a fast and a slow response would appear in the same trial is 0.64 x 0.19 = 0.12. This predicts that 12 responses should be mixed, whereas 11 were observed. The results of this analysis thus support the view that the two classes of response at this and several other similar NMJs were evoked independently. Unfortunately, these fibres were too rare to allow detailed analysis of the sites of origin of the two different classes of evoked response.
Response to repetitive stimulation. The results described so far relate to NMJs stimulated at 1 Hz. As a further test of the recovery of quantal release, in conditions more related to normal functioning, we studied the response to repetitive stimulation in some muscles 4 months after exposure to BoNT/A. Over the frequency range of 1–100 Hz, the properties of release at NMJs exposed to a single injection at BoNT/A were qualitatively similar to normal (Fig. 11). At frequencies below 50 Hz there was a depression of release during a train of 100 stimuli. This included both a rapid phase consisting of the first 20 or so responses followed by a much slower phase. At 50 and 100 Hz, these two phases of depression were preceded by a brief phase of potentiation. This resulted in EPPins amplitudes about 1.2 times the initial value at both treated and control NMJs.
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Recovery of neuromuscular transmission. It is clear from clinical experience that some degree of functional recovery is possible even after multiple injections of BoNT/A. However, we are unaware of any reports of systematic studies of functional recovery of quantal release after more than one exposure to BoNT/A. We therefore investigated the recovery of quantal content in the ETA muscles after two and three injections, given at intervals of roughly 3–4 months (Fig. 12). In each case, the standard BoNT/A injection initially caused a rapid and profound block of quantal release.
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The statistical strength of these conclusions was tested using ANCOVA. The outcomes of this analysis are: (1) the regressions of the three groups (1, 2 and 3 injections) are not homogeneous (P = 0.018), (2) the regressions of the data for two and three injections are not significantly different, (3) the regression of the data for one injection is significantly different from that for either two (P < 0.0001) or three (P << 0.0001) injections, and (4) the regression of the data for one injection is significantly different from that for the pooled data for two and three injections (P << 0.0001). It is thus clear that in these experiments, recovery of evoked quantal release after an injection of BoNT/A was significantly slower in those mice that had received one or two previous injections.
Structural abnormalities persist after multiple BoNT/A injections. Although no systematic studies have been made, available evidence indicates that structural abnormalities of motor innervation persist in humans for years after the last of a series of BoNT/A injections (Alderson et al. 1991). We were therefore interested to see whether NMJs in the mouse ETA whose function had recovered after two or three BoNT/A injections showed similarly persistent structural abnormalities. In spite of the clear functional recovery, the abnormalities of the anatomical features of motor innervation were still present months after a second or, as illustrated in Fig. 13, a third injection.
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At higher magnification, it was clear that the individual NMJs were also often very abnormal. Some examples from muscles at 124 days after a third injection of BoNT/A are shown in Fig. 13D–G. These show both persisting individual sprouts (Fig. 13D) and multiple lateral sprouts (Fig. 13F and G). At longer times after the final injection of BoNT/A (223 days), a number of very abnormal NMJs persisted (Fig. 13H–K). These were often associated with lateral branches of long myelinated axons coursing along the muscle fibres (Fig. 13I). Although NMJs from uninjected contralateral muscles were often rather complex (Fig. 13B), probably a result of the advanced age (476 days) (Barker & Ip, 1966; Tuffery, 1971; Cardasis & LaFontaine, 1987), they only rarely had sprouts.
In summary, it is clear that although substantial recovery of quantal release occurs in ETA muscles after two or three injection of BoNT/A, striking structural abnormalities of the motor axons and NMJs may persist for many months.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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An important finding of our study of the mouse ETA is that the region of the original NMJ plays a much greater role in the initial recovery from the blocking effects of BoNT/A than previously thought. In fact, our estimates of quantal release from new synaptic contacts suggest that they play a relatively minor role in functional recovery. A second important finding is that after several exposures to BoNT/A, while neuromuscular transmission can be restored to near normal levels, it does so more slowly than after a single exposure and is accompanied by profound abnormalities in the structure of the intramuscular nerve and the NMJs. These findings raise some concerns about the cumulative effects of repeated exposure to BoNT/A in a therapeutic or cosmetic context. At the same time, they attest to the remarkable adaptive plasticity of the mammalian NMJ.
Initial recovery
Timing of recovery. The time course of recovery of quantal release after administration of BoNT/A is similar in the ETA to that described for sternomastoid (de Paiva et al. 1999) but is somewhat faster than that reported for other mouse muscles (soleus, EDL, LAL) where quantal content reached 50% of its normal value in 10–14 weeks (Tonge, 1974; Santafe et al. 2000). In making these comparisons, it is important to bear in mind that different preparations of BoNT/A were used, and possibly different effective doses, and that different methods were used to estimate quantal content. In spite of these differences, the time required for all these mouse muscles to recover after BoNT/A is roughly similar to the duration of effectiveness of a single injection in man. It should be noted, however, that because of the high safety factor for neuromuscular transmission (Wood & Slater, 2001), patients may sense significant functional recovery when transmission is still substantially depressed. What of course differs between mice and men is the fraction of the lifespan required for recovery.
Several studies have noticed the ‘weaker’ sprouting response evoked by BoNT/A in functionally fast muscles (Duchen, 1970; Brown et al. 1980; Caroni et al. 1997; Frey et al. 2000). It is reasonable to suppose that that the pattern of short, high frequency bursts characteristic of fast motor units (Hennig & Lomo, 1985), which is known to be particularly effective in suppressing many effects of muscle inactivity (Windisch et al. 1998), might also suppress axonal sprouting and thus hinder recovery from BoNT/A (Brown et al. 1980). However, it is clear from both our observations of the ETA and previous studies of the LAL (Juzans et al. 1996a) that rapid and extensive sprouting can occur in an almost exclusively fast muscle, and that functional recovery can also be relatively fast. As discussed below, one possible explanation for this may be that fast muscles tend to make new sprouts within the area of the original NMJ.
Contribution of new synaptic contacts to recovery. Our conclusions concerning the role of new synaptic contacts to functional recovery from BoNT/A differ substantially from those of the only previous study to investigate this point (de Paiva et al. 1999; Meunier et al. 2003). Because the conclusions of that study have been cited in a number of subsequent articles (Meunier et al. 2002; Foran et al. 2003; Dolly & Aoki, 2006; Osborne et al. 2007), it is important to consider the possible origins of these differences.
de Paiva et al. (1999) used FM1-43 uptake to assess nerve terminal function. In many situations, a good correspondence between the intensity of FM1-43 labelling and quantal release has been demonstrated (Cochilla et al. 1999). However, in the present situation, this method has certain limitations. One is its inherent sensitivity. During the early stages of recovery after BoNT/A, the intensity of quantal release we observed at the sites of the original NMJs is only a few per cent of normal. While electrophysiological recording is capable of detecting the effects of individual quanta, the fluorescence signals associated with such low levels of release would be difficult to detect. Further, it is important to note that the lowest levels of FM1-43 labelling of the original terminals documented by de Paiva et al. remained at 10–25% of the normal value (see their Fig. 1h; de Paiva et al. 1999). However in studies, including the present one, where quantal release has been measured directly, it has initially fallen to less than 1% of normal. This suggests that the intensity of FM1-43 fluorescence may not accurately reflect the low levels of quantal release that follow BoNT/A intoxication.
An important further assumption made by de Paiva et al. 1999 is that the FM1-43 signal from the entire length of the sprouts reflects physiologically significant transmitter release. Only transmitter quanta released within a few micrometres of AChR clusters can have any impact on membrane excitation. However, it is clear from de Paiva et al. and from this study that AChR clusters occupy only a small fraction of the total length of the sprouts (cf. Figs 3 and 13). Thus, while membrane cycling may occur all along the sprouts as they grow and extend lateral branches, much of this may not be relevant for quantal release of ACh. Our electrophysiological approach, while not measuring ACh release directly, does indicate ‘effective’ release, i.e. that which leads to membrane depolarization.
The influence of terminal sprouting on quantal release from the mammalian NMJs has been investigated in another context, that of recovery after chronic block of nerve impulses. Using a cuff containing tetrodotoxin to block the rat sciatic nerve, Tsujimoto et al. (1990) observed an increase in quantal content before any sprouting was detectable. At later times, the quantal content was no greater at those NMJs where sprouting had occurred than where it had not. They concluded that sprouting and increased quantal content are not causally related in the conditions of their experiments. By implication, a mechanism must exist by which quantal content can be raised within the confines of the original NMJ. A similar conclusion was reached in studies of increased quantal release in the absence of any enlargement of the NMJ in myasthenia gravis and an animal model of it (Plomp et al. 1992, 1995). The mechanism of the up-regulation of quantal release in these situations is unknown. It is therefore impossible to know whether a similar mechanism might be responsible for the recovery of the original NMJs after exposure to BoNT/A. However, it should be noted that in these other examples there is no indication of structural alterations to the proteins involved in exocytosis, or to the resulting need to replace them, that characterize the response to BoNT/A.
Changes in synaptic events during recovery. Previous studies of NMJs blocked by BoNT/A have reported an increase in the number of spontaneous mEPPs with abnormally large amplitudes and long time courses (Thesleff & Molgo, 1983; Kim et al. 1984), sometimes referred to as ‘giant’ or ‘slow’ mEPPs. Much less has been reported about the properties of evoked EPPs and EPCs. While it has been stated that the rise time of individual evoked EPPs remained normal during the recovery period (Kim et al. 1984), evidence has also been presented of a slowing of the decay phase of EPCs 1 week after exposure to BoNT/A (Sellin & Thesleff, 1981). In the present study we observed a striking slowing of the time course of the evoked responses recorded during recovery. This slowing resulted from increases in the latency, synaptic delay and the rise and decay times of the EPPs. These changes reached a peak just as transmission began to be effective and were largely reversed when the quantal content was still only 20–30% of normal. It may be that these changes were missed in earlier studies which concentrated more on changes in the first week after exposure to BoNT/A.
The nature of these changes in EPP time course is likely to be multifactorial. Both the prolonged synaptic delay and the slower rise time of the EPP suggest alterations of the release process. These may well be related to recovery from the effects of the cleavage of SNAP-25 (Sakaba et al. 2005). In addition, there is evidence that during recovery from BoNT/A, L- and N-type voltage-gated calcium channels are present in the nerve terminal along with the P/Q-type channels normally present (Day et al. 1997; Santafe et al. 2000; Pagani et al. 2004). It is possible that the L- and N-type channels are not as closely associated with the active zone (Urbano et al. 2003) as the P/Q-channels and this may lead to an increase in latency and/or rise time of the EPP. Little is known about the sequence of events in this process of nerve terminal maturation or how it is regulated.
On the postsynaptic side, the period of inactivity imposed by the action of BoNT/A almost certainly results in reduction of AChE activity (Hall, 1973) at the original synaptic sites and increase in the expression of 
-AChR subunits and hence slow, ‘fetal’ AChRs (Klarsfeld & Changeux, 1985; Goldman et al. 1988; Witzemann et al. 1991; Koltgen et al. 1994; Missias et al. 1996). Both of these postsynaptic changes would be likely to lead to the prolongation of the falling phase of the EPP. However, they would not be expected to have a major impact on the rising phase. AChE appears at new synaptic sites soon after the sprouts grow out (Duchen, 1970) and would be expected to hasten the falling phase of synaptic currents and potentials at the new sites. How, if at all, the slow EPPs we observed correspond to the slow mEPPs reported previously is unclear. Although we found a correlation between the time courses of EPPs and mEPPs from the same fibres, the numbers of mEPPs was too small to be confident of this relationship.
It was particular striking that more than one population of EPPs could be recorded from some single muscle fibres (Fig. 10). The very slow rise time of the ‘slow’ components strongly suggests local variations in the properties of the nerve terminals. It may be that the various synaptic spots, potentially both new and old, on a given muscle fibre acquire ‘mature’ properties at different times. The increase in quantal content associated with recovery of the ETA lasts at least 8–10 weeks. Previous studies have shown that axonal sprouts may continue to grow for a similar length of time (Juzans et al. 1996a; Santafe et al. 2000). It is therefore likely that new contacts continue to be made throughout this period, though possibly at a progressively slower rate as muscle activity returns to normal. This suggests that synaptic contacts established both early and late in the recovery process might coexist on the same muscle fibre, and might have relatively fast and slow EPPs associated with them.
By the end of the main recovery process, 3–4 months after exposure to BoNT/A, the functional properties of mouse ETA NMJs that we examined had essentially returned to normal. The observation that the response to repetitive stimulation is qualitatively similar to that in untreated mice, showing both rapid transient potentiation at high frequency followed by depression, suggests that both calcium dynamics and synaptic vesicle pools in the motor nerve terminals recover their normal properties after treatment with BoNT/A. Not enough experiments of this kind were performed to know whether the differences between the curves in Fig. 11 represent statistically significant differences.
Recovery from multiple exposures to BoNT/A
Ours is the only study we are aware of that has examined systematically the state of neuromuscular
