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Recovery from botulinum neurotoxin poisoning in vivo
Neuroscience 139 (2006) 629 – 637
RECOVERY FROM BOTULINUM NEUROTOXIN POISONING IN VIVO J. E. KELLER*
BoNT/C and /D have never been known to cause botulism in humans, yet these two serotypes cause significant disease among livestock and water fowl (Duncan and Jensen, 1976; Hubalek et al., 1991; Tucker et al., 2002; Centers for Disease Control and Prevention, 1998). Poisoning by any one of these neurotoxins impairs communication between nerve and muscle. In humans, the resulting paralysis caused by BoNT/A or B tends to be protracted regardless of the route of entry. This makes treating the disease difficult but has led to using purified BoNT preparations to treat several human neuromuscular disorders (Hughes et al., 1981; Woodruff et al., 1992; Sloop et al., 1997; Brashear et al., 1999). In studies of healthy volunteers, local i.m. injection of medical-grade BoNT/A had a prolonged effect, with muscle function gradually returning over 12 months. Paralysis from BoNT/B endured for up to four months (Sloop et al., 1997; Brin et al., 1999). BoNT/E has not been used to treat human muscle disorders because of its relatively short duration of action (Whittaker et al., 1964; Sellin et al., 1983a). However, its prevalence in causing human botulism has placed much interest on its biological properties (Koenig et al., 1964) and recently it has been reported as having beneficial effects for reducing epileptic seizures in rats (Costantin et al., 2005). The BoNTs are 150 kDa proteins that enter motor nerve terminals by receptor-mediated endocytosis (Habermann and Dreyer, 1986; Coffield et al., 1994; Jahn and Niemann, 1994). A proteolytic domain of the toxin crosses the endosomal membrane by threading through a toxininduced pore leading to the cytosol (Boquet and Duflot, 1982; Koriazova and Montal, 2003) where it targets one of two synaptic proteins (Schiavo et al., 1992; Simpson, 2004). Proteolysis by BoNT/A, B or E is limited to either synaptosomal membrane associated protein of 25 kDa or vesicle-associated membrane protein 2, where each BoNT cuts its substrate at one serotype-specific bond. The resulting cleavage disrupts proper synaptic vesicle trafficking during neurotransmission (Pevsner and Scheller, 1994; Fasshauer et al., 1998) and consequently causes muscle paralysis. Systemic poisoning can often become life threatening. However, controlled injection of BoNT/A or B directly into muscle tissue generally does not cause illness and has become a useful medical procedure for preferentially paralyzing overactive nerve groups (Jankovic and Brin, 1991; Hallett, 1999). Few studies have measured recovery from BoNT intoxication because quantifying muscle activity over the course of many weeks is difficult. Several studies indicate that the longest duration of paralysis is in the order of BoNT/A⬎B⬎E (Sellin et al., 1983; Sloop et al., 1997; Eleopra et al., 1998; Adler et al., 2001; Jurasinski et al.,
Laboratory of Bacterial Toxins, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, 29 Lincoln Drive, HFM 434, Room 122, Bethesda, MD 20892, USA
Abstract—Botulinum neurotoxins cause the disease botulism, which is characterized by prolonged muscle paralysis. In contrast, injections of low doses of purified botulinum neurotoxins do not cause systemic illness but produce localized muscle paralysis that is beneficial for treating several human medical disorders involving uncontrollable muscle contraction. Optimizing the therapeutic efficacy while diminishing adverse reactions requires precise knowledge of toxin potency as well as a clear understanding of how each toxin causes disease. A novel in vivo mouse assay has been used to correlate toxin dosage with the duration of muscle paralysis. Voluntary running activity performed by mice was proportional to the amount of toxin injected into the hind limbs and the subsequent rate of recovery over the ensuing days or weeks was a function of botulinum neurotoxin serotype A or B concentration. Botulinum neurotoxin A produced longer paralysis than botulinum neurotoxin B consistent with human observations. A third serotype, botulinum neurotoxin E, had the shortest duration of action, but unlike the other two toxins, dosage did not influence recovery time. Botulinum neurotoxin A recovery appeared biphasic with the initial phase about two-fold faster than the final phase. Over four weeks, muscle activity had gradually improved following the highest botulinum neurotoxin A dose, reaching about half of the normal running activity. Lower botulinum neurotoxin A doses led to incrementally faster and complete recovery. Persistence of maximum paralysis was exponentially related to botulinum neurotoxin A dosage, with a doubling of the paralysis time occurring with every 25% increase of the toxin concentration. In contrast, the rate of recovery from botulinum neurotoxin B was monophasic relative to toxin dosage and the duration of maximum paralysis was linear relative to dosage. Combinations of botulinum neurotoxin A and B and botulinum neurotoxin A and E were tested and shown to exacerbate paralysis compared with individually administered serotypes. © 2006 Published by Elsevier Ltd on behalf of IBRO. Key words: botulism, botulinum neurotoxin, paralysis, recovery, potency assay.
Botulism is a neuromuscular disease characterized by progressive muscle weakness leading to paralysis, and possible death from respiratory failure. Of the seven known botulinum neurotoxins (BoNTs), only three routinely cause human disease, BoNT serotypes A, B or E. Since 1950, serotype F has been attributed to five cases, whereas, *Tel: ⫹1-301-402-4418; fax: ⫹1-301-402-2776. E-mail address: [email protected] (J. E. Keller). Abbreviations: BoNT, botulinum neurotoxin; EDB, extensor digitorum brevis; EDL, extensor digitorum longus; LD50, dose that will kill 50% of mice injected within a four day period. 0306-4522/06$30.00⫹0.00 © 2006 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.12.029
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2001; Foran et al., 2003). However, the most common potency value for BoNT activity is derived from lethality titration curves where groups of mice are exposed to the toxin by i.p. injection and monitored for four days (Schantz and Johnson, 1990). The dose of toxin that kills 50% of the mice is one standard lethal-dose unit (LD50). In addition to LD50 measurement, two laboratory methods exist for monitoring recovery following localized i.m. injection of toxin. In the first method, in situ or in vitro nerve-muscle preparations can be used to measure either muscle contraction or neuronal electrical properties; both measurements require skilled surgical techniques to properly expose the nerve tissue, and this approach requires dozens of animals to compile useful data (Cull-Candy et al., 1976; Sellin et al., 1983). A less complicated model system entails monitoring the toe-spread reflex in living mice or rats (Aoki, 2001; Jurasinski et al., 2001; Meunier et al., 2003). This method relies upon visually assigning a numerical value for legfoot-toe kicking that occurs upon lifting animals by the tail. As a result, large numbers of animals are required to compensate for the subjective manner and high variability of assigning values. Both approaches are difficult to implement when testing multiple conditions such as different dosages using several BoNTs. Retaining the mouse for BoNT measurement is advantageous because sensitivity to the three BoNT serotypes is similar to human responses and the mouse LD50 assay is currently the most sensitive method used for detecting and measuring the potencies of all BoNTs (Schantz and Johnson, 1990). This study describes a novel in vivo method for assessing the effects of sub-lethal BoNT injection into the mouse hind limb. Toxin potency and duration of paralysis were measured by recording the nightly running activity of mice on exercise wheels. This approach reduced the number of animals needed compared with other methods and has allowed comparison of the biological actions of BoNT/A, B and E at several dosages. In addition to causing unique dose-dependent symptoms, recovery of muscle function was distinct for each BoNT, which improves the current understanding of the physiological long-term effects caused by BoNT poisoning.
EXPERIMENTAL PROCEDURES Animals BALB/cJ male mice at five weeks old (18 –20 g) were obtained from NCT (Frederick, MD, USA). All aspects of the experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, NIH Publication No. 80-23. The research had prior approval from the CBER Animal Care-and-Use Committee. A minimal number of animals was used in this study and every effort was made to reduce their suffering. Groups of eight animals were housed with free access to exercise wheels (Penn-Plax, Inc., Garden City, NY, USA) according to previous descriptions (Allen et al., 2001). Each wheel with a diameter of 11.5 cm was fitted with a magnet and micro-computer that recorded the number of revolutions, speed and total distance (Cat Eye, Co., Ltd., Boulder, CO, USA). At three-day increments animals were divided into groups or four then two per cage. Rooms were maintained with a 12-h light/dark
cycle, 20 –22 °C. Use of the wheels, feeding and drinking were ad libitum. Distance data were collected at 24 h intervals.
Toxin injection procedure Purified BoNT complexes were used in this study. BoNT/A (WAKO Chemicals, Richmond, VA, USA) was stored as a 1 mg/mL stock solution (2.0⫻107 ipLD50/mg). BoNT/B and E (WAKO Chemicals) were activated with trypsin and aliquoted as described previously (Ohishi and Sakaguchi, 1977; Keller et al., 1999). Potency of each was reported by the manufacturer to be 3.0⫻107 and 1.0⫻107 ipLD50 per mg of BoNT/B and E, respectively. Actual potency after trypsin treatment was 2.7⫻107 and 1.0⫻107 LD50 per mg of BoNT/B and BoNT/E. Aliquots of each toxin preparation were stored at ⫺80 °C. Dilution of each toxin was performed on the day of the experiment using sterile physiological saline containing 0.5% bovine serum albumin. Animals were lightly sedated with 2% halothane and the injection area was moistened with 70% isopropanol prior to administering toxin. I.m. injections of 30 L were given directly into the largest, central region of the gastrocnemius (calf) muscle mass. Since the animals had been running for 10 –14 days prior to injection, this injection volume did not cause visible swelling of the leg, as happened with non-running animals (Meunier et al., 2003). Because physical tissue trauma enhances the recovery rate from BoNT/A, syringes with a 32-gauge diameter were used to reduce the likelihood of injury.
Statistical analysis Compiled distances from three or more cages are represented as average determinations regardless of the number of animals within each cage. Error bars represent one standard deviation unit. Linear and non-linear regression analyses were performed using Prism 3.0 (Graphpad, San Diego, CA, USA). In the case of recovery times, data during paralysis onset were excluded from regression analysis. Linear regression analysis of BoNT/A used the standard equation: y⫽mx⫹b. Nonlinear analysis used the following equations: (BoNT/A) y⫽(1⫺exp(⫺k⫻t))⫻M⫹c. Y is the distance (km); M, the plateau maximum was predicted at time (t), with a first-order rate constant, k or for (BoNT/B): y⫽B0⫹((T0⫺B0)/ 1⫹10C). B0 and T0 represented the lower and upper limit values of the curve. The constant, C, was log (EC50⫺t)⫻H. Where EC50, t and H represent the 50% recovery point at time, t, which was influenced by a cooperativity coefficient, H.
RESULTS Gastrointestinal symptoms were observed in response to i.m. injection of BoNT/A and B when the dose exceeded 0.7 LD50. BoNT/A i.m. injection into the hind limb elicited diarrhea, whereas, BoNT/B reduced fecal accumulation within the cages, suggesting sub-lethal i.m. administered BoNT/A or B can selectively poison different autonomic neurons of the GI tract. Interestingly, BoNT/E toxicity was very sharp; animals did not display systemic signs of illness even at 0.9 LD50 units but all died within 18 h if given 1.0 LD50. At all doses of BoNT/A, B or E tested, animals demonstrated an ability to move, eat, drink and groom regardless of the toxin type. Animal housing was examined to optimize the distance and consistency of voluntary running activity. Mice were active almost exclusively during the 12 h dark cycle (⬎95%) with only slight running displayed during the daytime. Initially eight animals, then subsequently smaller groups were tested. Housing three or more animals within
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Fig. 1. Nightly running activity. (A) Mice were housed in various sized groups with access to two exercise wheels in each cage. The average distance (km) per mouse was calculated from running values collected over three days. Data were compiled from at least four cages for each condition. (B) The best paradigm required two mice co-housed with access to two running wheels. Mice that demonstrated at least 5 km per night were monitored and distances were recorded daily. Data from four cages (, □, , Œ) are shown with each symbol representing individual data points recorded at 24 h intervals. (C) Two animals in each cage were treated as indicated and running activity was collected over three days. Injection of BoNT/A (0.2 LD50 units) reduced running activity by about three-fold compared with untreated animals (n⫽4). Animals injected with either saline or BoNT/A combined with antitoxin did not demonstrate reduced running over a period of three days.
a cage was counterproductive since individually each animal ran approximately five hours per night (Konhilas et al., 2004; Lightfoot et al., 2004; Irani et al., 2005), thus limiting wheel access during the 12 h active period (Fig. 1A). This was resolved by placing two animals together, which eliminated aggressive competition that often developed within larger groups. When single animals were tested, running
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became sporadic with increasing variation (Fig. 1A). Based on these data, the experimental design consisted of two mice in a single cage; running distances were reported as the average between the two animals. Pairs of animals that ran consistently near 6 km per night were used for further experimentation (Fig. 1B) (Lightfoot et al., 2004; Irani et al., 2005). The running distances remained generally constant over a period of several days and were not affected by injection of saline or BoNT/A premixed with mouse antitoxin (Fig. 1C). Injection of BoNTs into the hind limb reduced running distances when toxin was administered over a four- to five-fold range of sub-lethal doses. The effects caused by each toxin are described below in terms of onset of paralysis, duration of total paralysis (zero running) and the rate of recovery after total paralysis subsided. Running distances were recorded for up to four weeks following injection of BoNT/A (Fig. 2A). Paralysis onset was similar for all doses tested with the maximum effect occurring within 48 h of injection. At the lowest dose, 0.14 LD50 unit, the total running distance was reduced by 85%. Recovery began on the third day with gradual improvement leading to normal physical activity about seven days after injection. Higher doses of BoNT/A led to complete cessation of running that persisted for many days relative to toxin dosage. The rate of recovery following total paralysis was similarly influenced by higher amounts of toxin. The highest dose tested, 0.8 LD50 units, produced eight to nine days of total inactivity followed by about 50% recovery (⬃3 km) 28 days later. Reducing BoNT/A by two-fold to 0.4 LD50 units resulted in only one day of about 95% inhibition of running and recovery began the following day. Full recovery was achieved within 15 days of the injection of 0.4 LD50 units. Regression analysis using a single exponential rate constant predicted that 90% recovery required 11, 24.1, 45.3 or 61 days post-injection for the four doses of toxin tested, which overestimated the actual recovery times observed for the two lowest BoNT/A doses. Since recovery from the highest dose of BoNT/A appeared to follow a nearly linear path from day 12 to day 30, the recovery profile for the BoNT/A poisoning was evaluated using two linear equations overlapping at day 19. Although BoNT/A dosage clearly influences the duration of total paralysis, the ensuing recovery may entail a two-part process. Recovery proceeded with a faster, initial phase until 33–38% of normal running was achieved after three to seven days, depending on the dose of toxin. A second recovery phase was 2.4-fold slower than the initial phase (Fig. 3C), which was too slow to allow full recovery from the highest dose of BoNT/A during the time of the experiment. However, extrapolating from the lower doses, full recovery from 0.8 LD50 units would require 48⫾8 days. Both linear rates of recovery were directly related to toxin dosage, and the analysis predicts that each rate becomes zero if a lethal amount of toxin were injected (Fig. 3C). Similar experiments using BoNT/B and E yielded results that were generally distinct from BoNT/A (Fig. 2B and 2C). BoNT/B, like BoNT/A, produced a dose-dependent duration of total paralysis. However, once total paralysis
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Fig. 2. Dose-response of running distance versus BoNT dosage. (A) Animals in groups of two were injected with BoNT/A (i.m. LD50 units): 0.14 (□), 0.40 (⽧), 0.6 (), or 0.8 (). One i.m. LD50 unit equals 0.06 mL of 1.25 pM BoNT/A. Error bars are averages of triplicate determinations except for 0.8 LD50 units (n⫽6). Non-linear regression analysis included data only after maximum paralysis subsided. The equation was y⫽(1⫺exp(⫺k⫻t))⫻M⫹c, as described in the Experimental Procedures. (B) Dose-response of running distance versus BoNT/B (i.m. LD50 units): 0.30 LD50 units (‘), 0.60 (), 0.90 (). Regression analysis for BoNT/B data used the equation: y⫽Bo⫹((To⫺Bo)/1⫹10C) described in the Experimental Procedures section. Error bars are averages of triplicate determinations. (C) BoNT/E determinations were done in duplicate using 0.4 LD50 units (□,Œ) or 0.90 (,). Each symbol from the BoNT/E trial represents a single determination. The arrow indicates the day of injection.
had subsided, the recovery phase proceeded at approximately the same rate for all BoNT/B concentrations. Unlike BoNT/A, recovery from BoNT/B was best represented by a sigmoidal curve representing early, middle and late phases of recovery that begin within several days of disease onset. Full recovery to 100% normal running activity occurred eight to nine days after total paralysis regardless of toxin concentration. The lowest concentration of BoNT/B tested
(0.3 LD50 units) demonstrated a delayed onset time, which peaked four to five days post-injection with about 90% blockade in running; the higher doses of BoNT/B completely inhibited running two to three days after injection. As with BoNT/A, the duration of maximum paralysis from BoNT/B was dose-dependent but unlike BoNT/A, the recovery rate was influenced to a lesser extent by BoNT/B dose (Fig. 2B). Injection of BoNT/E produced short-term paralysis relative to either BoNT/A or B (Fig. 2C). The concentration of BoNT/E did not influence the onset of muscle weakness nor did it alter the duration of paralysis. Toxin action persisted for up to seven days after injection regardless of the BoNT/E dose. This is similar to other studies that have examined BoNT/E in cultured neurons (Keller et al., 1999) and in rat muscle tissue (Sellin et al., 1983a; Adler et al., 2001), where recovery was complete within two weeks. In addition to confirming the brevity of duration, mouse running demonstrated that paralysis onset, duration of maximal blockade and the rate of recovery were generally independent of BoNT/E dose. The degree of paralysis observed on the second day after injection was the only symptom clearly influenced by the amount of toxin. Graphical depiction of the duration of total paralysis versus toxin concentration shows an exponential relationship between BoNT/A concentration and muscle inactivity (Fig. 4). In comparison, the duration of paralysis caused by BoNT/B is linear with toxin concentration producing similar paralysis to BoNT/A when both toxins were at relatively low levels but at higher BoNT/B concentrations, paralysis was brief compared with BoNT/A. BoNT/E, unlike BoNT/A and B, had practically no effect on the time of muscle inactivity. These observations are consistent with clinical observations of pharmaceutical-grade BoNT injections used to treat several medical disorders in human patients (Sloop et al., 1997; Eleopra et al., 1998). To examine if BoNTs can influence each other when simultaneously injected, combinations were tested and the results compared with previously reported observations (Adler et al., 2001; Meunier et al., 2003). BoNT/A (0.5 LD50 units) was combined with either BoNT/B or E. This dose of BoNT/A, as suggested by Fig. 2 and Fig. 4, produced maximum paralysis for two to three days (Fig. 5). Injection of BoNT/B at 0.3 LD50 units produced paralysis developing over several days leading to about a 90% reduction of running four days after injection but did not cause complete paralysis. Animals recovered fully within two to three weeks from the individual injections of BoNT/B and /A, respectively. In contrast, the AB mixture caused complete paralysis in less than 12 h compared with the slower onset times observed for the separate toxins (Fig. 2 and 5). Running activity from the AB combination remained suppressed by more than 90% for seven days followed by biphasic recovery over several weeks. Running activity returned to about half of the normal distance at the end of four weeks. A similar experiment was performed by mixing BoNT/A and E (Fig. 5). In this situation the onset of paralysis was not affected by the combination. Recovery from
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Fig. 3. Recovery rates from BoNT/A paralysis. Data from Fig. 2A are graphed demonstrating two linear recovery phases for the return of running activity. Both phases intersect at 33–38% (dashed line) recovery (Frames A and B). The rapid and slow rates are depicted as linear lines. Frame C demonstrates the dose-dependent relationship of the rapid () and slow () phases.
BoNT/E was complete within seven days but the extent of the recovery from the AE combination was incomplete compared with BoNT/A alone, and reached a plateau where running distances remained depressed one month later. Paralysis caused by BoNT/A alone had subsided completely in three weeks.
DISCUSSION Botulism has long been associated with food poisoning where the primary symptom, muscle paralysis, develops slowly over several days and may persist for many weeks or months. The disease is derived from the ingestion of
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Fig. 4. Comparison of BoNT serotype duration. (A) Duplicate data for BoNT/A (), BoNT/B (), and BoNT/E (‘) was normalized to a percentage scale. BoNT/A and /B traces represent 0.9 LD50 units, BoNT/E trace is 0.8 LD50 units. (B) The duration of total paralysis (zero running) was compiled and graphed relative to each toxin dose. BoNT/A (□) was fit using nonlinear regression analysis, whereas BoNT/B (Œ) and BoNT/E () were fit using linear regression analysis.
BoNT, which eventually enters nerve terminals where it proteolytically cuts certain nerve terminal proteins controlling synaptic vesicle function (Hayashi et al., 1994; McMahon and Sudhof, 1995; Schiavo et al., 2000; Simpson, 2004). This, in turn, causes a gradual blockade of neurotransmission, which stops muscle function. Death may result depending on the severity of symptoms and the timing of medical intervention, however, should a patient survive beyond the initial onset, recovery is very likely. The duration of the recovery phase in human cases can range from several days to many months (Ball et al., 1979; Colebatch et al., 1989; Shapiro et al., 1998). The precise reason(s) for the variable recovery times are not clearly known because natural occurrences of botulism rarely reveal when the poisoning occurred or how much toxin was consumed. In the current study, running activity from mice injected with BoNT/A, B or E has provided useful data for understanding how each neurotoxin causes botulism and how recovery proceeds after poisoning. Complete and partial paralysis produced by BoNT/A, B and E persisted in the order of A⬎B⬎E (Fig. 4) in agreement with results compiled from previous studies (Sellin et al., 1983a,b; Jurasinski et al., 2001; Sheridan et al., 2005). Previously, ex vivo or in vivo nerve-muscle analysis has been used to examine the duration of BoNT symptoms but these studies tended to produce minimal data because of the inherent difficulties
of the experimental approach. Generally, this required large numbers of animals which were monitored at broad time intervals after poisoning (Simpson, 1973, 1978; Polak et al., 1981; Sellin, 1981; Sellin and Thesleff, 1981; Adler et al., 2000, 2001), thus greatly limiting the conditions that have been examined. Those methods have not been used to assess variable BoNT/A, B or E dosages on muscle paralysis, for example, or to measure the ensuing recovery on a daily basis. Running activity in response to i.m. injection allowed simultaneous testing of many dosages from the three BoNTs and demonstrated that each toxin has distinct characteristics that differentially influence the duration of paralysis and the rate of recovery. The persistence of symptoms from BoNT/A poisoning is exacerbated with small increases in toxin exposure, whereas paralysis caused by BoNT/B and /E is influenced relatively less by toxin dosage. Whether BoNT/A recovery encompasses a single exponential rate or a two-part linear process is unclear at this time, however, full recovery from BoNT/A poisoning is considered to entail several steps, each proceeding with a separate rate. Some of these factors are: the rate of toxin degradation within poisoned nerve terminals, the rate of repair of the poisoned nerve terminals, the rate of new (non-poisoned) nerve terminal growth, recruitment of resting nerve terminals that escaped poisoning and a return of normal muscle mass. In this study the multifunctional requirements for recovery from BoNT/A seem to be distinguished from the simpler recovery profiles for BoNT/B and /E but the individual processes are not clearly depicted in the data.
Fig. 5. Co-injection of toxin mixtures. Duplicate determinations were tested using 0.5 LD50 units of BoNT/A mixed with either 0.3 LD50 units of BoNT/B (Top frame) or 0.5 LD50 units of BoNT/E (Lower frame). In both circumstances the rate of recovery was reduced when two serotypes were present compared with the recovery rate for either serotype individually.
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The running assay has revealed several unknown characteristics of BoNT/A, B and E while confirming several previously known properties. The similarities to human cases of botulism provide continued support for the mouse model as a good predictor of toxin action in human tissue. Because this approach can quantitatively monitor recovery, the running assay can benefit in vivo research that is aimed at developing or screening BoNT antagonists to stimulate the return of normal muscle function after poisoning. Although this assay relies upon mice, the separate findings for recovery from BoNT/A and B are consistent with the duration of paralysis produced with controlled, low-dose injections used to treat human muscle disorders. In this sense, the different recovery phases observed for the individual toxins in the present study have the potential to improve our understanding of how each BoNT acts in human tissue, which consequently may help develop alternative or improved BoNT regimens. This research was extended to examine the effect of BoNT combinations. Two previous in vivo studies using rodents have reported opposite findings for BoNT/A and E mixtures (Adler et al., 2001; Meunier et al., 2003). To attempt to provide additional data, running distances were measured after injecting combinations of BoNT/A and B or BoNT/A and E. When BoNT/A was combined with BoNT/B the resulting paralysis developed more rapidly and persisted much longer than the individually injected BoNTs (Fig. 5A). Combination of BoNT/A and E in similar ratios prolonged paralysis compared with BoNT/A alone. This final observation is in agreement with Adler et al. (2001) where BoNT/A and E were found to act independently following injection of A, then E into rat extensor digitorum longus (EDL) muscle. In that study, muscle contractions were elicited by electrical stimulation of the poisoned nerve-muscle group. Muscle force had fully recovered within one month after injection of BoNT/E but was 70% depressed following BoNT/A injection, and about 80% depressed by the AE combination. This trend is identical to the running data presented here. The in vivo results from both studies support data obtained from cultured neurons showing that BoNT/A and E do not influence each other at the molecular level within nerve terminals (Keller et al., 1999). Results from human volunteers (Eleopra et al., 1998) suggest that co-injection of BoNT/A and E into the extensor digitorum brevis (EDB) muscle resulted in a shortened duration of paralysis. The EDB is a slow-twitch muscle group, whereas, the EDL tested in rats and the gastrocnemius group tested here are fast-twitch muscle groups. The duration of paralysis in each type of muscle is strongly influenced by innervation properties. Comparison studies have shown that the slow-twitch soleus muscle in mice regains normal contractile properties within several days after injection of BoNT/A but the fast-twitch gastrocnemius muscle group recovered only partially over several weeks (Duchen, 1971; Brown et al., 1980). The present study targeted the fast-twitch gastrocnemius mouse muscle, and produced results similar the rat EDL muscle study, both results are consistent with these muscle groups having
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fast-twitch electrical properties. The contrasting results between human and rodent studies may entail species variation, tissue types, size of muscle groups and experimental designs. Rats are naturally resistant to BoNT/B so BoNT/A and B combinations were not attempted in the rat EDL study, and were not mentioned in the human EDB study. The apparent additive effect observed in mice by mixing sub-optimal doses of BoNT/A and BoNT/B has not been reported elsewhere and may represent compounded disruption of synaptic vesicle trafficking due to each toxin cleaving different synaptic proteins (Blasi et al., 1993; Montecucco and Schiavo, 1993). Although the results from the BoNT/A and B combination in mice imply that human patients can benefit from such a mixture, direct extrapolation of the mouse results to human clinical application should not be done with the present study for several reasons. Firstly, this research was not designed to evaluate the mouse model as a substitute for human clinical trials, but was undertaken to compare the progression of paralysis caused by each toxin in vivo. These data will serve as a framework for testing drugs intended to restore normal nerve-muscle function after BoNT poisoning. Secondly, human dosages cannot be directly estimated from mouse injections primarily because of animal size, injection volumes and different sensitivities of specific muscle tissues to each toxin. And thirdly, the toxins used here were laboratory-grade preparations, which were manufactured by different methods and have different degrees of purity compared with BoNTs used to treat patients. These variables prevent direct comparisons between different toxin preparations. Therefore, the data presented here can, at best, only estimate the effect of pharmaceutical-grade products in human patients. Comparison of the three BoNT serotypes commonly associated with causing human disease, shows that running activity decreases in direct proportion to the amount of toxin injected. Furthermore, each of the three BoNTs demonstrates distinct biological effects on both the duration of paralysis and the manner in which recovery proceeds. BoNT/A concentration affects at least two aspects of muscle function: duration of maximum paralysis followed by a prolonged and relatively slow rate of recovery. In contrast, BoNT/B concentration primarily influences the duration of maximum paralysis while slightly prolonging the subsequent return of normal muscle action. BoNT/E concentration does not greatly affect the duration of maximum paralysis or the time to recovery but reduces only the extent of muscle activity after the initial poisoning. These distinct variations represent biochemical properties unique to each BoNT, and suggest that each toxin influences different biological process(es) required for a return of normal synaptic function. Acknowledgments—The many conversations within the F.D.A. Center for Biologics Evaluation and Research (CBER) and the Center for Drug Evaluation and Research (CDER) were tremendously welcomed and appreciated. In particular, the help and guidance from Drs. W. F. Vann, PhD and A. Rosenberg, MD, were of great value.
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REFERENCES Adler M, Capacio B, Deshpande SS (2000) Antagonism of botulinum toxin A-mediated muscle paralysis by 3, 4-diaminopyridine delivered via osmotic minipumps. Toxicon 38:1381–1388. Adler M, Keller JE, Sheridan RE, Deshpande SS (2001) Persistence of botulinum neurotoxin A demonstrated by sequential administration of serotypes A and E in rat EDL muscle. Toxicon 39:233–243. Allen DL, Harrison BC, Maass A, Bell ML, Byrnes WC, Leinwand LA (2001) Cardiac and skeletal muscle adaptations to voluntary wheel running in the mouse. J Appl Physiol 90:1900 –1908. Aoki KR (2001) A comparison of the safety margins of botulinum neurotoxin serotypes A, B, and F in mice. Toxicon 39:1815–1820. Ball AP, Hopkinson RB, Farrell ID, Hutchison JG, Paul R, Watson RD, Page AJ, Parker RG, Edwards CW, Snow M, Scott DK, LeoneGanado A, Hastings A, Ghosh AC, Gilbert RJ (1979) Human botulism caused by Clostridium botulinum type E: the Birmingham outbreak. Q J Med 48:473– 491. Blasi J, Chapman ER, Link E, Binz T, Yamasaki S, De Camilli P, Sudhof TC, Niemann H, Jahn R (1993) Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25. Nature 365:160–163. Boquet P, Duflot E (1982) Tetanus toxin fragment forms channels in lipid vesicles at low pH. Proc Natl Acad Sci U S A 79:7614 –7618. Brashear A, Lew MF, Dykstra DD, Comella CL, Factor SA, Rodnitzky RL, Trosch R, Singer C, Brin MF, Murray JJ, Wallace JD, WillmerHulme A, Koller M (1999) Safety and efficacy of NeuroBloc (botulinum toxin type B) in type A-responsive cervical dystonia. Neurology 53:1439 –1446. Brin MF, Lew MF, Adler CH, Comella CL, Factor SA, Jankovic J, O’Brien C, Murray JJ, Wallace JD, Willmer-Hulme A, Koller M (1999) Safety and efficacy of NeuroBloc (botulinum toxin type B) in type A-resistant cervical dystonia. Neurology 53:1431–1438. Brown MC, Holland RL, Ironton R (1980) Nodal and terminal sprouting from motor nerves in fast and slow muscles of the mouse. J Physiol 306:493–510. Centers for Disease Control and Prevention (1998) Botulism in the United States, 1899 –1996. Handbook for epidemiologists, clinicians, and laboratory workers. Atlanta, GA: Centers for Disease Control and Prevention. Coffield JA, Considine RV, Simpson LL (1994) Clostridial neurotoxins in the age of molecular medicine. Trends Microbiol 2:67– 69; discussion 69 –72. Colebatch JG, Wolff AH, Gilbert RJ, Mathias CJ, Smith SE, Hirsch N, Wiles CM (1989) Slow recovery from severe foodborne botulism. Lancet 2:1216 –1217. Costantin L, Bozzi Y, Richichi C, Viegi A, Antonucci F, Funicello M, Gobbi M, Mennini T, Rossetto O, Montecucco C, Maffei L, Vezzani A, Caleo M (2005) Antiepileptic effects of botulinum neurotoxin E. J Neurosci 25:1943–1951. Cull-Candy SG, Lundh H, Thesleff S (1976) Effects of botulinum toxin on neuromuscular transmission in the rat. J Physiol 260:177–203. Duchen LW (1971) An electron microscopic study of the changes induced by botulinum toxin in the motor end-plates of slow and fast skeletal muscle fibres of the mouse. J Neurol Sci 14:47– 60. Duncan RM, Jensen WL (1976) A relationship between avian carcasses and living invertebrates in the epizootiology of avian botulism. J Wildl Dis 12:116 –126. Eleopra R, Tugnoli V, Rossetto O, De Grandis D, Montecucco C (1998) Different time courses of recovery after poisoning with botulinum neurotoxin serotypes A and E in humans. Neurosci Lett 256:135–138. Fasshauer D, Eliason WK, Brunger AT, Jahn R (1998) Identification of a minimal core of the synaptic SNARE complex sufficient for reversible assembly and disassembly. Biochemistry 37:10354–10362. Foran PG, Mohammed N, Lisk GO, Nagwaney S, Lawrence GW, Johnson E, Smith L, Aoki KR, Dolly JO (2003) Evaluation of the therapeutic usefulness of botulinum neurotoxin B, C1, E, and F compared with the long lasting type A. Basis for distinct durations
of inhibition of exocytosis in central neurons. J Biol Chem 278: 1363–1371. Habermann E, Dreyer F (1986) Clostridial neurotoxins: handling and action at the cellular and molecular level. Curr Top Microbiol Immunol 129:93–179. Hallett M (1999) One man’s poison: clinical applications of botulinum toxin. N Engl J Med 341:118 –120. Hayashi T, McMahon H, Yamasaki S, Binz T, Hata Y, Sudhof TC, Niemann H (1994) Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J 13:5051–5061. Hubalek Z, Pellantova J, Hudec K, Halouzka J, Chytil J, Machacek P, Sebela M, Kubicek F (1991) [Botulism in birds living in an aquatic environment in Nove Mlyny in the Breclav District]. Vet Med (Praha) 36:57– 63. Hughes JM, Blumenthal JR, Merson MH, Lombard GL, Dowell VR Jr, Gangarosa EJ (1981) Clinical features of types A and B food-borne botulism. Ann Intern Med 95:442– 445. Irani BG, Xiang Z, Moore MC, Mandel RJ, Haskell-Luevano C (2005) Voluntary exercise delays monogenetic obesity and overcomes reproductive dysfunction of the melanocortin-4 receptor knockout mouse. Biochem Biophys Res Commun 326:638 – 644. Jahn R, Niemann H (1994) Molecular mechanisms of clostridial neurotoxins. Ann N Y Acad Sci 733:245–255. Jankovic J, Brin MF (1991) Therapeutic uses of botulinum toxin. N Engl J Med 324:1186 –1194. Jurasinski CV, Lieth E, Dang Do AN, Schengrund CL (2001) Correlation of cleavage of SNAP-25 with muscle function in a rat model of botulinum neurotoxin type A induced paralysis. Toxicon 39:1309–1315. Keller JE, Neale EA, Oyler G, Adler M (1999) Persistence of botulinum neurotoxin action in cultured spinal cord cells. FEBS Lett 456: 137–142. Koenig MG, Spickard A, Cardella MA, Rogers DE (1964) Clinical and laboratory observations on type E botulism in man. Medicine (Baltimore) 43:517–545. Konhilas JP, Maass AH, Luckey SW, Stauffer BL, Olson EN, Leinwand LA (2004) Sex modifies exercise and cardiac adaptation in mice. Am J Physiol Heart Circ Physiol 287:H2768 –H2776. Koriazova LK, Montal M (2003) Translocation of botulinum neurotoxin light chain protease through the heavy chain channel. Nat Struct Biol 10:13–18. Lightfoot JT, Turner MJ, Daves M, Vordermark A, Kleeberger SR (2004) Genetic influence on daily wheel running activity level. Physiol Genomics 19:270 –276. McMahon HT, Sudhof TC (1995) Synaptic core complex of synaptobrevin, syntaxin, and SNAP25 forms high affinity alpha-SNAP binding site. J Biol Chem 270:2213–2217. Meunier FA, Lisk G, Sesardic D, Dolly JO (2003) Dynamics of motor nerve terminal remodeling unveiled using SNARE-cleaving botulinum toxins: the extent and duration are dictated by the sites of SNAP-25 truncation. Mol Cell Neurosci 22:454 – 466. Montecucco C, Schiavo G (1993) Tetanus and botulism neurotoxins: a new group of zinc proteases. Trends Biochem Sci 18:324 –327. Ohishi I, Sakaguchi G (1977) Activation of botulinum toxins in the absence of nicking. Infect Immunol 17:402– 407. Pevsner J, Scheller RH (1994) Mechanisms of vesicle docking and fusion: insights from the nervous system. Curr Opin Cell Biol 6:555–560. Polak RL, Sellin LC, Thesleff S (1981) Acetylcholine content and release in denervated or botulinum poisoned rat skeletal muscle. J Physiol 319:253–259. Schantz EJ, Johnson EA (1990) Dose standardisation of botulinum toxin. Lancet 335:421 Schiavo G, Matteoli M, Montecucco C (2000) Neurotoxins affecting neuroexocytosis. Physiol Rev 80:717–766. Schiavo G, Rossetto O, Santucci A, DasGupta BR, Montecucco C (1992) Botulinum neurotoxins are zinc proteins. J Biol Chem 267:23479 –23483.
J. E. Keller / Neuroscience 139 (2006) 629 – 637 Sellin LC (1981) The action of botulinum toxin at the neuromuscular junction. Med Biol 59:11–20. Sellin LC, Kauffman JA, Dasgupta BR (1983a) Comparison of the effects of botulinum neurotoxin types A and E at the rat neuromuscular junction. Med Biol 61:120 –125. Sellin LC, Thesleff S, Dasgupta BR (1983b) Different effects of types A and B botulinum toxin on transmitter release at the rat neuromuscular junction. Acta Physiol Scand 119:127–133. Sellin LC, Thesleff S (1981) Pre- and post-synaptic actions of botulinum toxin at the rat neuromuscular junction. J Physiol 317: 487– 495. Shapiro RL, Hatheway C, Swerdlow DL (1998) Botulism in the United States: a clinical and epidemiologic review. Ann Intern Med 129: 221–228. Sheridan RE, Smith TJ, Adler M (2005) Primary cell culture for evaluation of botulinum neurotoxin antagonists. Toxicon 45:377–382. Simpson LL (1973) The interaction between divalent cations and botulinum toxin type A in the paralysis of the rat phrenic nervehemidiaphragm preparation. Neuropharmacology 12:165–176.
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Simpson LL (1978) Pharmacological studies on the subcellular site of action of botulinum toxin type A. J Pharmacol Exp Ther 206: 661– 669. Simpson LL (2004) Identification of the major steps in botulinum toxin action. Annu Rev Pharmacol Toxicol 44:167–193. Sloop RR, Cole BA, Escutin RO (1997) Human response to botulinum toxin injection: type B compared with type A. Neurology 49: 189 –194. Tucker N, Sobel J, Maslanka SE, Griffin PM, Tauxe RV (2002) Surveillance for botulism: summary of 2001 data, pp 1–11 [Centers for Disease Control and Prevention web site]. http://www.cdc.gov/ncidod/ dbmd/diseaseinfo/botulism_a.htm. Whittaker RL, Gilbertson RB, Garrett AS Jr (1964) Botulism, type E: report of eight simultaneous cases. Ann Intern Med 61:448 – 454. Woodruff BA, Griffin PM, McCroskey LM, Smart JF, Wainwright RB, Bryant RG, Hutwagner LC, Hatheway CL (1992) Clinical and laboratory comparison of botulism from toxin types A, B, and E in the United States, 1975–1988. J Infect Dis 166:1281–1286.
(Accepted 1 December 2005) (Available online 20 February 2006)
RECOVERY FROM BOTULINUM NEUROTOXIN POISONING IN VIVO J. E. KELLER*
BoNT/C and /D have never been known to cause botulism in humans, yet these two serotypes cause significant disease among livestock and water fowl (Duncan and Jensen, 1976; Hubalek et al., 1991; Tucker et al., 2002; Centers for Disease Control and Prevention, 1998). Poisoning by any one of these neurotoxins impairs communication between nerve and muscle. In humans, the resulting paralysis caused by BoNT/A or B tends to be protracted regardless of the route of entry. This makes treating the disease difficult but has led to using purified BoNT preparations to treat several human neuromuscular disorders (Hughes et al., 1981; Woodruff et al., 1992; Sloop et al., 1997; Brashear et al., 1999). In studies of healthy volunteers, local i.m. injection of medical-grade BoNT/A had a prolonged effect, with muscle function gradually returning over 12 months. Paralysis from BoNT/B endured for up to four months (Sloop et al., 1997; Brin et al., 1999). BoNT/E has not been used to treat human muscle disorders because of its relatively short duration of action (Whittaker et al., 1964; Sellin et al., 1983a). However, its prevalence in causing human botulism has placed much interest on its biological properties (Koenig et al., 1964) and recently it has been reported as having beneficial effects for reducing epileptic seizures in rats (Costantin et al., 2005). The BoNTs are 150 kDa proteins that enter motor nerve terminals by receptor-mediated endocytosis (Habermann and Dreyer, 1986; Coffield et al., 1994; Jahn and Niemann, 1994). A proteolytic domain of the toxin crosses the endosomal membrane by threading through a toxininduced pore leading to the cytosol (Boquet and Duflot, 1982; Koriazova and Montal, 2003) where it targets one of two synaptic proteins (Schiavo et al., 1992; Simpson, 2004). Proteolysis by BoNT/A, B or E is limited to either synaptosomal membrane associated protein of 25 kDa or vesicle-associated membrane protein 2, where each BoNT cuts its substrate at one serotype-specific bond. The resulting cleavage disrupts proper synaptic vesicle trafficking during neurotransmission (Pevsner and Scheller, 1994; Fasshauer et al., 1998) and consequently causes muscle paralysis. Systemic poisoning can often become life threatening. However, controlled injection of BoNT/A or B directly into muscle tissue generally does not cause illness and has become a useful medical procedure for preferentially paralyzing overactive nerve groups (Jankovic and Brin, 1991; Hallett, 1999). Few studies have measured recovery from BoNT intoxication because quantifying muscle activity over the course of many weeks is difficult. Several studies indicate that the longest duration of paralysis is in the order of BoNT/A⬎B⬎E (Sellin et al., 1983; Sloop et al., 1997; Eleopra et al., 1998; Adler et al., 2001; Jurasinski et al.,
Laboratory of Bacterial Toxins, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, Food and Drug Administration, 29 Lincoln Drive, HFM 434, Room 122, Bethesda, MD 20892, USA
Abstract—Botulinum neurotoxins cause the disease botulism, which is characterized by prolonged muscle paralysis. In contrast, injections of low doses of purified botulinum neurotoxins do not cause systemic illness but produce localized muscle paralysis that is beneficial for treating several human medical disorders involving uncontrollable muscle contraction. Optimizing the therapeutic efficacy while diminishing adverse reactions requires precise knowledge of toxin potency as well as a clear understanding of how each toxin causes disease. A novel in vivo mouse assay has been used to correlate toxin dosage with the duration of muscle paralysis. Voluntary running activity performed by mice was proportional to the amount of toxin injected into the hind limbs and the subsequent rate of recovery over the ensuing days or weeks was a function of botulinum neurotoxin serotype A or B concentration. Botulinum neurotoxin A produced longer paralysis than botulinum neurotoxin B consistent with human observations. A third serotype, botulinum neurotoxin E, had the shortest duration of action, but unlike the other two toxins, dosage did not influence recovery time. Botulinum neurotoxin A recovery appeared biphasic with the initial phase about two-fold faster than the final phase. Over four weeks, muscle activity had gradually improved following the highest botulinum neurotoxin A dose, reaching about half of the normal running activity. Lower botulinum neurotoxin A doses led to incrementally faster and complete recovery. Persistence of maximum paralysis was exponentially related to botulinum neurotoxin A dosage, with a doubling of the paralysis time occurring with every 25% increase of the toxin concentration. In contrast, the rate of recovery from botulinum neurotoxin B was monophasic relative to toxin dosage and the duration of maximum paralysis was linear relative to dosage. Combinations of botulinum neurotoxin A and B and botulinum neurotoxin A and E were tested and shown to exacerbate paralysis compared with individually administered serotypes. © 2006 Published by Elsevier Ltd on behalf of IBRO. Key words: botulism, botulinum neurotoxin, paralysis, recovery, potency assay.
Botulism is a neuromuscular disease characterized by progressive muscle weakness leading to paralysis, and possible death from respiratory failure. Of the seven known botulinum neurotoxins (BoNTs), only three routinely cause human disease, BoNT serotypes A, B or E. Since 1950, serotype F has been attributed to five cases, whereas, *Tel: ⫹1-301-402-4418; fax: ⫹1-301-402-2776. E-mail address: [email protected] (J. E. Keller). Abbreviations: BoNT, botulinum neurotoxin; EDB, extensor digitorum brevis; EDL, extensor digitorum longus; LD50, dose that will kill 50% of mice injected within a four day period. 0306-4522/06$30.00⫹0.00 © 2006 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.12.029
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2001; Foran et al., 2003). However, the most common potency value for BoNT activity is derived from lethality titration curves where groups of mice are exposed to the toxin by i.p. injection and monitored for four days (Schantz and Johnson, 1990). The dose of toxin that kills 50% of the mice is one standard lethal-dose unit (LD50). In addition to LD50 measurement, two laboratory methods exist for monitoring recovery following localized i.m. injection of toxin. In the first method, in situ or in vitro nerve-muscle preparations can be used to measure either muscle contraction or neuronal electrical properties; both measurements require skilled surgical techniques to properly expose the nerve tissue, and this approach requires dozens of animals to compile useful data (Cull-Candy et al., 1976; Sellin et al., 1983). A less complicated model system entails monitoring the toe-spread reflex in living mice or rats (Aoki, 2001; Jurasinski et al., 2001; Meunier et al., 2003). This method relies upon visually assigning a numerical value for legfoot-toe kicking that occurs upon lifting animals by the tail. As a result, large numbers of animals are required to compensate for the subjective manner and high variability of assigning values. Both approaches are difficult to implement when testing multiple conditions such as different dosages using several BoNTs. Retaining the mouse for BoNT measurement is advantageous because sensitivity to the three BoNT serotypes is similar to human responses and the mouse LD50 assay is currently the most sensitive method used for detecting and measuring the potencies of all BoNTs (Schantz and Johnson, 1990). This study describes a novel in vivo method for assessing the effects of sub-lethal BoNT injection into the mouse hind limb. Toxin potency and duration of paralysis were measured by recording the nightly running activity of mice on exercise wheels. This approach reduced the number of animals needed compared with other methods and has allowed comparison of the biological actions of BoNT/A, B and E at several dosages. In addition to causing unique dose-dependent symptoms, recovery of muscle function was distinct for each BoNT, which improves the current understanding of the physiological long-term effects caused by BoNT poisoning.
EXPERIMENTAL PROCEDURES Animals BALB/cJ male mice at five weeks old (18 –20 g) were obtained from NCT (Frederick, MD, USA). All aspects of the experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, NIH Publication No. 80-23. The research had prior approval from the CBER Animal Care-and-Use Committee. A minimal number of animals was used in this study and every effort was made to reduce their suffering. Groups of eight animals were housed with free access to exercise wheels (Penn-Plax, Inc., Garden City, NY, USA) according to previous descriptions (Allen et al., 2001). Each wheel with a diameter of 11.5 cm was fitted with a magnet and micro-computer that recorded the number of revolutions, speed and total distance (Cat Eye, Co., Ltd., Boulder, CO, USA). At three-day increments animals were divided into groups or four then two per cage. Rooms were maintained with a 12-h light/dark
cycle, 20 –22 °C. Use of the wheels, feeding and drinking were ad libitum. Distance data were collected at 24 h intervals.
Toxin injection procedure Purified BoNT complexes were used in this study. BoNT/A (WAKO Chemicals, Richmond, VA, USA) was stored as a 1 mg/mL stock solution (2.0⫻107 ipLD50/mg). BoNT/B and E (WAKO Chemicals) were activated with trypsin and aliquoted as described previously (Ohishi and Sakaguchi, 1977; Keller et al., 1999). Potency of each was reported by the manufacturer to be 3.0⫻107 and 1.0⫻107 ipLD50 per mg of BoNT/B and E, respectively. Actual potency after trypsin treatment was 2.7⫻107 and 1.0⫻107 LD50 per mg of BoNT/B and BoNT/E. Aliquots of each toxin preparation were stored at ⫺80 °C. Dilution of each toxin was performed on the day of the experiment using sterile physiological saline containing 0.5% bovine serum albumin. Animals were lightly sedated with 2% halothane and the injection area was moistened with 70% isopropanol prior to administering toxin. I.m. injections of 30 L were given directly into the largest, central region of the gastrocnemius (calf) muscle mass. Since the animals had been running for 10 –14 days prior to injection, this injection volume did not cause visible swelling of the leg, as happened with non-running animals (Meunier et al., 2003). Because physical tissue trauma enhances the recovery rate from BoNT/A, syringes with a 32-gauge diameter were used to reduce the likelihood of injury.
Statistical analysis Compiled distances from three or more cages are represented as average determinations regardless of the number of animals within each cage. Error bars represent one standard deviation unit. Linear and non-linear regression analyses were performed using Prism 3.0 (Graphpad, San Diego, CA, USA). In the case of recovery times, data during paralysis onset were excluded from regression analysis. Linear regression analysis of BoNT/A used the standard equation: y⫽mx⫹b. Nonlinear analysis used the following equations: (BoNT/A) y⫽(1⫺exp(⫺k⫻t))⫻M⫹c. Y is the distance (km); M, the plateau maximum was predicted at time (t), with a first-order rate constant, k or for (BoNT/B): y⫽B0⫹((T0⫺B0)/ 1⫹10C). B0 and T0 represented the lower and upper limit values of the curve. The constant, C, was log (EC50⫺t)⫻H. Where EC50, t and H represent the 50% recovery point at time, t, which was influenced by a cooperativity coefficient, H.
RESULTS Gastrointestinal symptoms were observed in response to i.m. injection of BoNT/A and B when the dose exceeded 0.7 LD50. BoNT/A i.m. injection into the hind limb elicited diarrhea, whereas, BoNT/B reduced fecal accumulation within the cages, suggesting sub-lethal i.m. administered BoNT/A or B can selectively poison different autonomic neurons of the GI tract. Interestingly, BoNT/E toxicity was very sharp; animals did not display systemic signs of illness even at 0.9 LD50 units but all died within 18 h if given 1.0 LD50. At all doses of BoNT/A, B or E tested, animals demonstrated an ability to move, eat, drink and groom regardless of the toxin type. Animal housing was examined to optimize the distance and consistency of voluntary running activity. Mice were active almost exclusively during the 12 h dark cycle (⬎95%) with only slight running displayed during the daytime. Initially eight animals, then subsequently smaller groups were tested. Housing three or more animals within
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Fig. 1. Nightly running activity. (A) Mice were housed in various sized groups with access to two exercise wheels in each cage. The average distance (km) per mouse was calculated from running values collected over three days. Data were compiled from at least four cages for each condition. (B) The best paradigm required two mice co-housed with access to two running wheels. Mice that demonstrated at least 5 km per night were monitored and distances were recorded daily. Data from four cages (, □, , Œ) are shown with each symbol representing individual data points recorded at 24 h intervals. (C) Two animals in each cage were treated as indicated and running activity was collected over three days. Injection of BoNT/A (0.2 LD50 units) reduced running activity by about three-fold compared with untreated animals (n⫽4). Animals injected with either saline or BoNT/A combined with antitoxin did not demonstrate reduced running over a period of three days.
a cage was counterproductive since individually each animal ran approximately five hours per night (Konhilas et al., 2004; Lightfoot et al., 2004; Irani et al., 2005), thus limiting wheel access during the 12 h active period (Fig. 1A). This was resolved by placing two animals together, which eliminated aggressive competition that often developed within larger groups. When single animals were tested, running
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became sporadic with increasing variation (Fig. 1A). Based on these data, the experimental design consisted of two mice in a single cage; running distances were reported as the average between the two animals. Pairs of animals that ran consistently near 6 km per night were used for further experimentation (Fig. 1B) (Lightfoot et al., 2004; Irani et al., 2005). The running distances remained generally constant over a period of several days and were not affected by injection of saline or BoNT/A premixed with mouse antitoxin (Fig. 1C). Injection of BoNTs into the hind limb reduced running distances when toxin was administered over a four- to five-fold range of sub-lethal doses. The effects caused by each toxin are described below in terms of onset of paralysis, duration of total paralysis (zero running) and the rate of recovery after total paralysis subsided. Running distances were recorded for up to four weeks following injection of BoNT/A (Fig. 2A). Paralysis onset was similar for all doses tested with the maximum effect occurring within 48 h of injection. At the lowest dose, 0.14 LD50 unit, the total running distance was reduced by 85%. Recovery began on the third day with gradual improvement leading to normal physical activity about seven days after injection. Higher doses of BoNT/A led to complete cessation of running that persisted for many days relative to toxin dosage. The rate of recovery following total paralysis was similarly influenced by higher amounts of toxin. The highest dose tested, 0.8 LD50 units, produced eight to nine days of total inactivity followed by about 50% recovery (⬃3 km) 28 days later. Reducing BoNT/A by two-fold to 0.4 LD50 units resulted in only one day of about 95% inhibition of running and recovery began the following day. Full recovery was achieved within 15 days of the injection of 0.4 LD50 units. Regression analysis using a single exponential rate constant predicted that 90% recovery required 11, 24.1, 45.3 or 61 days post-injection for the four doses of toxin tested, which overestimated the actual recovery times observed for the two lowest BoNT/A doses. Since recovery from the highest dose of BoNT/A appeared to follow a nearly linear path from day 12 to day 30, the recovery profile for the BoNT/A poisoning was evaluated using two linear equations overlapping at day 19. Although BoNT/A dosage clearly influences the duration of total paralysis, the ensuing recovery may entail a two-part process. Recovery proceeded with a faster, initial phase until 33–38% of normal running was achieved after three to seven days, depending on the dose of toxin. A second recovery phase was 2.4-fold slower than the initial phase (Fig. 3C), which was too slow to allow full recovery from the highest dose of BoNT/A during the time of the experiment. However, extrapolating from the lower doses, full recovery from 0.8 LD50 units would require 48⫾8 days. Both linear rates of recovery were directly related to toxin dosage, and the analysis predicts that each rate becomes zero if a lethal amount of toxin were injected (Fig. 3C). Similar experiments using BoNT/B and E yielded results that were generally distinct from BoNT/A (Fig. 2B and 2C). BoNT/B, like BoNT/A, produced a dose-dependent duration of total paralysis. However, once total paralysis
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Fig. 2. Dose-response of running distance versus BoNT dosage. (A) Animals in groups of two were injected with BoNT/A (i.m. LD50 units): 0.14 (□), 0.40 (⽧), 0.6 (), or 0.8 (). One i.m. LD50 unit equals 0.06 mL of 1.25 pM BoNT/A. Error bars are averages of triplicate determinations except for 0.8 LD50 units (n⫽6). Non-linear regression analysis included data only after maximum paralysis subsided. The equation was y⫽(1⫺exp(⫺k⫻t))⫻M⫹c, as described in the Experimental Procedures. (B) Dose-response of running distance versus BoNT/B (i.m. LD50 units): 0.30 LD50 units (‘), 0.60 (), 0.90 (). Regression analysis for BoNT/B data used the equation: y⫽Bo⫹((To⫺Bo)/1⫹10C) described in the Experimental Procedures section. Error bars are averages of triplicate determinations. (C) BoNT/E determinations were done in duplicate using 0.4 LD50 units (□,Œ) or 0.90 (,). Each symbol from the BoNT/E trial represents a single determination. The arrow indicates the day of injection.
had subsided, the recovery phase proceeded at approximately the same rate for all BoNT/B concentrations. Unlike BoNT/A, recovery from BoNT/B was best represented by a sigmoidal curve representing early, middle and late phases of recovery that begin within several days of disease onset. Full recovery to 100% normal running activity occurred eight to nine days after total paralysis regardless of toxin concentration. The lowest concentration of BoNT/B tested
(0.3 LD50 units) demonstrated a delayed onset time, which peaked four to five days post-injection with about 90% blockade in running; the higher doses of BoNT/B completely inhibited running two to three days after injection. As with BoNT/A, the duration of maximum paralysis from BoNT/B was dose-dependent but unlike BoNT/A, the recovery rate was influenced to a lesser extent by BoNT/B dose (Fig. 2B). Injection of BoNT/E produced short-term paralysis relative to either BoNT/A or B (Fig. 2C). The concentration of BoNT/E did not influence the onset of muscle weakness nor did it alter the duration of paralysis. Toxin action persisted for up to seven days after injection regardless of the BoNT/E dose. This is similar to other studies that have examined BoNT/E in cultured neurons (Keller et al., 1999) and in rat muscle tissue (Sellin et al., 1983a; Adler et al., 2001), where recovery was complete within two weeks. In addition to confirming the brevity of duration, mouse running demonstrated that paralysis onset, duration of maximal blockade and the rate of recovery were generally independent of BoNT/E dose. The degree of paralysis observed on the second day after injection was the only symptom clearly influenced by the amount of toxin. Graphical depiction of the duration of total paralysis versus toxin concentration shows an exponential relationship between BoNT/A concentration and muscle inactivity (Fig. 4). In comparison, the duration of paralysis caused by BoNT/B is linear with toxin concentration producing similar paralysis to BoNT/A when both toxins were at relatively low levels but at higher BoNT/B concentrations, paralysis was brief compared with BoNT/A. BoNT/E, unlike BoNT/A and B, had practically no effect on the time of muscle inactivity. These observations are consistent with clinical observations of pharmaceutical-grade BoNT injections used to treat several medical disorders in human patients (Sloop et al., 1997; Eleopra et al., 1998). To examine if BoNTs can influence each other when simultaneously injected, combinations were tested and the results compared with previously reported observations (Adler et al., 2001; Meunier et al., 2003). BoNT/A (0.5 LD50 units) was combined with either BoNT/B or E. This dose of BoNT/A, as suggested by Fig. 2 and Fig. 4, produced maximum paralysis for two to three days (Fig. 5). Injection of BoNT/B at 0.3 LD50 units produced paralysis developing over several days leading to about a 90% reduction of running four days after injection but did not cause complete paralysis. Animals recovered fully within two to three weeks from the individual injections of BoNT/B and /A, respectively. In contrast, the AB mixture caused complete paralysis in less than 12 h compared with the slower onset times observed for the separate toxins (Fig. 2 and 5). Running activity from the AB combination remained suppressed by more than 90% for seven days followed by biphasic recovery over several weeks. Running activity returned to about half of the normal distance at the end of four weeks. A similar experiment was performed by mixing BoNT/A and E (Fig. 5). In this situation the onset of paralysis was not affected by the combination. Recovery from
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Fig. 3. Recovery rates from BoNT/A paralysis. Data from Fig. 2A are graphed demonstrating two linear recovery phases for the return of running activity. Both phases intersect at 33–38% (dashed line) recovery (Frames A and B). The rapid and slow rates are depicted as linear lines. Frame C demonstrates the dose-dependent relationship of the rapid () and slow () phases.
BoNT/E was complete within seven days but the extent of the recovery from the AE combination was incomplete compared with BoNT/A alone, and reached a plateau where running distances remained depressed one month later. Paralysis caused by BoNT/A alone had subsided completely in three weeks.
DISCUSSION Botulism has long been associated with food poisoning where the primary symptom, muscle paralysis, develops slowly over several days and may persist for many weeks or months. The disease is derived from the ingestion of
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Fig. 4. Comparison of BoNT serotype duration. (A) Duplicate data for BoNT/A (), BoNT/B (), and BoNT/E (‘) was normalized to a percentage scale. BoNT/A and /B traces represent 0.9 LD50 units, BoNT/E trace is 0.8 LD50 units. (B) The duration of total paralysis (zero running) was compiled and graphed relative to each toxin dose. BoNT/A (□) was fit using nonlinear regression analysis, whereas BoNT/B (Œ) and BoNT/E () were fit using linear regression analysis.
BoNT, which eventually enters nerve terminals where it proteolytically cuts certain nerve terminal proteins controlling synaptic vesicle function (Hayashi et al., 1994; McMahon and Sudhof, 1995; Schiavo et al., 2000; Simpson, 2004). This, in turn, causes a gradual blockade of neurotransmission, which stops muscle function. Death may result depending on the severity of symptoms and the timing of medical intervention, however, should a patient survive beyond the initial onset, recovery is very likely. The duration of the recovery phase in human cases can range from several days to many months (Ball et al., 1979; Colebatch et al., 1989; Shapiro et al., 1998). The precise reason(s) for the variable recovery times are not clearly known because natural occurrences of botulism rarely reveal when the poisoning occurred or how much toxin was consumed. In the current study, running activity from mice injected with BoNT/A, B or E has provided useful data for understanding how each neurotoxin causes botulism and how recovery proceeds after poisoning. Complete and partial paralysis produced by BoNT/A, B and E persisted in the order of A⬎B⬎E (Fig. 4) in agreement with results compiled from previous studies (Sellin et al., 1983a,b; Jurasinski et al., 2001; Sheridan et al., 2005). Previously, ex vivo or in vivo nerve-muscle analysis has been used to examine the duration of BoNT symptoms but these studies tended to produce minimal data because of the inherent difficulties
of the experimental approach. Generally, this required large numbers of animals which were monitored at broad time intervals after poisoning (Simpson, 1973, 1978; Polak et al., 1981; Sellin, 1981; Sellin and Thesleff, 1981; Adler et al., 2000, 2001), thus greatly limiting the conditions that have been examined. Those methods have not been used to assess variable BoNT/A, B or E dosages on muscle paralysis, for example, or to measure the ensuing recovery on a daily basis. Running activity in response to i.m. injection allowed simultaneous testing of many dosages from the three BoNTs and demonstrated that each toxin has distinct characteristics that differentially influence the duration of paralysis and the rate of recovery. The persistence of symptoms from BoNT/A poisoning is exacerbated with small increases in toxin exposure, whereas paralysis caused by BoNT/B and /E is influenced relatively less by toxin dosage. Whether BoNT/A recovery encompasses a single exponential rate or a two-part linear process is unclear at this time, however, full recovery from BoNT/A poisoning is considered to entail several steps, each proceeding with a separate rate. Some of these factors are: the rate of toxin degradation within poisoned nerve terminals, the rate of repair of the poisoned nerve terminals, the rate of new (non-poisoned) nerve terminal growth, recruitment of resting nerve terminals that escaped poisoning and a return of normal muscle mass. In this study the multifunctional requirements for recovery from BoNT/A seem to be distinguished from the simpler recovery profiles for BoNT/B and /E but the individual processes are not clearly depicted in the data.
Fig. 5. Co-injection of toxin mixtures. Duplicate determinations were tested using 0.5 LD50 units of BoNT/A mixed with either 0.3 LD50 units of BoNT/B (Top frame) or 0.5 LD50 units of BoNT/E (Lower frame). In both circumstances the rate of recovery was reduced when two serotypes were present compared with the recovery rate for either serotype individually.
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The running assay has revealed several unknown characteristics of BoNT/A, B and E while confirming several previously known properties. The similarities to human cases of botulism provide continued support for the mouse model as a good predictor of toxin action in human tissue. Because this approach can quantitatively monitor recovery, the running assay can benefit in vivo research that is aimed at developing or screening BoNT antagonists to stimulate the return of normal muscle function after poisoning. Although this assay relies upon mice, the separate findings for recovery from BoNT/A and B are consistent with the duration of paralysis produced with controlled, low-dose injections used to treat human muscle disorders. In this sense, the different recovery phases observed for the individual toxins in the present study have the potential to improve our understanding of how each BoNT acts in human tissue, which consequently may help develop alternative or improved BoNT regimens. This research was extended to examine the effect of BoNT combinations. Two previous in vivo studies using rodents have reported opposite findings for BoNT/A and E mixtures (Adler et al., 2001; Meunier et al., 2003). To attempt to provide additional data, running distances were measured after injecting combinations of BoNT/A and B or BoNT/A and E. When BoNT/A was combined with BoNT/B the resulting paralysis developed more rapidly and persisted much longer than the individually injected BoNTs (Fig. 5A). Combination of BoNT/A and E in similar ratios prolonged paralysis compared with BoNT/A alone. This final observation is in agreement with Adler et al. (2001) where BoNT/A and E were found to act independently following injection of A, then E into rat extensor digitorum longus (EDL) muscle. In that study, muscle contractions were elicited by electrical stimulation of the poisoned nerve-muscle group. Muscle force had fully recovered within one month after injection of BoNT/E but was 70% depressed following BoNT/A injection, and about 80% depressed by the AE combination. This trend is identical to the running data presented here. The in vivo results from both studies support data obtained from cultured neurons showing that BoNT/A and E do not influence each other at the molecular level within nerve terminals (Keller et al., 1999). Results from human volunteers (Eleopra et al., 1998) suggest that co-injection of BoNT/A and E into the extensor digitorum brevis (EDB) muscle resulted in a shortened duration of paralysis. The EDB is a slow-twitch muscle group, whereas, the EDL tested in rats and the gastrocnemius group tested here are fast-twitch muscle groups. The duration of paralysis in each type of muscle is strongly influenced by innervation properties. Comparison studies have shown that the slow-twitch soleus muscle in mice regains normal contractile properties within several days after injection of BoNT/A but the fast-twitch gastrocnemius muscle group recovered only partially over several weeks (Duchen, 1971; Brown et al., 1980). The present study targeted the fast-twitch gastrocnemius mouse muscle, and produced results similar the rat EDL muscle study, both results are consistent with these muscle groups having
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fast-twitch electrical properties. The contrasting results between human and rodent studies may entail species variation, tissue types, size of muscle groups and experimental designs. Rats are naturally resistant to BoNT/B so BoNT/A and B combinations were not attempted in the rat EDL study, and were not mentioned in the human EDB study. The apparent additive effect observed in mice by mixing sub-optimal doses of BoNT/A and BoNT/B has not been reported elsewhere and may represent compounded disruption of synaptic vesicle trafficking due to each toxin cleaving different synaptic proteins (Blasi et al., 1993; Montecucco and Schiavo, 1993). Although the results from the BoNT/A and B combination in mice imply that human patients can benefit from such a mixture, direct extrapolation of the mouse results to human clinical application should not be done with the present study for several reasons. Firstly, this research was not designed to evaluate the mouse model as a substitute for human clinical trials, but was undertaken to compare the progression of paralysis caused by each toxin in vivo. These data will serve as a framework for testing drugs intended to restore normal nerve-muscle function after BoNT poisoning. Secondly, human dosages cannot be directly estimated from mouse injections primarily because of animal size, injection volumes and different sensitivities of specific muscle tissues to each toxin. And thirdly, the toxins used here were laboratory-grade preparations, which were manufactured by different methods and have different degrees of purity compared with BoNTs used to treat patients. These variables prevent direct comparisons between different toxin preparations. Therefore, the data presented here can, at best, only estimate the effect of pharmaceutical-grade products in human patients. Comparison of the three BoNT serotypes commonly associated with causing human disease, shows that running activity decreases in direct proportion to the amount of toxin injected. Furthermore, each of the three BoNTs demonstrates distinct biological effects on both the duration of paralysis and the manner in which recovery proceeds. BoNT/A concentration affects at least two aspects of muscle function: duration of maximum paralysis followed by a prolonged and relatively slow rate of recovery. In contrast, BoNT/B concentration primarily influences the duration of maximum paralysis while slightly prolonging the subsequent return of normal muscle action. BoNT/E concentration does not greatly affect the duration of maximum paralysis or the time to recovery but reduces only the extent of muscle activity after the initial poisoning. These distinct variations represent biochemical properties unique to each BoNT, and suggest that each toxin influences different biological process(es) required for a return of normal synaptic function. Acknowledgments—The many conversations within the F.D.A. Center for Biologics Evaluation and Research (CBER) and the Center for Drug Evaluation and Research (CDER) were tremendously welcomed and appreciated. In particular, the help and guidance from Drs. W. F. Vann, PhD and A. Rosenberg, MD, were of great value.
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REFERENCES Adler M, Capacio B, Deshpande SS (2000) Antagonism of botulinum toxin A-mediated muscle paralysis by 3, 4-diaminopyridine delivered via osmotic minipumps. Toxicon 38:1381–1388. Adler M, Keller JE, Sheridan RE, Deshpande SS (2001) Persistence of botulinum neurotoxin A demonstrated by sequential administration of serotypes A and E in rat EDL muscle. Toxicon 39:233–243. Allen DL, Harrison BC, Maass A, Bell ML, Byrnes WC, Leinwand LA (2001) Cardiac and skeletal muscle adaptations to voluntary wheel running in the mouse. J Appl Physiol 90:1900 –1908. Aoki KR (2001) A comparison of the safety margins of botulinum neurotoxin serotypes A, B, and F in mice. Toxicon 39:1815–1820. Ball AP, Hopkinson RB, Farrell ID, Hutchison JG, Paul R, Watson RD, Page AJ, Parker RG, Edwards CW, Snow M, Scott DK, LeoneGanado A, Hastings A, Ghosh AC, Gilbert RJ (1979) Human botulism caused by Clostridium botulinum type E: the Birmingham outbreak. Q J Med 48:473– 491. Blasi J, Chapman ER, Link E, Binz T, Yamasaki S, De Camilli P, Sudhof TC, Niemann H, Jahn R (1993) Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25. Nature 365:160–163. Boquet P, Duflot E (1982) Tetanus toxin fragment forms channels in lipid vesicles at low pH. Proc Natl Acad Sci U S A 79:7614 –7618. Brashear A, Lew MF, Dykstra DD, Comella CL, Factor SA, Rodnitzky RL, Trosch R, Singer C, Brin MF, Murray JJ, Wallace JD, WillmerHulme A, Koller M (1999) Safety and efficacy of NeuroBloc (botulinum toxin type B) in type A-responsive cervical dystonia. Neurology 53:1439 –1446. Brin MF, Lew MF, Adler CH, Comella CL, Factor SA, Jankovic J, O’Brien C, Murray JJ, Wallace JD, Willmer-Hulme A, Koller M (1999) Safety and efficacy of NeuroBloc (botulinum toxin type B) in type A-resistant cervical dystonia. Neurology 53:1431–1438. Brown MC, Holland RL, Ironton R (1980) Nodal and terminal sprouting from motor nerves in fast and slow muscles of the mouse. J Physiol 306:493–510. Centers for Disease Control and Prevention (1998) Botulism in the United States, 1899 –1996. Handbook for epidemiologists, clinicians, and laboratory workers. Atlanta, GA: Centers for Disease Control and Prevention. Coffield JA, Considine RV, Simpson LL (1994) Clostridial neurotoxins in the age of molecular medicine. Trends Microbiol 2:67– 69; discussion 69 –72. Colebatch JG, Wolff AH, Gilbert RJ, Mathias CJ, Smith SE, Hirsch N, Wiles CM (1989) Slow recovery from severe foodborne botulism. Lancet 2:1216 –1217. Costantin L, Bozzi Y, Richichi C, Viegi A, Antonucci F, Funicello M, Gobbi M, Mennini T, Rossetto O, Montecucco C, Maffei L, Vezzani A, Caleo M (2005) Antiepileptic effects of botulinum neurotoxin E. J Neurosci 25:1943–1951. Cull-Candy SG, Lundh H, Thesleff S (1976) Effects of botulinum toxin on neuromuscular transmission in the rat. J Physiol 260:177–203. Duchen LW (1971) An electron microscopic study of the changes induced by botulinum toxin in the motor end-plates of slow and fast skeletal muscle fibres of the mouse. J Neurol Sci 14:47– 60. Duncan RM, Jensen WL (1976) A relationship between avian carcasses and living invertebrates in the epizootiology of avian botulism. J Wildl Dis 12:116 –126. Eleopra R, Tugnoli V, Rossetto O, De Grandis D, Montecucco C (1998) Different time courses of recovery after poisoning with botulinum neurotoxin serotypes A and E in humans. Neurosci Lett 256:135–138. Fasshauer D, Eliason WK, Brunger AT, Jahn R (1998) Identification of a minimal core of the synaptic SNARE complex sufficient for reversible assembly and disassembly. Biochemistry 37:10354–10362. Foran PG, Mohammed N, Lisk GO, Nagwaney S, Lawrence GW, Johnson E, Smith L, Aoki KR, Dolly JO (2003) Evaluation of the therapeutic usefulness of botulinum neurotoxin B, C1, E, and F compared with the long lasting type A. Basis for distinct durations
of inhibition of exocytosis in central neurons. J Biol Chem 278: 1363–1371. Habermann E, Dreyer F (1986) Clostridial neurotoxins: handling and action at the cellular and molecular level. Curr Top Microbiol Immunol 129:93–179. Hallett M (1999) One man’s poison: clinical applications of botulinum toxin. N Engl J Med 341:118 –120. Hayashi T, McMahon H, Yamasaki S, Binz T, Hata Y, Sudhof TC, Niemann H (1994) Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J 13:5051–5061. Hubalek Z, Pellantova J, Hudec K, Halouzka J, Chytil J, Machacek P, Sebela M, Kubicek F (1991) [Botulism in birds living in an aquatic environment in Nove Mlyny in the Breclav District]. Vet Med (Praha) 36:57– 63. Hughes JM, Blumenthal JR, Merson MH, Lombard GL, Dowell VR Jr, Gangarosa EJ (1981) Clinical features of types A and B food-borne botulism. Ann Intern Med 95:442– 445. Irani BG, Xiang Z, Moore MC, Mandel RJ, Haskell-Luevano C (2005) Voluntary exercise delays monogenetic obesity and overcomes reproductive dysfunction of the melanocortin-4 receptor knockout mouse. Biochem Biophys Res Commun 326:638 – 644. Jahn R, Niemann H (1994) Molecular mechanisms of clostridial neurotoxins. Ann N Y Acad Sci 733:245–255. Jankovic J, Brin MF (1991) Therapeutic uses of botulinum toxin. N Engl J Med 324:1186 –1194. Jurasinski CV, Lieth E, Dang Do AN, Schengrund CL (2001) Correlation of cleavage of SNAP-25 with muscle function in a rat model of botulinum neurotoxin type A induced paralysis. Toxicon 39:1309–1315. Keller JE, Neale EA, Oyler G, Adler M (1999) Persistence of botulinum neurotoxin action in cultured spinal cord cells. FEBS Lett 456: 137–142. Koenig MG, Spickard A, Cardella MA, Rogers DE (1964) Clinical and laboratory observations on type E botulism in man. Medicine (Baltimore) 43:517–545. Konhilas JP, Maass AH, Luckey SW, Stauffer BL, Olson EN, Leinwand LA (2004) Sex modifies exercise and cardiac adaptation in mice. Am J Physiol Heart Circ Physiol 287:H2768 –H2776. Koriazova LK, Montal M (2003) Translocation of botulinum neurotoxin light chain protease through the heavy chain channel. Nat Struct Biol 10:13–18. Lightfoot JT, Turner MJ, Daves M, Vordermark A, Kleeberger SR (2004) Genetic influence on daily wheel running activity level. Physiol Genomics 19:270 –276. McMahon HT, Sudhof TC (1995) Synaptic core complex of synaptobrevin, syntaxin, and SNAP25 forms high affinity alpha-SNAP binding site. J Biol Chem 270:2213–2217. Meunier FA, Lisk G, Sesardic D, Dolly JO (2003) Dynamics of motor nerve terminal remodeling unveiled using SNARE-cleaving botulinum toxins: the extent and duration are dictated by the sites of SNAP-25 truncation. Mol Cell Neurosci 22:454 – 466. Montecucco C, Schiavo G (1993) Tetanus and botulism neurotoxins: a new group of zinc proteases. Trends Biochem Sci 18:324 –327. Ohishi I, Sakaguchi G (1977) Activation of botulinum toxins in the absence of nicking. Infect Immunol 17:402– 407. Pevsner J, Scheller RH (1994) Mechanisms of vesicle docking and fusion: insights from the nervous system. Curr Opin Cell Biol 6:555–560. Polak RL, Sellin LC, Thesleff S (1981) Acetylcholine content and release in denervated or botulinum poisoned rat skeletal muscle. J Physiol 319:253–259. Schantz EJ, Johnson EA (1990) Dose standardisation of botulinum toxin. Lancet 335:421 Schiavo G, Matteoli M, Montecucco C (2000) Neurotoxins affecting neuroexocytosis. Physiol Rev 80:717–766. Schiavo G, Rossetto O, Santucci A, DasGupta BR, Montecucco C (1992) Botulinum neurotoxins are zinc proteins. J Biol Chem 267:23479 –23483.
J. E. Keller / Neuroscience 139 (2006) 629 – 637 Sellin LC (1981) The action of botulinum toxin at the neuromuscular junction. Med Biol 59:11–20. Sellin LC, Kauffman JA, Dasgupta BR (1983a) Comparison of the effects of botulinum neurotoxin types A and E at the rat neuromuscular junction. Med Biol 61:120 –125. Sellin LC, Thesleff S, Dasgupta BR (1983b) Different effects of types A and B botulinum toxin on transmitter release at the rat neuromuscular junction. Acta Physiol Scand 119:127–133. Sellin LC, Thesleff S (1981) Pre- and post-synaptic actions of botulinum toxin at the rat neuromuscular junction. J Physiol 317: 487– 495. Shapiro RL, Hatheway C, Swerdlow DL (1998) Botulism in the United States: a clinical and epidemiologic review. Ann Intern Med 129: 221–228. Sheridan RE, Smith TJ, Adler M (2005) Primary cell culture for evaluation of botulinum neurotoxin antagonists. Toxicon 45:377–382. Simpson LL (1973) The interaction between divalent cations and botulinum toxin type A in the paralysis of the rat phrenic nervehemidiaphragm preparation. Neuropharmacology 12:165–176.
637
Simpson LL (1978) Pharmacological studies on the subcellular site of action of botulinum toxin type A. J Pharmacol Exp Ther 206: 661– 669. Simpson LL (2004) Identification of the major steps in botulinum toxin action. Annu Rev Pharmacol Toxicol 44:167–193. Sloop RR, Cole BA, Escutin RO (1997) Human response to botulinum toxin injection: type B compared with type A. Neurology 49: 189 –194. Tucker N, Sobel J, Maslanka SE, Griffin PM, Tauxe RV (2002) Surveillance for botulism: summary of 2001 data, pp 1–11 [Centers for Disease Control and Prevention web site]. http://www.cdc.gov/ncidod/ dbmd/diseaseinfo/botulism_a.htm. Whittaker RL, Gilbertson RB, Garrett AS Jr (1964) Botulism, type E: report of eight simultaneous cases. Ann Intern Med 61:448 – 454. Woodruff BA, Griffin PM, McCroskey LM, Smart JF, Wainwright RB, Bryant RG, Hutwagner LC, Hatheway CL (1992) Clinical and laboratory comparison of botulism from toxin types A, B, and E in the United States, 1975–1988. J Infect Dis 166:1281–1286.
(Accepted 1 December 2005) (Available online 20 February 2006)