the structure and mode of action of different botulinum toxins

ORIGINAL ARTICLE

The structure and mode of action of different botulinum toxins

J. O. Dollya and K. R. AokibaInternational Centre for Neurotherapeutics, Dublin City University, Glasnevin, Dublin, Ireland; and bNeurotoxin Research Program,

Department of Biological Sciences, Allergan Inc., Irvine, CA, USA

Keywords:

botulinum toxin, sensitive

factor attachment protein-

25, sensitive factor

attachment protein recep-

tor

Received 30 August 2006

Accepted 15 September 2006

The seven serotypes (A–G) of botulinum neurotoxin (BoNT) are proteins produced by

Clostridium botulinum and have multifunctional abilities: (i) they target cholinergic

nerve endings via binding to ecto-acceptors (ii) they undergo endocytosis/transloca-

tion and (iii) their light chains act intraneuronally to block acetylcholine release. The

fundamental process of quantal transmitter release occurs by Ca2+-regulated exocy-

tosis involving sensitive factor attachment protein-25 (SNAP-25), syntaxin and syn-

aptobrevin. Proteolytic cleavage by BoNT-A of nine amino acids from the C-terminal

of SNAP-25 disables its function, causing prolonged muscle weakness. This unique

combination of activities underlies the effectiveness of BoNT-A haemagglutinin

complex in treating human conditions resulting from hyperactivity at peripheral

cholinergic nerve endings. In vivo imaging and immunomicroscopy of murine muscles

injected with type A toxin revealed that the extended duration of action results from

the longevity of its protease, persistence of the cleaved SNAP-25 and a protracted time

course for the remodelling of treated nerve–muscle synapses. In addition, an appli-

cation in pain management has been indicated by the ability of BoNT to inhibit

neuropeptide release from nociceptors, thereby blocking central and peripheral pain

sensitization processes. The widespread cellular distribution of SNAP-25 and the

diversity of the toxin’s neuronal acceptors are being exploited for other therapeutic

applications.

Introduction

Botulinum toxin was first identified in 1897 as the

product of the anaerobic bacterium Clostridium botuli-

num and the causative agent of botulism food poisoning

[1]. The development of a purification method for the

toxin began in 1944 and led to crystallization of the type

A serotype [2]. The toxin’s ability to disrupt neuro-

transmitter release at the neuromuscular junction,

thereby reducing muscle activity, resulted in the initi-

ation of investigations on the potential clinical appli-

cations for botulinum toxin by A. Scott in 1968. These

studies culminated in Food and Drug Administration

approval being granted in 1989 for the type A toxin

complex (specifically BOTOX�, Allergan Inc., Cali-

fornia, USA) treatment of individuals with involuntary

muscle disorders, such as strabismus, blepharospasm

and hemifacial spasm. Subsequently, botulinum toxin

has been used for a wide range of conditions charac-

terized by involuntary muscle contraction. More recent

findings have shown that, in addition to its effects at the

neuromuscular junction, botulinum toxin also has

additional clinical applications as a result of its ability

to block the responses of autonomic and sensory

nerves.

The botulinum neurotoxin family

Seven immunologically distinct forms of botulinum

neurotoxin (BoNT) exist, designated as serotypes A, B,

C1, D, E, F and G [3]. All toxin serotypes are synthes-

ized as a single-chain polypeptide of molecular mass

approximately 150 kDa. When produced by the bac-

terium, the toxin molecule associates with additional

non-toxic proteins to form a range of macromolecular

complexes between 300 and 900 kDa. To gain maximum

biological activity, the 150 kDa toxin polypeptide must

be cleaved into a 100 kDa heavy chain and a 50 kDa

light chain, which remain connected by a disulphide

bond and non-covalent interactions. All toxin serotypes

act by preventing acetylcholine release at the peripheral

nerve endings, thus inducing temporary denervation

and relaxation of muscles.

All BoNT serotypes are highly specific in their

targeting of cholinergic nerves, as a result of the

C-terminal region of the toxin binding with high affinity

to ecto-acceptors on the pre-synaptic motor nerve

endings [4]. Application of radiolabelled BoNT to

nerve–muscle preparations showed discrete localization

Correspondence: J. Oliver Dolly, International Centre for Neuro-

therapeutics, Dublin City University, Glasnevin, Dublin 9, Ireland

(tel.: +353 (0) 1 700 7757; fax: +353 (0) 1 700 7758;

e-mail: [email protected]).

� 2006 EFNS 1

European Journal of Neurology 2006, 13 (Suppl. 4): 1–9

of toxin to the motor neurones [5]. After binding,

BoNT is taken up into the motor neurone, via the

process of acceptor-mediated endocytosis [6]. Following

internalization, the light chain is translocated into

the cytoplasm of the motor neurone and mediates the

toxin’s activity. BoNT selectively blocks acetylcholine

release at the neuromuscular junction, thereby abol-

ishing the motor end-plate potential [7]. This effect is

highly specific and non-cholinergic neurotransmission is

not compromised [8].

Recent observations that BoNT may prevent

migraine in some patients prompted re-evaluation of

the neuronal selectivity of its actions, as it was accepted

that the mechanism of the headache pain was inde-

pendent of any muscle contractions [3]. Subsequent

studies have identified a potential antinociceptive

mechanism for BoNT-A on sensory pain nerves [9]. The

biochemical mechanism in sensory nerves (e.g. cleavage

of a SNARE protein target) is identical to that seen

with motor neurones, with the peripheral terminal

being the site of action for both nerves. The difference

in the pharmacological effect is related to the peripheral

release of neuropeptides, which act to sensitize the pain

nerve endings, leading to increased sensitivity of the

terminal to activation [e.g. the direction of pharmaco-

logical effect is periphery to the central nervous system

(CNS)]. In contrast, the action of BoNT on the motor

nerve is to block neurotransmission from the CNS to

the muscle (e.g. direction of pharmacological effect is

CNS to the periphery). In summary, the intracellular

biochemical mechanism and target of BoNT is the same

for a motor nerve or a sensory pain nerve and is

localized to the peripheral terminal of the nerve.

The administration of BoNT has also been shown to

block acetylcholine release in post-ganglionic fibres of

the parasympathetic division of the autonomic nervous

system. This suggests possible clinical roles for BoNT in

the treatment of conditions such as achalasia, hyper-

hidrosis and hypersalivation. In vitro studies have

shown that BoNT-A blocks cholinergic excitatory

transmission in the guinea pig ileum [10] and that radio-

labelled toxin binds to cholinergic nerves in the mouse

ileum [4].

Although the effects of BoNT are mediated by dis-

ruption of acetylcholine release, early studies showed

that introduction of the toxin directly into any nerve

cell could block the release of any neurotransmitter.

These findings indicate that BoNT acts on ubiquitous

intracellular targets required for neurotransmitter

release and that the mechanism of the process is com-

mon to all tissues [8,11]. The in vivo cholinergic specif-

icity of the toxin was solely mediated by the presence of

suitable ecto-acceptors at the motor nerve ending that

mediate toxin binding. Recent reports have indicated

that the synaptic vesicle protein SV2 is responsible for

the binding of BoNT to neurones [12,13].

The molecular basis of botulinum toxinactivity at the neuromuscular junction

Characterization of the molecular basis of BoNT action

was facilitated by the finding that introduction of the

isolated toxin light chain directly into the nerve ending

was sufficient to block acetylcholine release [14]. BoNT

light chains were identified as having zinc-dependent

protease activity and to specifically target a group of

proteins known as SNAREs, involved in mediating

neurotransmitter release from the motor nerve ending

[15,16].

The release of neurotransmitter involves a complex,

multistep process in which stimulation of the nerve

leads to membrane depolarization, which activates

voltage-dependent calcium channels, effecting an influx

of calcium into the nerve terminal. A resultant

increased concentration of calcium causes synaptic

vesicles to fuse with the plasma membrane, thereby

releasing neurotransmitter at the neuromuscular junc-

tion [17]. The SNARE proteins mediate the fusion of

the synaptic vesicle with the plasma membrane and

are classified as the vesicle-associated SNAREs

(v-SNAREs) and the target membrane SNAREs

(t-SNAREs) located at the plasma membrane

[Fig. 1(a)]. The v-SNARE is a protein known as synap-

tobrevin or vesicle-associated membrane protein

(VAMP) that is attached to the synaptic vesicle mem-

brane via its C-terminal region. The t-SNAREs com-

prise two proteins both located at the plasmalemma:

synaptosomal protein with a molecular weight of

25 kDa (SNAP-25) and syntaxin. When the synaptic

vesicle approaches the plasma membrane, the three

proteins interact to form a ternary complex, in which

Syntaxin SNAP-25

PM

Transmitter-containing vesicle

C

N

N

C

N

C

Synaptobrevin

4-Helix bundle

v-SNARE

t-SNAREs

(a)

(b)

Figure 1 The site of action of botulinum toxin light chains within

the SNARE complex. (Adapted from Dolly [17].)

2 J. O. Dolly and K. R. Aoki

� 2006 EFNS European Journal of Neurology, 13 (Suppl. 4): 1–9

two domains from SNAP-25 and one domain each

from synaptobrevin and syntaxin form a four-helix

bundle [Fig. 1(b)]. This complex serves as the SNARE

and plays an essential role in exocytosis, by bringing the

vesicle membrane into close apposition with the plasma

membrane. Once the SNARE complex has formed, it

facilitates avid binding of soluble N-ethylmaleimide

SNAP, which, in turn, allows the association of N-

ethylmaleimide sensitive factor. The latter is an ATPase

enzyme and the cleavage of ATP provides the energy to

dissociate the SNARE complex and allows exocytic

processes to continue.

The light chains of the different BoNT serotypes all

act to disable the SNARE complex by selectively clea-

ving the different SNARE components. BoNT-A

cleaves SNAP-25 by removing nine amino acids from

the C-terminus, whereas BoNT-E cleaves 26 amino

acids from the C-terminus of SNAP-25. The light chains

of the other BoNT serotypes cleave syntaxin (BoNT-C1)

and synaptobrevin (BoNT-B, BoNT-D, BoNT-F and

BoNT-G) at various locations [Fig. 1(b)] [17]. Proteo-

lytic cleavage of the SNARE components serves to

inactivate the individual proteins and disrupt the func-

tioning of the SNARE complex, thereby preventing

exocytosis. However, the ability of the various BoNT

light chain serotypes to act at different cleavage sites

within the SNARE components confers subtle differ-

ences in the resulting inhibition of neurotransmitter

release and clinical applications of the toxins [18].

The clinical utility of botulinum toxinserotype A

One of the major differences seen between the effects of

the toxin serotypes is the duration of the blockade of

neurotransmitter release. The clinical muscle relaxa-

tion seen with type A BoNT complex persists for

4–6 months, which is longer than is seen with other

serotypes. BoNT-A light chain effects cleavage of nine

C-terminal amino acids from SNAP-25 resulting in a

197-amino-acid product. In an animal model, the

inhibition of neurotransmitter release by BoNT-A

persisted for approximately 28 days [19]. In contrast,

deletion of an additional 17 amino acids from SNAP-25

by BoNT-E (giving a 180-amino-acid product) results

in transient paralysis of approximately 5 days duration.

Understanding of the molecular basis of action of the

different serotypes has helped to elucidate the mecha-

nisms behind BoNT-A’s prolonged clinical effect.

A major factor in the persistence of the blockade of

neurotransmitter release by BoNT-A is the intracellular

stability of the toxin’s proteolytic activity. In an

experimental model, the treatment of chromaffin cells

with BoNT-A resulted in a blockade of exocytosis over

at least 3 weeks. In an attempt to overcome this

blockade, the gene-encoding SNAP-25 was over-

expressed in chromaffin cells but could not restore

exocytosis, indicative of persistent intracellular protease

activity [20]. Only over-expression of a mutant form of

SNAP-25, resistant to cleavage by BoNT-A light chain,

was able to rescue exocytic function. Further, pulse-

chase labelling experiments confirmed that BoNT-A

proteolytic activity persists in cerebellar granular neu-

rones for over 31 days [18]. In contrast, the proteolytic

activity of the other toxin serotypes showed shorter

durations: BoNT-C1 for 25 days, BoNT-B for 10 days,

BoNT-F for 2 days and BoNT-E for 0.8 days. Studies

have shown that the BoNT-A light chain has motifs

that promote sequestration at the plasma membrane,

which seemingly protects it from normal degradative

processes [21,22].

The BoNT-A cleavage product, SNAP-25A, is also

very stable and persists for extended periods at the

motor nerve terminal. Confocal microscopy was used to

image the motor nerve and the presence of SNAP-25Aand SNAP-25E using labelled antibodies [19]. Figure 2

images mouse nerve terminals using a motor end-plate

marker (red) and SNAP-25A/E (green), following

treatment with either BoNT-A [Fig. 2(a)] or BoNT-E

[Fig. 2(b)]. Following treatment with BoNT-A, SNAP-

25A accumulated in the motor nerve terminals and

persisted for at least 40 days after toxin administration.

In contrast, the accumulation of SNAP-25E was con-

siderably more short-lived, being apparent 2 days after

toxin administration but not at 7 days. In addition, it

has been shown that the blockade of neurotransmitter

release is maintained by an antagonistic effect mediated

by SNAP-25A on the function of full-length SNAP-25.

Expression of a gene-encoding-truncated SNAP-25

(amino acids 1–197) in chromaffin cells is sufficient to

block exocytosis, indicating that SNAP-25A is able to

antagonize the SNARE function of native SNAP-25

[20].

These properties of the BoNT-A toxin serotype make

it ideally suited for clinical use, as the toxin persists at

the plasma membrane of the motor nerve terminal

affecting long-term proteolysis of SNAP-25. Not only

does the cleavage of SNAP-25 blocks the function of

SNARE complex but also the truncated SNAP-25Aproduct is antagonistic and inhibits the function of

unproteolysed SNAP-25.

Synaptic plasticity associated with botulinumtoxin treatment

A key clinical factor when considering the action of

BoNT-A at the neuromuscular junction is that the

blockade of neurotransmitter release occurs without

Structure and mode of action of botulinum toxins 3


atrophy of the nerve endings. In vivo imaging of the

neurological changes occurring in the mouse following

application of BoNT-A has shown that affected motor

nerve endings retain viability and undergo extensive

nerve sprouting and end-plate remodelling [23].

Figure 3 images the nerve terminals in the mouse

sternomastoid muscle following intramuscular injection

of BoNT-A complex. The body of the nerve was char-

acterized by the uptake of the fluorescent vital dye

4-(4-diethyl aminostyryl)-N-methylpyridinium iodide

(green) and vesicle recycling was demonstrated by

labelling with the exo-endocytic marker N-(3-triethyl

ammonium propyl)-4-(4-(dibutylamino)styryl) pyridi-

nium dibromide (FMI-43, red). Prior to the adminis-

tration of the toxin (day 0), the green vital dye staining

and FMI-43 co-localize in the motor nerve endings,

resulting in an overall yellow colouration. At day 14,

only the green colouration was associated with the

motor nerve endings, indicating that exocytic activity

had been lost. However, by day 14, long �nerve sprouts�were apparent extending from the original motor nerve

and these stained with both the green and the red (the

exoendocytic marker) dyes. Later, acetylcholine recep-

tors clustered on the muscle membrane adjacent to

where the red dye was localized on the nerve plasma

membrane. At day 28, the original motor end plate

remained inactive, but the nerve sprouts had extended

further and were able to transmit nerve impulses,

causing muscle twitch and showing low-level recovery

of function at the neuromuscular junction. By day 63,

the nerve sprouts had stopped growing but retained

exocytic activity. At 91 days after the toxin injection,

the original nerve terminal had regained exocytic

function and the extensive network of nerve sprouts

had retracted, with these findings correlating with full

recovery of nerve-evoked muscle contraction. The

motor end-plate remodelling and formation of fun-

ctional extra-junctional synapses is thought to be

important in allowing recovery of the original motor

end plate and provides evidence of the synaptic plasti-

city associated with the neuromuscular junction.

Sensory effects of botulinum toxin

During early investigations, it was noted that admin-

istration of BoNT-A complex often resulted in a

considerable reduction in pain and that pain relief

often preceded or exceeded the clinical reduction in

muscle tone. Various pre-clinical studies have subse-

quently shown that the toxin is able to inhibit neu-

ropeptide release from primary nociceptive afferent

C fibres and possibly from lightly myelinated A delta

fibres [24]. As these nociceptors are primarily located

in the skin and the toxin is administered in the peri-

phery, a localized effect is postulated for the pain relief

mechanism.

Ove

rlay

SN

AP

-25 A

Ove

rlay

SN

AP

-25 E

(b)

(a)

Figure 2 The persistence of SNAP-25A at

the mouse motor end plate. (Reproduced

from Meunier [19].)



The proposed mechanism for action of the toxin on

nociceptors is summarized in Fig. 4 [3]. Peripheral pain

stimulus is detected by the nociceptive C fibres and the

resulting action potential transmits a signal to the CNS.

The signal provokes release of neurotransmitters such

as glutamate and peptides in the CNS, within the dorsal

root ganglion. There is most probably an antidromic

activation signal, which travels down one of the per-

ipheral branches of the sensory nerve to cause auto- or

heterosensitization. The antidromic signal initiates the

release of glutamate and peptides by the peripheral

nerve terminals, which further stimulate all nerve ter-

minals in the vicinity. This localized stimulation of

nociceptors results in peripheral sensitization and in

additional afferent signals being transmitted to the

CNS, whereby more glutamate and peptides are

released, affecting central sensitization. These processes

result in a cyclical exacerbation of the pain response,

and in a chronic state, the CNS misinterprets external

signals, such as touch and elevated temperatures, as

d0

d14

d28 Bar =10 µm

Bar =10 µm

d 0

d 63

d 91Figure 3 BoNT-A-induced nerve sprout-

ing reproduced from de Paiva [23].

Figure 4 Model of the neurological basis

of the pain response.



pain. It is proposed that BoNT acts by blocking neu-

rotransmitter release at the peripheral nerves by dis-

rupting SNARE-dependent exocytic processes. The

blockade of glutamate and peptide release in the

periphery prevents the development of peripheral

sensitization. As this disrupts transmission of the

additional activation signals to the CNS, the toxin

indirectly blocks the process of central sensitization.

Various experimental models support this hypothesis

for the toxin’s pain reduction.

The formalin-induced pain model

In the formalin-induced pain model, a subcutaneous

injection of formalin into the rat footpad elicits quan-

tifiable behavioural responses indicative of pain, such as

shaking and licking the treated paw [25]. With this

model, the pain response occurred as an initial acute

phase (phase I) and a prolonged second phase (phase

II), characterized by localized inflammation and central

sensitization. The pre-treatment of the footpad with

BoNT-A complex resulted in a dose-dependent reduc-

tion in pain responses, at toxin doses too low to cause

muscle weakness [26]. This effect was only apparent

during the phase II pain response, indicating that the

toxin was not exerting a localized anaesthetic effect on

the nociceptors. Introduction of a dialysis probe into

the footpad showed that the toxin blocked the forma-

lin-induced release of glutamate during phase II of the

pain response.

In addition to limiting peripheral sensitization, there is

also evidence that type A toxin reduces subsequent CNS

sensitization. In the formalin-induced pain model, the

spinal wide dynamic range neurones show increased

activity during the phase I and II pain responses. The

phase II increase in the activity of wide dynamic range

neurones can be blocked by pre-treatment of the footpad

with BoNT-A complex [27,9]. In addition, this pre-

treatment resulted in a dose-dependent reduction in the

proportion of dorsal horn neuronal cells expressing the

c-fosgene, amarkerof second-orderneuronal stimulation.

The capsaicin model

In a second model, capsaicin was injected into the

plantar rat footpad surface and pain was assessed

according to the magnitude of pressure stimulus or

temperature elevation required to induce a paw with-

drawal response [28,29]. Enhanced sensitivity is thought

to reflect secondary thermal hyperalgesia or mechanical

allodynia. Blood flow measurement and mapping of the

wide dynamic range neurone receptive field within the

footpad can also be assessed. The injection of capsaicin

to the rat footpad affected an expansion in the wide

dynamic range neurone’s receptive field that was almost

completely blocked by pre-treatment with BoNT-A

complex [30]. This correlated with a reduced frequency

of foot withdrawal in response to pressure and an

increased latency period before foot withdrawal in

response to elevated temperature [31]. The ability of the

toxin to abolish the enhanced pressure and temperature

sensitivity following capsaicin injection or sciatic nerve

transaction has been confirmed by other investigators

[32,33]. A reduction in blood flow in the capsaicin-

treated footpad was also seen following pre-treatment

with BoNT-A complex, and occurred at doses consid-

erably lower than those required to inhibit mechanical

allodynia.

Capsaicin binds to the vanilloid/heat receptor

TRPV1 located on sensory nerve endings. The

TRPV1 receptor transduces noxious chemical and

thermal signals and stimulates the local release of

substance P. The Ferrer-Montiel laboratory [34] has

shown that TRPV1 surface expression in vitro is

increased with inflammatory mediator stimulation.

The mechanism by which TRPV1 is transferred to the

plasma membrane involves SNARE-dependent exo-

cytosis and is inhibited by BoNT-A treatment. As

the peripheral sensitization mediated by capsaicin

involves upregulation of the number of TRPV1

receptors on the surface of sensory neurones, the

ability of BoNT-A to block exocytosis may contribute

to the reduced pain responses seen in animal models.

These findings are supported by reports that patients

with intractable detrusor over activity show elevated

levels of TRPVI receptors in bladder biopsy samples

and that these levels are reduced by treatment with

BoNT-A complex [35].

The diabetic neuropathy model

The experimental diabetic neuropathy model uses

streptozocin to induce mechanical allodynia in rats,

with this model thought to act via nerves other than the

large myelinated afferent fibres [36–38]. Once allodynia

is established, determined by a paw withdrawal

threshold of less than 6 g, the ability of type A toxin to

reverse the effect is determined. The administration of

BoNT-A reverses this established allodynia in the ipsi-

lateral but not in the contralateral paw (Fig. 5) [31,38].

In contrast, administration of gabapentin reversed

allodynia in both paws and required daily treatments

for a sustained effect.

Clinical pain models

A number of human volunteer studies have considered

the use of BoNT in pain management, but with



conflicting results [39–43]. Careful consideration needs

to be given as to how such studies are conducted to

optimize the timing of (i) the challenge event; (ii) toxin

treatment; (iii) the dose employed; (iv) the pain model

used and (v) the coincidence of pain stimuli and toxin

treatment areas. Negative results in a human clinical

pain model may not represent the toxin’s effectiveness

in a patient if (i) the treated areas and the �pain� area do

not coincide; (ii) the peripheral pain source is predom-

inately driven by the sensory neurone and (iii) the

timing between the toxin treatment and the stimulus are

inappropriate.

Conclusions

Botulinum neurotoxin is a potent neurotoxin that

inhibits neurotransmitter release by disrupting the

functioning of the SNARE complex, required for exo-

cytosis of synaptic vesicles. The different BoNT sero-

types exhibit differences in their toxic properties,

depending on how they disrupt SNARE complex for-

mation. The characteristics of BoNT-A mean that it is

ideally suited to clinical use, as it exerts a long-acting

blockade of acetylcholine release at the neuromuscular

junction. This, in turn, effects long-lasting muscle

paralysis, over a period of 4–6 months in humans. This

optimal blockade of vesicle exocytosis is the result of

the multifunctional activity of the toxin, which persists

for extended periods at the neuronal plasma membrane,

inactivates SNAP-25, thereby disabling the exocytosis

machinery and producing a truncated form of SNAP-

25A that inhibits the formation of functional SNARE

complexes.

The ability of BoNT-A to inhibit neuromuscular

transmission is well characterized; however, a role for

the toxin in blocking neurotransmitter release by

sensory neurones is increasingly being recognized.

Pre-clinical studies have shown BoNT to inhibit glu-

tamate and neuropeptide release in the periphery,

which disrupts both peripheral and central sensitiza-

tion in response to chronic pain stimuli. The

molecular basis of the pain-inhibiting properties of

type A toxin is in common with the mechanism by

which the toxin affects muscle relaxation. As yet the

clinical relevance of these pre-clinical results remains

to be established, but the results tie in with obser-

vations concerning pain relief.

Further understanding of the molecular basis of the

action of both BoNT-A and other toxin serotypes is

likely to unveil novel clinical applications for this family

of molecules.

Acknowledgement

The production of this article was supported by an

unrestricted educational grant from Allergan.

Declaration of interest

Prof. J. Oliver Dolly received an honorarium from

Ashley Communications for lecturing on botulinum

toxin and has a research contract from Allergan Inc. for

basic science studies on botulinum toxins. Dr K. Roger

Aoki is an employee of Allergan Inc., which manufac-

tures and sells BOTOX�.

References

1. Schantz EJ, Johnson EA. Botulinum toxin: the storyof its development for the treatment of human disease.Perspectives in Biology and Medicine 1997; 40: 317–327.

2. Schantz EJ, Johnson EA. Properties and use of botulinumtoxin and other microbial neurotoxins in medicine.Microbiological Reviews 1992; 56: 80–99.

Figure 5 Inhibition of established allody-

nia in the diabetic neuropathy model.



3. Aoki KR. Evidence for antinociceptive activity of botul-inum toxin type A in pain management. Headache 2003;43(Suppl. 1): S9–S15.

4. Black JD, Dolly JO. Selective location of acceptors forbotulinum neurotoxin A in the central and peripheralnervous systems. Neuroscience 1987; 23: 767–779.

5. Dolly JO, Black J, Williams RS, Melling J. Acceptorsfor botulinum neurotoxin reside on motor nerve terminalsand mediate its internalization. Nature 1984; 307: 457–460.

6. Black JD, Dolly JO. Interaction of 125I-labeled botuli-num neurotoxins with nerve terminals. II. Autoradio-graphic evidence for its uptake into motor nerves byacceptor-mediated endocytosis. Journal of Cell Biology1986; 103: 535–544.

7. Dolly JO, Lande S, Wray DW. The effects of in vitroapplication of purified botulinum neurotoxin at mousemotor nerve terminals. Journal of Physiology 1987; 386:475–484.

8. Poulain B, Tauc L, Maisey EA, Wadsworth JD, MohanPM, Dolly JO. Neurotransmitter release is blockedintracellularly by botulinum neurotoxin, and this requiresuptake of both toxin polypeptides by a process mediatedby the larger chain. Proceedings of the National Academyof Sciences of the United States of America 1988; 85: 4090–4094.

9. Aoki KR. Review of a proposed mechanism for theantinociceptive action of botulinum toxin type A.Neurotoxicology 2005; 26: 785–793.

10. MacKenzie I, Burnstock G, Dolly JO. The effects ofpurified botulinum neurotoxin type A on cholinergic,adrenergic and non-adrenergic, atropine-resistant auto-nomic neuromuscular transmission. Neuroscience 1982; 7:997–1006.

11. Ashton AC, Dolly JO. Characterization of the inhibitoryaction of botulinum neurotoxin type A on the release ofseveral transmitters from rat cerebrocortical synapto-somes. Journal of Neurochemistry 1988; 50: 1808–1816.

12. Dong M, Yeh F, Tepp WH et al. SV2 is the proteinreceptor for botulinum neurotoxin A. Science 2006; 312:592–596.

13. Mahrhold S, Rummel A, Bigalke H, Davletov B, Binz T.The synaptic vesicle protein 2C mediates the uptake ofbotulinum neurotoxin A into phrenic nerves. FEBS Let-ters 2006; 580: 2011–2014.

14. de Paiva A, Dolly JO. Light chain of botulinum neuro-toxin is active in mammalian motor nerve terminals whendelivered via liposomes. FEBS Letters 1990; 277: 171–174.

15. Schiavo G, Benfenati F, Poulain B et al. Tetanus andbotulinum-B neurotoxins block neurotransmitter releaseby proteolytic cleavage of synaptobrevin. Nature 1992;359: 832–835.

16. Schiavo G, Rossetto O, Santucci A, DasGupta BR,Montecucco C. Botulinum neurotoxins are zinc proteins.The Journal of Biological Chemistry 1992; 267: 23479–23483.

17. Dolly O. Synaptic transmission: inhibition of neuro-transmitter release by botulinum toxins. Headache 2003;43(Suppl. 1): S16–S24.

18. Foran PG, Mohammed N, Lisk GO et al. Evaluation ofthe therapeutic usefulness of botulinum neurotoxin B, C1,E and F compared with the long lasting type A. Basis fordistinct durations of inhibition of exocytosis in centralneurons. The Journal of Biological Chemistry 2003; 278:1363–1371.

19. Meunier FA, Lisk G, Sesardic D, Dolly JO. Dynamics ofmotor nerve terminal remodeling unveiled using SNARE-cleaving botulinum toxins: the extent and duration aredictated by the sites of SNAP-25 truncation. Molecularand Cellular Neurosciences 2003; 22: 454–466.

20. O’Sullivan GA, Mohammed N, Foran PG, LawrenceGW, Oliver Dolly J. Rescue of exocytosis in botulinumtoxin A-poisoned chromaffin cells by expression of clea-vage-resistant SNAP-25. Identification of the minimalessential C-terminal residues. The Journal of BiologicalChemistry 1999; 274: 36897–36904.

21. Fernandez-Salas E, Ho H, Garay P, Steward LE, AokiKR. Is the light chain subcellular localization animportant factor in botulinum toxin duration of action?Movement Disorders 2004; 19(Suppl. 8): S23–S34.

22. Fernandez-Salas E, Steward LE, Ho H et al. Plasmamembrane localization signals in the light chain of bot-ulinum neurotoxin. Proceedings of the National Academyof Sciences of the United States of America 2004; 101:

3208–3213.23. de Paiva A, Meunier FA, Molgo J, Aoki KR, Dolly JO.

Functional repair of motor endplates after botulinumneurotoxin type A poisoning: biphasic switch of synapticactivity between nerve sprouts and their parent terminals.Proceedings of the National Academy of Sciences of theUnited States of America 1999; 96: 3200–3205.

24. Aoki KR. Pharmacology and immunology of botulinumtoxin serotypes. Journal of Neurology 2001; 248(Suppl. 1):3–10.

25. Wheeler-Aceto H, Porreca F, Cowan A. The rat pawformalin test: comparison of noxious agents. Pain 1990;40: 229–238.

26. Cui M, Khanijou S, Rubino J, Aoki KR. Subcutaneousadministration of botulinum toxin A reduces formalin-induced pain. Pain 2004; 107: 125–133.

27. Cui M, Li Z, You S, Khanijou S, Aoki KR. Mechanismsof the antinociceptive effect of subcutaneous Botox:inhibition of peripheral and central nociceptive process-ing. Naunyn Schmiedeberg’s Archives of Pharmacology2002; 365(Suppl. 2): R17.

28. Wu J, Fang L, Lin Q, Willis WD. Nitric oxide synthase inspinal cord central sensitization following intradermalinjection of capsaicin. Pain 2001; 94: 47–58.

29. Hargreaves K, Dubner R, Brown F, Flores C, Joris J. Anew and sensitive method for measuring thermal noci-ception in cutaneous hyperalgesia. Pain 1988; 32: 77–88.

30. Cui M, Li Z, Khanijou S, Rubino J, You S, Aoki R. Sub-cutaneous administration of BOTOX� inhibits capsaicin-induced thermal hyperalgesia and expansion of hornneuronal receptive field area.Society ofNeurosciencePosterF101. Program No. 812.13. 2003.

31. Francis J, You S, Satorius A et al. Analgesic propertiesand mechanism of action of botulinum toxin type A(BOTOX�). Toxins Meeting (Online) 2005, Abstract 30.

32. Bach-Rojecky L, Lackovic Z. Antinociceptive effect ofbotulinum toxin type a in rat model of carrageenan andcapsaicin induced pain. Croatian Medical Journal 2005;46: 201–208.

33. Bach-Rojecky L, Relja M, Lackovic Z. Botulinum toxintype A in experimental neuropathic pain. Journal ofNeural Transmission 2005; 112: 215–219.

34. Morenilla-Palao C, Planells-Cases R, Garcia-Sanz N,Ferrer-Montiel A. Regulated exocytosis contributes toprotein kinase C potentiation of vanilloid receptor



activity. The Journal of Biological Chemistry 2004; 279:25665–25672.

35. Apostolidis A, Popat R, Yiangou Y et al. Decreasedsensory receptors P2X3 and TRPV1 in suburothelialnerve fibers following intradetrusor injections of botuli-num toxin for human detrusor overactivity. Journal ofUrology 2005; 174: 977–983.

36. Dixon WJ. Efficient analysis of experimental observa-tions. Annual Review of Pharmacological Toxicology 1980;20: 441–462.

37. Chaplan SR, Bach FW, Pogrel JW, Chung JM, YakshTL. Quantitative assessment of tactile allodynia in the ratpaw. Journal of Neuroscience Methods 1994; 53: 55–63.

38. You S, Satorius A, Ardila C et al. Subcutaneous botuli-num neurotoxin type A inhibits streptozotocin (STZ)-induced chronic pain in a rat model of peripheral diabeticneuropathy. Society of Neuroscience 2005, Abstract514.28.

39. Voller B, Sycha T, Gustorff B et al. A randomized, dou-ble-blind, placebo controlled study on analgesic effects ofbotulinum toxin A. Neurology 2003; 61: 940–944.

40. Blersch W, Schulte-Mattler WJ, Przywara S, May A,Bigalke H, Wohlfarth K. Botulinum toxin A and thecutaneous nociception in humans: a prospective, double-blind, placebo-controlled, randomized study. Journal ofthe Neurological Sciences 2002; 205: 59–63.

41. Kramer HH, Schmidt K, Leis S, Schmelz M, Sommer C,Birklein F. Angiotensin converting enzyme has an inhib-itory role in CGRP metabolism in human skin. Peptides2006; 27(4):917–920.

42. Sycha T, Samal D, Chizh B et al. A lack of antinocicep-tive or antiinflammatory effect of botulinum toxin A in aninflammatory human pain model. Anesthesia and Anal-gesia 2006; 102: 509–516.

43. Gazerani P, Staahl C, Drewes AM, Arendt-Nielsen L. Theeffects of botulinum toxin type A on capsaicin-evokedpain, flare, and secondary hyperalgesia in an experimentalhuman model of trigeminal sensitisation. Pain 2006; 122:315–325.



the structure and mode of action of different botulinum toxins

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