Central Effects of Botulinum Toxin Type A:Evidence and Supposition

Antonio Curra, MD,1 Carlo Trompetto, MD,2 Giovanni Abbruzzese, MD,2 and Alfredo Berardelli, MD1*

1Dipartimento di Scienze Neurologiche and Istituto Neurologico Mediterraneo Neuromed IRCCS,Universita degli Studi di Roma “La Sapienza,” Italia

2Dipartimento di Neuroscienze, Oftalmologia e Genetica, Universita di Genova, Italia

Abstract: No convincing evidence exists that botulinum toxintype A (BT-A) injected intramuscularly at therapeutic doses inhumans acts directly on central nervous system (CNS) struc-tures. Nevertheless, several studies, using various approaches,strongly suggest that BT-A affects the functional organizationof the CNS indirectly through peripheral mechanisms. By act-ing at alpha as well as gamma motor endings, BT-A could alterspindle afferent inflow directed to spinal motoneurons or to thevarious cortical areas, thereby altering spinal as well as corticalmechanisms. Muscle afferent input is tightly coupled to motor

cortical output, so that the afferents from a stretched muscle goto cortical areas where they can excite neurons capable ofcontracting the same muscle. The BT-A–induced reduction inspindle signals could, therefore, alter the balance betweenafferent input and motor output, thereby changing corticalexcitability. © 2004 Movement Disorder Society

Key words: botulinum toxin type A; intramuscular injec-tion; central nervous system; cortical excitability; sensorimotorintegration; basic science; neurophysiology

Botulinum toxin type A (BT-A) is widely used to treata variety of clinical conditions characterized by musclehyperactivity. The clinical benefits of BT-A injectionsdepend primarily on the toxin’s peripheral action. Botu-linum toxin acts peripherally by inhibiting acetylcholine(ACh) release from the presynaptic neuromuscular ter-minals, thus weakening contraction of the muscle fibersresponsible for excessive involuntary movements. Al-though basic and clinical research has amply focused onthe toxin’s peripheral actions, BT-A acts also on centralnervous system (CNS) structures.1 Central mechanismsmight also help to explain why botulinum A injectionsoccasionally leave the injected muscles disproportion-ately weak.

Current knowledge, therefore, shows that intramuscu-larly injected BT-A acts peripherally. What we need now

is more information on the toxin’s possible central ac-tions when injected at therapeutic doses in humans, aswell as data to show whether these effects arise directlyor indirectly, or in both ways. Theoretically, locallyinjected BT-A could produce central effects directly, bybeing transported into central structures, or indirectly, byaltering central sensorimotor integration through a pe-ripheral mechanism.2 Here, we review the main experi-mental and clinical studies providing information on thecentral action of BT-A.

EXPERIMENTAL BACKGROUND

Experimental studies have provided ample evidencesupporting a central action of BT-A in humans. First,when injected into skeletal muscles, BT-A acts at theintrafusal as well the extrafusal neuromuscular junction.The toxin blocks the gamma motor endings of jaw mus-cles in the rat, reducing the spindle afferent dischargewithout altering muscle tension.3 These effects startwithin 80 minutes after the injection. A morphologicalstudy compared the effects of BT-A on extrafusal andintrafusal muscle fibers in the rat.4 The toxin caused fiberatrophy and spread of ACh staining in the end-plates,indicating parallel denervation of extrafusal and intra-

*Correspondence to: Alfredo Berardelli, MD, Dipartimento di Sci-enze Neurologiche, Universita degli Studi di Roma “La Sapienza,”Viale dell’Universita 30, 00185 Rome, Italy.E-mail: alfredo.berardelli@uniroma1.it.

DOI 10.1002/mds.20011Published online in Wiley InterScience (www.interscience.wiley.

com).

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fusal fibers. Evidence of a fusimotor denervation sug-gests that BT-A alters activity in muscle spindle affer-ents. The altered spindle afferent input, therefore, may beindirectly responsible for functional changes in centralmotor mechanisms at both spinal and supraspinal levels.

Second, evidence that intramuscularly injected BT-Ainfluences the spinal cord circuitry comes from a retro-grade-tracing study by Weigand and colleagues,5 whoshowed that approximately 48 hours after injecting ra-diolabeled BT-A into the cat gastrocnemius muscle, adistal–proximal gradient of radioactivity developed firstin the sciatic nerve, then in the ipsilateral spinal ventralroots, and ultimately in the spinal cord segments inner-vating the injected muscle. Radioactivity may alsospread to spinal cord segments contralateral to the injec-tion and in smaller amounts to the dorsal roots. Thesefindings suggest that BT-A is centrally transported onmotor and intrafusal afferent axons. Using neurophysio-logical techniques in the cat, others have confirmed thetoxin’s action on parts of the alpha-motoneuron somamembrane.6 In addition, BT-A, directly applied into thespinal cord of anaesthetized cats, decreases cholinergictransmission at Renshaw cells and reduces inhibition ofIa inhibitory interneurons, but when injected into thegastrocnemius muscle, the neurotoxin left these centralmechanisms unchanged.7

Studies in cultured cells have shown that the toxin canexert its effects directly and diffusely in CNS structures.In cell cultures, BT-A alters the activity of nerve termi-nals by blocking the release of various neurotransmitters,thus inhibiting cholinergic and noncholinergic synaptictransmission.8–10 Studies of animal brain tissue show thatbotulinum toxins bind preferentially to synaptosomesand to synapse-rich areas of the rat brain such as hip-pocampus and cerebellum.11–13

The question of central effects depends crucially onthe ability of BT-A to pass through the blood–brainbarrier. After intravenous injection of high doses ofBT-A marked with 125I, labeled toxin was detected bymeans of autoradiography and fluorescent labelling inthe brain parenchyma and blood vessels.14 In vivo evi-dence that the toxin diffuses through the blood streamand crosses the blood–brain barrier at the low therapeu-tic doses used in clinical applications is lacking.

NEUROPHYSIOLOGICAL ANDCLINICAL STUDIES

Effects of BT-A on Spinal Mechanisms

Information indicating a central action of BT-A comesfrom studies of the reciprocal inhibition between agonistand antagonist muscles. Reciprocal inhibition is nor-

mally analysed by conditioning the H reflex in forearmflexor muscles with a radial nerve stimulus delivered at arange of time intervals. In healthy subjects, reciprocalinhibition comprises an initial phase (conditioning-testinterval: 0 msec), reflecting a disynaptic pathway, fol-lowed by a second phase (conditioning-test intervals:10–30 msec), resulting from presynaptic mechanisms. Inpatients with upper limb dystonia, the second phase ofreciprocal inhibition between flexor and extensor mus-cles is abnormal. In a study of patients with arm dysto-nia, Priori and coworkers15 showed that the second,abnormal, phase of reciprocal inhibition was increased 3weeks after injection of BT-A in the dystonic forearmflexor muscles, whereas the first phase remained un-changed from pretreatment values. Botulinum toxintreatment also reduced the M wave and the H reflex by asimilar amount and left the H/M ratio unchanged. Thesefindings suggest a concurrent indirect effect of BT-A onspinal cord circuitry, probably through the presynapticblock of the intrafusal neuromuscular junction reducingthe spindle afferent input to the spinal cord and increas-ing presynaptic inhibition of flexor muscle afferents.Similar central changes were seen in a group of patientswith essential tremor.16 BT-A injection produced a sig-nificant functional improvement in tremor and, within 1month, restored the reduced presynaptic phase of recip-rocal inhibition almost to normal. These two studiesprovide evidence that BT-A injections alter the excitabil-ity of spinal cord circuitry by acting concurrently onextrafusal and intrafusal motor end-plates: action on in-trafusal end-plates decreases spindle afferent input to thespinal cord.

In contrast, in patients with upper limb spasticity, thereduction in both inhibitory phases of reciprocal inhibi-tion remained unchanged at various intervals after BT-Atreatment,17 suggesting that the toxin’s efficacy in spas-ticity depended mainly on its peripheral effects. Similarresults were reported by Panizza and colleagues,18 whofound no significant changes in the H/M ratio and in Hreflex presynaptic inhibition during vibration, althoughall the patients reported a subjective clinical improve-ment. A possible explanation for the contrasting effect ofBT-A injections on reciprocal inhibition in dystonia andspasticity is the different role of afferent feedback inthese clinical conditions.

Using transcranial magnetic stimulation (TMS), Pauriand coworkers19 investigated changes of in motor evokedpotentials (MEPs) to TMS of the leg area in patients withlower limb spasticity requiring BT-A injections in thecalf muscles. All patients benefited from the treatment,but MEP latency and central conduction time increasedsignificantly in the injected muscles 2 weeks after treat-

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ment. Pauri and coworkers19 tentatively attributed thesefindings to central change in spinal motoneuron respon-siveness to descending impulses from the corticospinaltracts.

Further evidence of possible changes in the spinal cordmachinery came recently from a study by Wohlfarth andcolleagues,20 showing that BT-A injected locally at ther-apeutic doses caused slight changes in the F-wave inremote nontreated muscles of patients with focal dysto-nia. One week after treatment, F-wave latencies weresignificantly prolonged (1–3 msec) and F-wave persis-tence decreased by approximately 20%. The investiga-tors suggested that the excitability of alpha-motoneuronssupplying nontreated muscles decreased because of re-duced muscle spindle activity or changes in recurrentinhibition. In addition, they postulated that the toxincould reach the motoneurons by means of the hematog-enous route (directly or indirectly) after uptake at thenerve endings and retrograde axonal transport.

Effects of BT-A on Brainstem Mechanisms

Studies in cranial dystonia failed to demonstratechanges in the excitability of brainstem reflexes afterlocal injections of BT-A. Blepharospasm, the most com-mon cranial dystonia, is characterized by involuntaryrecurrent spasms of both eyelids. Evidence of increasedexcitability of brainstem interneurons in blepharospasmhas been provided by studying the blink reflex recoverycycle.21 The blink reflex is tested by stimulating thesupraorbital nerve unilaterally while recording from theorbicularis oculi muscle bilaterally. Blink reflex record-ings show a first early response (R1), ipsilateral to thestimulated side, and a second bilateral late response (R2).While the R1 component is relayed centrally through anoligosynaptic (trigeminal–facial) arc, the R2 componentreflects a polysynaptic pathway and its recovery cycle isenhanced in patients with blepharospasm.21,22 The excit-ability of the blink reflex recovery cycle has been studiedin patients with blepharospasm before and after BT-Atreatment. After treatment, the amplitude of the R1 com-ponent was reduced, but the recovery cycle of the R2response remained abnormally enhanced even at the timeof maximal clinical benefit.22 The same results wereconfirmed by other groups who treated patients withblepharospasm unilaterally and recorded the blink reflexfrom the untreated orbicularis oculi muscle.23,24 The re-covery curve of the R2 component remained unchanged,although a clinical benefit was present bilaterally possi-bly because of toxin spread. Altogether, these studiessuggest that botulinum toxin treatment has little influ-ence upon the enhanced excitability of brainstem inter-neurons in patients with blepharospasm. In this line of

evidence, BT-A injections failed to show any effect onbrainstem auditory evoked potentials.25,26

Findings in other motor disorders nevertheless suggestotherwise. After treating adductor spasmodic dysphoniawith unilateral thyroarytenoid muscle injections ofBT-A, Bielamowicz and Ludlow27 observed that laryn-geal muscle bursts were reduced bilaterally, also in thenoninjected muscles. They, therefore, suggested that thebotulinum-induced improvement in speech symptomsmight reflect changes in a central pathophysiologicalmechanism.

Effects of BT-A on Cortical Mechanisms

Different approaches have been used to investigatepossible BT-A–induced changes in functional corticalorganization. Investigating the behaviour of long-latencyreflexes (LLR) elicited in the thenar muscles by electricalmedian nerve stimulation in a group of patients withfocal hand dystonia, Naumann and Reiners28 observedthat the late component (LLR2, occurring at approxi-mately 50 msec) was significantly reduced in amplitudeon the clinically affected side after botulinum toxin treat-ment. Because the LLR2 component is supposed to re-flect a cortical generator (involving the supplementarymotor area), the researchers suggested that BT-A injec-tions modify the afferent input coming from the injectedmuscles, thus modulating the cortical overflow observedin dystonia.

Similarly, recording somatosensory evoked responsesbefore and after treatment with BT-A for cervical dys-tonia, Kanovsky and coworkers29 showed that the am-plitude of the P22/N30 precentral component (recordedcontralaterally to the direction of head deviation) wassignificantly reduced after treatment, paralleling the clin-ical improvement in head position. These findings mightreflect changes in precentral cortex excitability second-ary to toxin-induced modulation of spindle afferent in-puts.

Evidence of changes in cortical organization afterBT-A injections has been provided also by studies withTMS. In a study designed to map the topography of theprimary motor cortex projections to the upper limb mus-cles in patients with writer’s cramp during a sustainedisometric contraction, Byrnes and coworkers30 deliveredTMS before and after BT-A injections in the affectedmuscles. Corticomotor representation differed in patientsrelative to normal subjects, patients’ cortical maps beingdistorted in shape and having extended lateral borders.Botulinum toxin treatment induced a clinical improve-ment in focal hand dystonia and concurrently reversedthe cortical map changes. When the clinical benefit inwriter’s cramp wore off, the cortical maps returned to

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their original topography. This study suggested thatchanges in the primary motor cortex of dystonic patientsmight be secondary to abnormal afferent inputs thatinjected BT-A may transiently modulate.

Another way to look at changes in cortical excitabilityis to use TMS with paired stimuli in a conditioning-testparadigm.31 In normal subjects, a conditioning sub-threshold transcranial magnetic stimulus over the motorcortex (set to such a low intensity that it elicits no MEPon its own) suppresses the motor potentials evoked by asubsequent suprathreshold test stimulus given at shortinterstimulus intervals (1 to 5 msec later). Inhibition ofthis kind is a purely cortical mechanism and is thought toreflect the activation of intracortical GABAergic inhibi-tory interneurons.31 Studies using paired-pulse TMShave shown that patients with dystonia have less intra-cortical inhibition than normal subjects,32 probably ow-ing to a defective cortical inhibitory system.33

The paired-pulse TMS technique has been used toinvestigate whether BT-A injections alter intracorticalinhibitory mechanisms in patients with upper limb dys-tonia.34 In all patients, BT-A injections reduced dystonicmovements in the arm. Before treatment, patientsshowed a reduced test response inhibition. One monthafter the injection, intracortical inhibition increased, re-turning to values seen in normal subjects. Three monthsafter treatment, values of intracortical inhibition droppedagain to pretreatment levels. This study suggests thatBT-A can transiently modify the excitability of the motorcortical areas by reorganizing inhibitory and excitatoryintracortical circuits. The effect probably results indi-rectly from the toxin’s peripheral action.

Finally, positron emission tomography (PET) has beenused to investigate possible changes in the pattern ofcortical activation induced by botulinum toxin injections.PET activation studies showed an overactivity of stria-tum and nonprimary motor areas and an underactivity ofthe primary motor cortex in patients with dystonia duringvoluntary movement. After BT-A injections, the im-provement in writing of patients with focal hand dystoniawas accompanied by an increased activation in the pari-etal cortex and caudal supplementary motor area, leavingthe pattern of activity in the primary motor cortex un-changed.35 These effects suggested a cortical reorgani-zation secondary to the deafferentation of alpha-motor-neurons or changes in motor strategy.

Another concept relevant to the potential targets ofBT-A action is brain plasticity. Brain plasticity has beenwidely demonstrated by functional studies using PET orTMS techniques. A reorganization of the human motorsystem can follow motor learning36 or lesions of periph-eral or central structures.37,38 In particular, transient deaf-

ferentation can result in short-term plasticity.39 Changesin peripheral feedback or its central integration play amajor role in the pathophysiology of movement disor-ders.2 Hence, some of the long-term clinical benefits ofBT-A treatment may also reflect plastic changes in motoroutput after the reorganization of synaptic density.

In conclusion, this review suggests that BT-A has acomplex mechanism of action. In addition to actingdirectly at the neuromuscular junction, the toxin mostprobably alters sensory inputs to the CNS, thus indirectlyinducing secondary central changes. We cannot yet saywhether a knowledge of the indirect central action ofbotulinum toxin will alter its future use in clinical prac-tice. A deeper understanding of this mechanism mayultimately lead to a more rational therapeutic use, andshould prompt questions for further research.

REFERENCES

1. Simpson LL. The origin, structure, and pharmacological activity ofbotulinum toxin. Pharmacol Rev 1981;33:155–188.

2. Abbruzzese G, Berardelli A. Sensorimotor integration in move-ment disorders. Mov Disord 2003;18:231–240.

3. Filippi GM, Errico P, Santarelli R, Bagolini B, Manni E. Botuli-num A toxin effects on rat jaw muscle spindles. Acta Otolaryngol1993;113:400–404.

4. Rosales RL, Arimura K, Takenaga S, Osame M. Extrafusal andintrafusal muscle effects in experimental botulinum toxin-A injec-tion. Muscle Nerve 1996;19:488–496.

5. Wiegand H, Erdmann G, Wellhoner HH. 125I-labelled botulinumA neurotoxin: pharmacokinetics in cats after intramuscular injec-tion. Naunyn Schmiedebergs Arch Pharmacol 1976;292:161–165.

6. Wiegand H, Welhoner HH. The action of botulinum A neurotoxinon the inhibition by antidromic stimulation of the lumbar mono-synaptic reflex. Naunyn Schmiedebergs Arch Pharmacol. 1977;298:235–238.

7. Hagenah R, Benecke R, Wiegand H. Effects of type A botulinumtoxin on the cholinergic transmission at spinal Renshaw cells andon the inhibitory action at Ia inhibitory interneurones. NaunynSchmiedebergs Arch Pharmacol 1977;299:267–272.

8. Janicki PK, Habermann E. Tetanus and botulinum toxins inhibit,and black widow spider venom stimulates the release of methio-nine-enkephalin-like material in vitro. J Neurochem 1983;41:395–402.

9. Bigalke H, Heller I, Bizzini B, Habermann E. Tetanus toxin andbotulinum A toxin inhibit release and uptake of various transmit-ters, as studied with particulate preparations from rat brain andspinal cord. Naunyn Schmiedebergs Arch Pharmacol 1981;316:244–251.

10. Habermann E, Muller H, Hidel M. Tetanus toxin and botulinum Aand C neurotoxins inhibit noradrenaline release from culturedmouse brain. J Neurochem 1988;51:522–527.

11. Kitamura M. Binding of botulinum neurotoxin to the synaptosomefraction of rat brain. Naunyn Schmiedebergs Arch Pharmacol1976;295:171–175.

12. Gundersen CB, Howard BD. The effects of botulinum toxin onacetylcholine metabolism in mouse brain slices and synaptosomes.J Neurochem 1978;31:1005–1013.

13. Black JD, Dolly JO. Selective location of acceptors for botulinumneurotoxin A in the central and peripheral nervous systems. Neu-roscience 1987;23:767–779.

14. Boroff DA, Chen GS. On the question of permeability of theblood-brain barrier to BoNT. Int Arch Allergy Appl Immunol1975;48:495–504.

EFFECTS OF BOTULINUM TOXIN TYPE A S63

Movement Disorders, Vol. 19, Suppl. 8, 2004

15. Priori A, Berardelli A, Mercuri B, Manfredi M. Physiologicaleffects produced by BoNT treatment of upper limb dystonia.Changes in reciprocal inhibition between forearm muscles. Brain1995;118:801–807.

16. Modugno N, Priori A, Berardelli A, Vacca L, Mercuri B, ManfrediM. BoNT restores presynaptic inhibition of group Ia afferents inpatients with essential tremor. Muscle Nerve 1998;21:1701–1705.

17. Girlanda P, Quartarone A, Sinicropi S, Nicolosi C, Roberto ML,Picciolo G, Macaione V, Battaglia F, Ruggeri M, Messina C.Botulinum toxin in upper limb spasticity: study of reciprocalinhibition between forearm muscles. Neuroreport 1997;8:3039–3044.

18. Panizza M, Castagna M, Di Summa A, Saibene L, Grioni G,Nilsson J. Functional and clinical changes in upper limb spasticpatients treated with botulinum toxin (BTX). Funct Neurol 2000;15:147–155.

19. Pauri F, Boffa L, Cassetta E, Pasqualetti P, Rossini PM. Botulinumtoxin type-A treatment in spastic paraparesis: a neurophysiologicalstudy. J Neurol Sci 2000;181:89–97.

20. Wohlfarth K, Schubert M, Rothe B, Elek J, Dengler R. RemoteF-wave changes after local botulinum toxin application. Clin Neu-rophysiol 2001;112:636–640.

21. Berardelli A, Rothwell JC, Day BL, Marsden CD. Pathophysiol-ogy of blepharospasm and oromandibular dystonia. Brain 1985;108:593–608.

22. Valls-Sole J, Tolosa ES, Ribera G. Neurophysiological observa-tions on the effects of botulinum toxin treatment in patients withdystonic blepharospasm. J Neurol Neurosurg Psychiatry 1991;54:310–313.

23. Girlanda P, Quartarone A, Sinicropi S, Nicolosi C, Messina C.Unilateral injection of botulinum toxin in blepharospasm: singlefiber electromyography and blink reflex study. Mov Disord 1996;11:27–31.

24. Grandas F, Traba A, Alonso F, Esteban A. Blink reflex recoverycycle in patients with blepharospasm unilaterally treated withbotulinum toxin. Clin Neuropharmacol 1998;21:307–311.

25. Behar M, Raju GB. Electrophysiological studies in patients withblepharospasm before and after botulinum toxin A therapy. J Neu-rol Sci 1996;135:74–77.

26. Ce P. Central effects of botulinum toxin: study of brainstemauditory evoked potentials. Eur J Neurol 2000;7:747.

27. Bielamowicz S, Ludlow CL. Effects of botulinum toxin on patho-physiology in spasmodic dysphonia. Ann Otol Rhinol Laryngol2000;109:194–203.

28. Naumann M, Reiners K. Long-latency reflexes of hand muscles inidiopathic focal dystonia and their modification by botulinumtoxin. Brain 1997;120:409–416.

29. Kanovsky P, Streitova H, Dufek J, Znojil V, Daniel P, Rektor I.Change in lateralization of the P22/N30 cortical component ofmedian nerve somatosensory evoked potentials in patients withcervical dystonia after successful treatment with botulinum toxinA. Mov Disord 1998;13:108–117.

30. Byrnes ML, Thickbroom GW, Wilson SA, Sacco P, Shipman JM,Stell R, Mastaglia FL. The corticomotor representation of upperlimb muscles in writer’s cramp and changes following botulinumtoxin injection. Brain 1998;121:977–988.

31. Kujirai T, Caramia MD, Rothwell JC, et al. Corticocortical inhi-bition in human motor cortex. J Physiol 1993;471:501–519.

32. Ridding MC, Sheean G, Rothwell JC, Inzelberg R, Kujirai T.Changes in the balance between motor cortical excitation andinhibition in focal, task specific dystonia. J Neurol NeurosurgPsychiatry 1995;59:493–498.

33. Berardelli A, Rothwell JC, Hallett M, Thompson PD, Manfredi M,Marsden CD. The pathophysiology of primary dystonia [review].Brain 1998;121:1195–1212.

34. Gilio F, Curra A, Lorenzano C, Modugno N, Manfredi M, Be-rardelli A. Effects of botulinum toxin type A on intracorticalinhibition in patients with dystonia. Ann Neurol 2000;48:20–26.

35. Ceballos-Baumann AO, Sheean G, Passingham RE, Marsden CD,Brooks DJ. Botulinum toxin does not reverse the cortical dysfunc-tion associated with writer’s cramp. A PET study. Brain 1997;120:571–582.

36. Pascual-Leone A, Grafman J, Hallett M. Modulation of corticalmotor output maps during development of implicit and explicitknowledge. Science 1994;263:1287–1289.

37. Traversa R, Cicinelli P, Bassi A, et al. Mapping of motor corticalreorganization after stroke. A brain stimulation study with focalmagnetic pulses. Stroke 1997;28:110–117.

38. Cohen LG, Bandinelli S, Topka HR, et al. Topographic maps ofhuman motor cortex in normal and pathological conditions: mirrormovements, amputations and spinal cord injuries. Electroencepha-logr Clin Neurophysiol Suppl 1991;43:36–50.

39. Cohen L, Ziemann U, Chen R, et al. Studies of neuroplasticity withtranscranial magnetic stimulation. J Clin Neurophysiol 1998;15:305–324.

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