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124 Current Pharmaceutical Biotechnology, 2013, 14, 124-130
Intracerebrally Applied Botulinum Neurotoxin in Experimental Neurosci-
ence
Alexander Hawlitschka1,*, Veronica Antipova1, Oliver Schmitt1, Martin Witt1, Reiner Benecke2,
Eilhard Mix2 and Andreas Wree1
1Department of Anatomy, University of Rostock, Germany; Department of Neurology, University of Rostock, Germany
Abstract The use of botulinum neurotoxins (BoNTs) for therapeutic purposes in neuromuscular disorders and peripheral
hypercholinergic conditions as well as in aesthetic medicine is widespread and common. BoNTs are also able to block the
release of a wide range of transmitters from presynaptic boutons. Therefore, application of BoNTs directly in the central
nervous system (CNS) is currently under study with respect to basic research and potentially as a new therapeutic strategy
of neurological diseases. Investigations concentrate on effects of intracerebral and intraspinal application of BoNTs in ro-
dents on the impact on spinal, nuclear, limbic and cortical neuronal circuits. In animal model first promising BoNT-
induced therapeutical benefit has been shown in the treatment of pain, epilepsy, stroke and Parkinson’s disease.
Keywords: Botulinum neurotoxin, dementia, epilepsy, hippocampus, ischemia, Parkinson’s disease, spinal cord, striatum.
INTRODUCTION
Botulinum neurotoxins (BoNTs) are synthesized by dif-
ferent strains of Clostridium botulinum, a species of gram-
positive, rod shaped, anaerobe bacteria [1, 2]. BoNTs cause
intense flaccid paralyse and dysfunction of the voluntary and
vegetative nervous system by blocking the release of acetyl-
choline and to a lesser degree of other neurotransmitters
from nerve terminals. Clinically, it leads to so called botu-
lism affecting humans as well as wild and domestic animals.
Men mostly acquire botulism from inadequately stored meat
products and preserved raw fish or home-prepared condi-
ments, vegetables and non-acid fruits. Cattle sicken often
after eating inadequately fermented silage. Death is primarily
caused by respiratory and/ or cardiac failure [3].
Mechanism of BoNT Action
Eight different subtypes of BoNTs (BoNT A, B, C1, C2,
D, E, F, G) are known, all of them are zinc-dependent metal-
loendopeptidases with partly different target substrates. All
of these substrates are components of the SNARE (soluble
N-ethylmaleimide-sensitive factor attachment protein recep-
tor) complex. The SNARE complex is a crucial component
of the vesicle membrane fusion apparatus in synapses that
consists of the following three proteins: synaptosomal-
associated protein-25 (SNAP-25), syntaxin and synaptobre-
vin (= vesicle-associated membrane protein-2, VAMP-2).
BoNT-A and -E cleave SNAP-25, BoNT-B, -D, -G and -F
cleave synaptobrevin, and BoNT-C cleaves SNAP-25 and
syntaxin [4, 5]. After synthesis of the primordial single-chain
form of BoNTs by Clostridium botulinum a post-
translational proteolytical cleavage in a light chain (~50
*Address correspondence to this author at the Department of Anatomy,
University of Rostock, Gertrudenstraße 9, D-18055 Rostock, Germany; Tel:
49-381-494-8400; Fax: 49-381-494-8402;
E-mail: alexander.hawlitschka@uni-rostock.de
kDa) and a heavy chain (~100 kDa) occurs and BoNTs be-
come active, whereby both chains remain associated by a
disulfide bridge. BoNTs possess binding domains in the C-
terminal part of their heavy chain, which binds specifically
to gangliosides and protein receptors of presynaptic mem-
branes [6]. These protein receptors are the synaptic vesicle
proteins SV2 for BoNT-A and synaptotagmin I and II for
BoNT-B and -G, respectively [7 -15].
The N-terminal part of the heavy chain permits the trans-
location of the whole BoNT-molecule through the presynap-
tic membrane by vesicle endocytosis. After intracellular
acidification of the vesicle, the ligh t chains split off and
reach the cytoplasm through a pore in the vesicle membrane
formed by the heavy chain based on pH-dependent confor-
mational changes. The light chain contains a zinc-dependent
metalloendopeptidase active region, which is responsible for
the cleavage of the specific SNARE components [16 -19].
Experimentally, the cleavage of the SNARE components by
BoNTs does not only result in an inhibition of the release of
acetylcholine. Additionally, the release of a series of other
neurotransmitters such as glutamate, noradrenaline, glycine,
serotonin and dopamine from synaptosomes is affected [14,
20]. Obviously the extent of the effect of BoNT-A on non-
cholinergic synaptic transmission depends on the BoNT con-
centration [21] and most probably also on the expression
pattern of the specific BoNT-A receptor SV2 [22].
Clinical Applications
Up to now, BoNT has been mostly applied in the periph-
ery and only a few effects were described in the CNS, e.g.,
alterations in H reflex [23], changes in cortical activity [24]
and appearance of pyramidal signs [25]. In the spinal cord,
early experiments of Benecke et al. [26] in cats showed that
only direct intraspinal application of BoNT-A reduced the
Renshaw cell response, whereas BoNT-A injection into the
1873-4316/13 $58.00+.00 © 2013 Bentham Science Publishers
Intracerebral Botulinum Neurotoxin Current P harmaceutical Biote chnology, 2013, Vol. 14, No. 1 125
ventral root did not affect cholinergic transmission on these
cells. However, retrograde transport of BoNT-A from the
periphery into the ventral root and the spinal cord [27, 28] as
well as passage of the blood brain barrier [29] were ob-
served. More recently, subsequent to intraspinal BoNT ap-
plication neuronal circuits were affected [30] and neuro-
physiological conspicuities have been reported [31-33]. An
affection of the brainstem circuitry in cats was shown after
injection of BoNT-A into the orbital lateral rectus muscle,
where the central changes of the discharge patterns of the
abducens motor neurons were thought to be due to a retro-
grade axonal transport of BoNT-A [34, 35].
Additionally, in vitro studies have shown that BoNTs
seems to have a much more profound effect on central neu-
rons than on peripheral ones, as revealed by a prolonged
proteolytic cleavage of SNAP-25 and inhibition of transmit-
ter release in central neurons [36, 37].
In summary, although BoNT injections into peripheral
structures are routinely performed since several decades,
only a few experimental BoNT applications were done di-
rectly into parts of the CNS. Various groups described the
application of BoNTs into the CNS as an appropriate method
to investigate basic neuronal mechanisms in the CNS such as
single neuron behaviour, neuronal circuitry, plasticity of the
developing and the adult brain, and the action of BoNTs it-
self [38-40].
BASIC RESEARCH WITH APPLICATION OF BONT
IN THE CNS
Spinal reflex arcs of cats, especially the role of the cho-
linergic transmission of Renshaw cells and that of Ia inhibi-
tory interneurons, were investigated by [38] by BoNT-A-
injection into the spinal cord and the ventral root. The inves-
tigators recorded extracellular field potentials of these cells
and observed a decrease in response of Renshaw cells after
BoNT application into the spinal cord, but not when BoNT
was injected peripherally (triceps surae muscle). Renshaw
cells themselves were still able to inhibit Ia inhibitory in-
terneurons after BoNT-application into the muscle. There-
fore, the authors draw the conclusion that BoNT affects pre-
dominantly synaptic transmission of the motoneurons.
The group of Pavone [41] investigated the effect of
stereotactic injection of BoNT-A and BoNT-B in different
concentrations into one lateral cerebral ventricle of CD1
male mice. A dose response curve for lethality was gener-
ated. The median lethal dose (LD50) for BoNT-A and
BoNT-B was extrapolated for 0.5-1.0 x 10-6 mg/kg body
weight. Two to six hours after injection of BoNT-A as well
as BoNT-B mice looked bristled. Six to 24 h later the eyelids
became partially or fully closed, whereby the contralateral
eyelid closed first and porphyrin accumulated around the
eyes. Also exophthalmos, dehydration, weight loss and a
massive decrease of body temperature were observed when
1.9 x 10-6 mg/kg body weight BoNT-A was injected. One
day after injection of this high dose the animals lost sensori-
motor reflexes and developed deadly dyspnea and heart fail-
ure. At lower doses the same symptoms were observed in
milder forms. In those cases mice recovered after 4-5 days.
Normalization of body temperature occurred after 2-3 days
and of body weight after 5 days. A slight difference between
BoNT-A and BoNT-B concerning the time period until death
in intraventricularly injected mice was observed (75 p g
BoNT-A per mouse: most animals died within the first 48 h
post injection; 75 pg BoNT-B per mouse: most animals died
within the first 24 h post injection; 7.5 pg BoNT-A per
mouse: first death case occurred not before 36 h post injec-
tion; 7.5 pg BoNT-B per mouse: first death case occurred not
before 60 h post injection).
Antonucci et al. [40] described the ability of BoNT-A to
reach certain regions of the CNS via a retrograde axonal
pathway when injected directly into specific brain areas and
even to be transported from the periphery into the CNS when
injected into a muscle. As a proof of the intracerebral migra-
tion of BoNT-A after focal application they measured the
content of cleaved SNAP-25 in different brain region of
C57BL/6N mice and Sprague Dawley rats. After a unilateral
injection of BoNT-A into the hippocampus of mice, they
found cleaved SNAP-25 not only in the injected site, but also
in the contralateral hippocampus, reciprocally connected to
the injected hippocampus. Furthermore, an injection of
BoNT-A into the superior colliculus of Sprague Dawley rats
led to a truncation of SNAP-25 in the contralateral retina and
in the ipsilateral visual cortex. When BoNT-A was injected
into the whisker muscle, cleaved SNAP-25 was detected in
the ipsilateral facial nucleus. These results argue for a retro-
grade axonal transport of BoNT-A from the presynaptic
membrane of axonal endings to the membrane of the respec-
tive nerve cell body and/ or the membrane of the dendrites.
Moreover, the authors mentioned the retrograde transport
from the tectal injection side to the retinal ganglia cells and
the transcytosis from the retina ganglion cells to the starburst
amacrine cells. After the first appearance of truncated
SNAP-25 in the retina following intratectal BoNT-A app lica-
tion they cut off the optic nerve to circumvent further retro-
grade transport via the optic nerve. In those animals, the
amount of truncated SNAP-25 continously increased. In this
way it could be shown that the accumulation of SNAP-25 in
the retina was due to a transport of BoNT-A and to its prote-
olytic activity into the retina rather than to a transport of
SNAP-25 itself.
BONT-INDUCED ANIMAL MODEL OF DEMENTIA
The application of BoNTs into specific brain regions
constituted innovative new animal models of several neuro-
logical diseases. It is well established that in Alzh eimer’s
disease the primary neuronal loss occurs among the cho-
linergic neurons in the enthorinal cortex [42-44]. In order to
establish an animal model which simulates the cholinergic
breakdown of the entorhinal cortex, Ando et al. [45] injected
BoNT-A into the entorhinal cortex (unilaterally and
bilaterally) of rats to block the cholinergic signal transmis-
sion. Subsequently, several memory and learning tests such
as T-maze test, Hebb-Williams maze test and AKON-1 maze
test were performed. BoNT-A-injected rats actually showed
impairment of learning. Cognitive functions were more af-
fected after bilateral application of BoNT-A than in unilater-
ally injected animals. Additionally, bilateral intracerebroven-
tricular injection of BoNT-A in rats impaired the water maze
performance for up to one year [46]. Beside basic research, a
focus of intracerebral BoNT application was layed on possi-
ble therapeutic options.
126 Current Pharmaceutical Biotechnology, 2013, Vol. 14, No. 1 Hawlitschka et al.
BONT INJECTION AS THERAPEUTIC OPTION
Pain Treatment
Recently, experiments concerning pain treatment by ap-
plication of BoNTs or BoNT derivatives into the CNS were
described [47]. Chaddock et al. [48] produced a catalytically
active endopeptidase derivative of BoNT-A, in which the C-
terminal domain of its heavy chain was replaced by
Erythrina cristagalli lectin to retarget BoNT-A to nocicep-
tive neurons and block the release of substance P and gly-
cine. To this aim, a proteolytic cleavage of BoNT-A was
performed to obtain a BoNT-A fragment with a light chain
connected by a disulfide bond with the N-terminal domain of
the heavy chain and Erythrina cristagalli lectin. This active
fragment of BoNT-A was injected into the dorsal horn of rats
to inhibit signal transduction of pain. By electrophysiological
recordings of C-fibre-evoked responses and behavioural pain
model an analgesic effect was detected that lasted at least
one month. Luvisetto et al. [39] tested the possibility to treat
inflammatory pain triggered by subcutaneous formalin injec-
tion into the dorsal side of the right hindpaw after injection
of BoNT-A and B into the lateral ventricles of mice. In con-
trast to peripheral injected BoNT, they found a significant
reduction of licking response in formalin-induced pain after
injection of BoNT. Also, Bach-Rojecky et al. [49] reported
an antinociceptive effect of BoNT-A when injected into the
lumbar cerebrospinal space of rats after inducing pain by
injection of acidic saline into the hind paw pad.
Treatment of Epilepsy
Since SNAP-25 is also necessary for the release of glu-
tamate from the presynaptic membrane, BoNT-E is able to
block glutamatergic signal transduction for a longer period
of time via the cleavage of SNAP-25. Antonucci et al. [50]
and Costantin et al. [51] obtained promising experimental
results in treatment of a kainic acid induced mesial temporal
lobe epilepsy model by injection of BoNT-E into the hippo-
campus of mice and rats. BoNT-E significantly reduced the
seizure incidence as observed clinically and by electroen-
cephalography (EEG). Also the duration of seizures was
reduced. However, the beneficial effect of BoNT-E was not
permament, but vanished after 3 weeks [50]. In kainate le-
sioned rats, a reduced loss of neurons after BoNT-E applica-
tion was found [51]. 35d-old rats received a BoNT-E injec-
tion into the left hippocampus and 2 days later a lesion with
kainate was performed. Afterwards, the EEG seizures were
recorded, the behaviour was assessed and the brains were
examined. As a result, BoNT-E treated rats had less EEG
seizures and a fourfold delay of the onset time to seizures
and a fifth of time spent in seizures, the duration of ictal epi-
sodes being reduced. Also the convulsive behaviour was
dramatically reduced. A negative effect on cognitive abilities
could not be observed.
Stroke Treatment
Ischemic brain damage is partly a result of massive re-
lease of excitatory transmitters such as glutamate. To prevent
this excessive transmitter release Antonucci et al. [52] ap-
plied BoNT-E 20 min after inducing ischemia in the hippo-
campus of rats by endothelin-1 injection into the same side.
They observed an increase of cell survival in the hippocam-
pus of BoNT-E injected rats.
Treatment of Parkinson’s Disease
In Parkinson’s disease there is a reduction or an absence
of dopaminergic inhibition of tonically active cholinergic
interneurons present in the striatum, which leads to an over-
activation of GABAergic medium spiny neurons and in con-
sequence to several motor dysfunctions. Blocking choliner-
gic overactivation of GABAergic neurons by systemic ad-
ministration of anticholinergic drugs is a common therapeu-
tic option in Parkinson’s disease, but it is hampered by sev-
eral side effects [53]. One possibility to circumvent these
side effects is a direct injection of BoNT-A into the striatum,
which was performed in our group [54]. Firstly, we applied
BoNT-A in doses of 100 pg, 1 ng and 2 ng into the right
striatum of naïv e adult rats (0 d) (Fig. 1). Brains were inves-
tigated 2 weeks and 1, 3, 6 and 12 months post injection. In
another series of experiments the right substantia nigra of the
animals was lesioned by injection of 6-hydroxydopamine (6-
OHDA) into the right medial forebrain bundle in order to
create hemiparkinsonian rats (Fig. 1). 4 weeks later animals
received BoNT-A into the right striatum.
Subsequently to behavioural tests, brains were studied by
Nissl-staing, image analysis of immunohistochemical stain-
ings, cell countings and immunoelectron microscopy. In rats
that were BoNT-A-injected into the right striatum, cell
counts revealed that up to 6 months the BoNT-A application
had no effect on the number of choline acetyltransferase
(ChAT)-positive neurons in the striatum and did not lead to a
cell loss. Interestingly, we found choline acetyltransferase-
and tyrosine hydroxylase (TH)-positive axonal swellings
with a diameter of about 2-9 m in the BoNT-A treated stri-
ata, which we named BoNT-A-induced varicosities (BiVs)
(Figs. 2 and 3). These varicosities were not detectable in the
contralateral striata or in untreated rats. BiVs were reactive
either for ChAT or for TH, but never localized for both
marker enzymes, and never stained for TH in the deaffer-
ented dopaminergic striatum of 6-OHDA-lesioned animals
(Fig. 3).
There was a strong positive correlation between the den-
sity of BiVs and the concentration of BoNT-A applied to the
rats, i.e. 1 or 2 ng BoNT led to more and larger BiVs than
100 pg BoNT [54]. The success of a BoNT-A treatment of
hemiparkinsonian rats was measured by the apomorphine-
induced rotation test and the cylinder test. Application of
apomorphine in hemiparkinsonian rats caused cyclings in the
direction contralateral to the side of the lesion. Application
of BoNT-A into the dopamine-depleted striatum, i.e. ipsilat-
eral to the 6-OHDA-injection, abolished completely spinning
of the animals in the apomorphine rotation test. This effect
was significant for a BoNT-A dose of 1 ng and a time span
up to six months after the BoNT-A-injection. To investigate
the asymmetric use of the forelimbs of hemiparkinsonian rats
we additionally performed the cylinder test. Hemiparkin-
sonian rats showed a clear paw preference (right paw usage).
Subsequent BoNT-A-injection caused a slight improvement
of the asymmetric forelimb usage at the dose of 2 ng,
whereas 1 ng BoNT-A and vehicle had no significant effect.
CONCLUSIONS AND OUTLOOK
Peripheral BoNT application represents an important
therapeutic option for movement disorders such as dystonia
Intracerebral Botulinum Neurotoxin Current Pharmaceutical Biotechnology, 2013, Vol. 14, No. 1 127
Fig. 1. Injection sites of BoNT-A and 6-OHDA. The injection sites of the BoNT-A application into the CPu are symbolized by yellow ellip-
ses, whereas the injection site of 6-OHDA into the medial forebrain bundle is shown by a white ellipse. Lateral view (A), dorsal view (B) of
translucent rat brain. CPu = caudatus putamen, SNpc = substantia nigra pars compacta.
Fig. (2). Immunohistochemical stained slices depicting a unilaterally BoNT-A treated rat brain. (A-E) Immunohistochemical reaction
for ChAT; (A) shows an overview (arrowheads mark the injection channel) and (B) and (C) show sections out of the BoNT-A injected CPu at
a low (B) and a higher (C) magnification. (D) is a low and (E) a higher magnified section from the non-injected CPu. Arrows point at large
cholinergic interneurons. A number of ChAT-positive varicosities (white peaks) in the BoNT-A treated CPu is clearly visible (B and C),
whereas no such varicosities are found in the contralateral side. (F and G) show immunohistochemical reactions for TH of a consecutive
section. In the injected CPu (F) TH-positive varicosities are also detectable (white peaks), whereas in the contralateral CPu (G) such struc-
tures are absent.
128 Current Pharmaceutical Biotechnology, 2013, Vol. 14, No. 1 Hawlitschka et al.
and spasticity as well as in aesthetic medicine. Nevertheless,
the possibility of central BoNT application as a potential
instrument of basic brain research and for new therapeutic
strategies of neurological diseases has been almost neglected
so far. But the existing findings of the last 10 years reveal an
exciting potential for new methods of circumscribed specific
signal blocking in the CNS for research purposes and treat-
ment options. This will include the development of animal
models of hypocholinergic disorders such as Huntington’s
disease and progressive supranuclear palsy [55].
Fig. (3). ChAT-positive varicosities, but no TH-positive varicosities
are present in the BoNT-A in jected CPu of a rat brain in which the
ipsilateral substantia nigra was previously lesioned by 6-OHDA.
(A) A slice of a unilaterally lesioned (6-OHDA) rat brain reacted
for TH is demonstrated. The CPu of the lesioned hemisphere (*)
shows no TH-positive immunoreactivity, whereas the contralateral
CPu is TH-positive. (B) An immunofluorescence double staining
against TH (green) and ChAT (red) of a consecutive section reveals
the appearance of ChAT-positive interneurons (arrows) and ChAT-
positive varicosities (white peaks), but no TH-positive structures in
a BoNT-A injected CPu.
Ahead of potential future therapeutic approaches a pro-
found investigation of side effects resulting from diffusion
and/or active transport of BoNT molecules remote of the
target region is necessary [56]. Thus, a lack of acetylcholine
in adjacent and distant brain regions caused by diffusion or
axonal transport of BoNTs from the injection site should be
excluded to prevent the occurrence of cognitive deficits [46].
Therefore, also the full range of cognitive tests has to be per-
formed with animals which received BoNT-A into the stria-
tum. A test series with intrastriatal BoNT application in pri-
mates, naïve healthy ones as well as animals with parkin-
sonian symptoms induced by lesion of the substantia nigra
has to be performed. For a better understanding of the
mechanism of action of intrastriatal BoNT application on the
entire basal ganglia complex and the whole brain, electro-
physiological experiments in animals and on brain slices are
warranted. Furthermore, striatal BoNT application in the 6-
OHDA hemiparkinsonian model of transgenic mice will con-
tribute to the exploration of pathologic signal pathways and
neural circuits [57]. Another approach will be autoradio-
graphic neurotransmitter receptor mapping by autoradiogra-
phy [58].
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flicts of interest.
ACKNOWLEDGEMENTS
Declared none.
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Received: November 10 , 2010 Revi sed: December 03, 2010 Accepted: January 25, 2011
