{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T15:44:57Z","timestamp":1790178297612,"version":"4.1.0"},"reference-count":62,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2018,4,25]],"date-time":"2018-04-25T00:00:00Z","timestamp":1524614400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"AIRC (Italian Association for Cancer Research)","award":["#IG18925"],"award-info":[{"award-number":["#IG18925"]}]},{"name":"Regione Toscana (\u201cProgramma Attuativo Regionale\u201d financed by FAS - now FSC)","award":["RONDA"],"award-info":[{"award-number":["RONDA"]}]},{"name":"CNR  project","award":["InterOmics"],"award-info":[{"award-number":["InterOmics"]}]},{"name":"CNR  project","award":["NanoMax"],"award-info":[{"award-number":["NanoMax"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Botulinum neurotoxins are metalloproteases that specifically cleave N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in synaptic terminals, resulting in a potent inhibition of vesicle fusion and transmitter release. The family comprises different serotypes (BoNT\/A to BoNT\/G). The natural target of these toxins is represented by the neuromuscular junction, where BoNTs block acetylcholine release. In this review, we describe the actions of botulinum toxins after direct delivery to the central nervous system (CNS), where BoNTs block exocytosis of several transmitters, with near-complete silencing of neural networks. The use of clostridial neurotoxins in the CNS has allowed us to investigate specifically the role of synaptic activity in different physiological and pathological processes. The silencing properties of BoNTs can be exploited for therapeutic purposes, for example to counteract pathological hyperactivity and seizures in epileptogenic brain foci, or to investigate the role of activity in degenerative diseases like prion disease. Altogether, clostridial neurotoxins and their derivatives hold promise as powerful tools for both the basic understanding of brain function and the dissection and treatment of activity-dependent pathogenic pathways.<\/jats:p>","DOI":"10.3390\/toxins10050175","type":"journal-article","created":{"date-parts":[[2018,4,25]],"date-time":"2018-04-25T11:15:39Z","timestamp":1524654939000},"page":"175","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":20,"title":["Exploiting Botulinum Neurotoxins for the Study of Brain Physiology and Pathology"],"prefix":"10.3390","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4333-6378","authenticated-orcid":false,"given":"Matteo","family":"Caleo","sequence":"first","affiliation":[{"name":"CNR Neuroscience Institute, via G. Moruzzi 1, 56124 Pisa, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura","family":"Restani","sequence":"additional","affiliation":[{"name":"CNR Neuroscience Institute, via G. Moruzzi 1, 56124 Pisa, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2018,4,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"899","DOI":"10.1152\/physrev.1994.74.4.899","article-title":"Quantal acetylcholine release at the vertebrate neuromuscular junction","volume":"74","year":"1994","journal-title":"Physiol. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"535","DOI":"10.1038\/nrmicro3295","article-title":"Botulinum neurotoxins: Genetic, structural and mechanistic insights","volume":"12","author":"Rossetto","year":"2014","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"200","DOI":"10.1124\/pr.116.012658","article-title":"Botulinum Neurotoxins: Biology, Pharmacology, and Toxicology","volume":"69","author":"Pirazzini","year":"2017","journal-title":"Pharmacol. Rev."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1031","DOI":"10.1113\/jphysiol.2012.242131","article-title":"Transsynaptic inhibition of spinal transmission by A2 botulinum toxin","volume":"591","author":"Akaike","year":"2013","journal-title":"J. Physiol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3894","DOI":"10.1128\/IAI.00536-13","article-title":"Characterization of botulinum neurotoxin A subtypes 1 through 5 by investigation of activities in mice, in neuronal cell cultures, and in vitro","volume":"81","author":"Whitemarsh","year":"2013","journal-title":"Infect. Immun."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Peck, M.W., Smith, T.J., Anniballi, F., Austin, J.W., Bano, L., Bradshaw, M., Cuervo, P., Cheng, L.W., Derman, Y., and Dorner, B.G. (2017). Historical Perspectives and Guidelines for Botulinum Neurotoxin Subtype Nomenclature. Toxins, 9.","DOI":"10.3390\/toxins9010038"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"717","DOI":"10.1152\/physrev.2000.80.2.717","article-title":"Neurotoxins affecting neuroexocytosis","volume":"80","author":"Schiavo","year":"2000","journal-title":"Physiol. Rev."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1146\/annurev.biochem.051908.125345","article-title":"Botulinum neurotoxin: A marvel of protein design","volume":"79","author":"Montal","year":"2010","journal-title":"Annu. Rev. Biochem."},{"key":"ref_9","first-page":"924","article-title":"Pharmacologic weakening of extraocular muscles","volume":"12","author":"Scott","year":"1973","journal-title":"Investig. Ophthalmol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1007\/BF03033939","article-title":"Action of botulinum neurotoxins in the central nervous system: Antiepileptic effects","volume":"9","author":"Bozzi","year":"2006","journal-title":"Neurotox. Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"592","DOI":"10.1126\/science.1123654","article-title":"SV2 is the protein receptor for botulinum neurotoxin A","volume":"312","author":"Dong","year":"2006","journal-title":"Science"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"995","DOI":"10.1038\/sj.embor.7400796","article-title":"Entering neurons: Botulinum toxins and synaptic vesicle recycling","volume":"7","author":"Verderio","year":"2006","journal-title":"EMBO Rep."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"19654","DOI":"10.1038\/srep19654","article-title":"Botulinum neurotoxin type-A enters a non-recycling pool of synaptic vesicles","volume":"6","author":"Harper","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Kroken, A.R., Blum, F.C., Zuverink, M., and Barbieri, J.T. (2017). Entry of Botulinum Neurotoxin Subtypes A1 and A2 into Neurons. Infect. Immun., 85.","DOI":"10.1128\/IAI.00795-16"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Restani, L., Giribaldi, F., Manich, M., Bercsenyi, K., Menendez, G., Rossetto, O., Caleo, M., and Schiavo, G. (2012). Botulinum Neurotoxins A and E Undergo Retrograde Axonal Transport in Primary Motor Neurons. PLoS Pathog., 12.","DOI":"10.1371\/journal.ppat.1003087"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1974","DOI":"10.1016\/j.celrep.2016.06.104","article-title":"Interneuronal Transfer and Distal Action of Tetanus Toxin and Botulinum Neurotoxins A and D in Central Neurons","volume":"16","author":"Vevea","year":"2016","journal-title":"Cell Rep."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"3689","DOI":"10.1523\/JNEUROSCI.0375-08.2008","article-title":"Long-distance retrograde effects of botulinum neurotoxin A","volume":"28","author":"Antonucci","year":"2008","journal-title":"J. Neurosci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1083","DOI":"10.1111\/j.1600-0854.2012.01369.x","article-title":"Botulinum neurotoxin A impairs neurotransmission following retrograde transynaptic transport","volume":"13","author":"Restani","year":"2012","journal-title":"Traffic"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1808","DOI":"10.1111\/j.1471-4159.1988.tb02482.x","article-title":"Characterization of the inhibitory action of botulinum neurotoxin type A on the release of several transmitters from rat cerebrocortical synaptosomes","volume":"50","author":"Ashton","year":"1988","journal-title":"J. Neurochem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"142","DOI":"10.1111\/j.1600-0854.2006.00520.x","article-title":"Traffic of botulinum toxins A and E in excitatory and inhibitory neurons","volume":"8","author":"Verderio","year":"2007","journal-title":"Traffic"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1002\/cne.21505","article-title":"Heterogeneous expression of SNAP-25 in rat and human brain","volume":"506","author":"Garbelli","year":"2008","journal-title":"J. Comp. Neurol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"599","DOI":"10.1016\/S0896-6273(04)00077-7","article-title":"SNAP-25 Modulation of Calcium Dynamics Underlies Differences in GABAergic and Glutamatergic Responsiveness to Depolarization","volume":"41","author":"Verderio","year":"2004","journal-title":"Neuron"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"159","DOI":"10.3389\/fncel.2015.00159","article-title":"Accelerated intoxication of GABAergic synapses by botulinum neurotoxin A disinhibits stem cell-derived neuron networks prior to network silencing","volume":"9","author":"Beske","year":"2015","journal-title":"Front. Cell. Neurosci."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"7190","DOI":"10.1523\/JNEUROSCI.17-19-07190.1997","article-title":"Ca2+ or Sr2+ partially rescues synaptic transmission in hippocampal cultures treated with botulinum toxin A and C, but not tetanus toxin","volume":"17","author":"Capogna","year":"1997","journal-title":"J. Neurosci."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1979","DOI":"10.1126\/science.1096202","article-title":"Regulation of dendritic protein synthesis by miniature synaptic events","volume":"304","author":"Sutton","year":"2004","journal-title":"Science"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1943","DOI":"10.1523\/JNEUROSCI.4402-04.2005","article-title":"Antiepileptic effects of botulinum neurotoxin E","volume":"25","author":"Costantin","year":"2005","journal-title":"J. Neurosci."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"388","DOI":"10.1016\/j.expneurol.2007.11.012","article-title":"Botulinum neurotoxin E (BoNT\/E) reduces CA1 neuron loss and granule cell dispersion, with no effects on chronic seizures, in a mouse model of temporal lobe epilepsy","volume":"210","author":"Antonucci","year":"2008","journal-title":"Exp. Neurol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1016\/j.tibs.2005.05.002","article-title":"SNARE complexes and neuroexocytosis: How many, how close?","volume":"30","author":"Montecucco","year":"2005","journal-title":"Trends Biochem. Sci."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"13476","DOI":"10.1074\/jbc.M010992200","article-title":"The role of the synaptic protein snap-25 in the potency of botulinum neurotoxin type A","volume":"276","author":"Keller","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Caleo, M., Restani, L., Vannini, E., Siskova, Z., Al-Malki, H., Morgan, R., O\u2019Connor, V., and Perry, V.H. (2012). The role of activity in Synaptic degeneration in a protein misfolding disease, prion disease. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0041182"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"147","DOI":"10.4161\/pri.23327","article-title":"Silencing synapses: A route to understanding synapse degeneration in chronic neurodegenerative disease","volume":"7","author":"Caleo","year":"2013","journal-title":"Prion"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1016\/j.nbd.2010.09.017","article-title":"Intrastriatal botulinum toxin abolishes pathologic rotational behaviour and induces axonal varicosities in the 6-OHDA rat model of Parkinson\u2019s disease","volume":"41","author":"Wree","year":"2011","journal-title":"Neurobiol. Dis."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1016\/j.brainres.2017.09.016","article-title":"Intrastriatally injected botulinum neurotoxin-A differently effects cholinergic and dopaminergic fibers in C57BL\/6 mice","volume":"1676","author":"Hawlitschka","year":"2017","journal-title":"Brain Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"446","DOI":"10.1016\/j.tins.2005.06.001","article-title":"Beyond BOTOX: Advantages and limitations of individual botulinum neurotoxins","volume":"28","author":"Davletov","year":"2005","journal-title":"Trends Neurosci."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1097\/01.fbp.0000129746.14753.1e","article-title":"Central injection of botulinum neurotoxins: Behavioural effects in mice","volume":"15","author":"Luvisetto","year":"2004","journal-title":"Behav. Pharmacol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1016\/j.brainres.2006.01.117","article-title":"Botulinum neurotoxins and formalin-induced pain: Central vs. peripheral effects in mice","volume":"1082","author":"Luvisetto","year":"2006","journal-title":"Brain Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1016\/j.pain.2003.10.008","article-title":"Subcutaneous administration of botulinum toxin A reduces formalin-induced pain","volume":"107","author":"Cui","year":"2004","journal-title":"Pain"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"234","DOI":"10.1016\/j.pbb.2009.08.012","article-title":"Central origin of the antinociceptive action of botulinum toxin type A","volume":"94","year":"2009","journal-title":"Pharmacol. Biochem. Behav."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1016\/j.pneurobio.2014.06.001","article-title":"Botulinum toxin A, brain and pain","volume":"119\u2013120","author":"Matak","year":"2014","journal-title":"Prog. Neurobiol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"4530","DOI":"10.1523\/JNEUROSCI.0772-07.2007","article-title":"Transient synaptic silencing of developing striate cortex has persistent effects on visual function and plasticity","volume":"27","author":"Caleo","year":"2007","journal-title":"J. Neurosci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1016\/B978-0-444-63327-9.00001-1","article-title":"Balancing plasticity\/stability across brain development","volume":"207","author":"Takesian","year":"2013","journal-title":"Prog. Brain Res."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"519","DOI":"10.1002\/jnr.10443","article-title":"Animal model of dementia induced by entorhinal synaptic damage and partial restoration of cognitive deficits by BDNF and carnitine","volume":"70","author":"Ando","year":"2002","journal-title":"J. Neurosci. Res."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Spalletti, C., Alia, C., Lai, S., Panarese, A., Conti, S., Micera, S., and Caleo, M. (2017). Combining robotic training and inactivation of the healthy hemisphere restores pre-stroke motor patterns in mice. Elife, 6.","DOI":"10.7554\/eLife.28662"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"344","DOI":"10.1016\/j.nurt.2009.01.017","article-title":"Convection-enhanced delivery in the treatment of epilepsy","volume":"6","author":"Rogawski","year":"2009","journal-title":"Neurotherapeutics"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"528","DOI":"10.1124\/jpet.113.205070","article-title":"Long-lasting attenuation of amygdala-kindled seizures after convection-enhanced delivery of botulinum neurotoxins A and B into the amygdala in rats","volume":"346","author":"Gasior","year":"2013","journal-title":"J. Pharmacol. Exp. Ther."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"232","DOI":"10.1016\/j.jconrel.2017.07.028","article-title":"Strategies to enhance the distribution of nanotherapeutics in the brain","volume":"267","author":"Zhang","year":"2017","journal-title":"J. Control. Release"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1111\/bpa.12082","article-title":"Convection-enhanced drug delivery to the brain: Therapeutic potential and neuropathological considerations","volume":"24","author":"Barua","year":"2014","journal-title":"Brain Pathol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"2147","DOI":"10.1046\/j.1460-9568.2003.02662.x","article-title":"Synaptic changes characterize early behavioural signs in the ME7 model of murine prion disease","volume":"17","author":"Cunningham","year":"2003","journal-title":"Eur. J. Neurosci."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1016\/j.neuroscience.2014.07.030","article-title":"Role of blood-brain barrier in temporal lobe epilepsy and pharmacoresistance","volume":"277","author":"Aronica","year":"2014","journal-title":"Neuroscience"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"963","DOI":"10.1111\/j.1528-1167.2008.01983.x","article-title":"Intrahippocampal infusion of botulinum neurotoxin E (BoNT\/E) reduces spontaneous recurrent seizures in a mouse model of mesial temporal lobe epilepsy","volume":"50","author":"Antonucci","year":"2009","journal-title":"Epilepsia"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1016\/j.neuroscience.2010.04.059","article-title":"Acute neuroprotection by the synaptic blocker botulinum neurotoxin E in a rat model of focal cerebral ischaemia","volume":"169","author":"Antonucci","year":"2010","journal-title":"Neuroscience"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1097\/WNR.0b013e32801b3cbb","article-title":"BoNT\/E prevents seizure-induced activation of caspase 3 in the rat hippocampus","volume":"18","author":"Manno","year":"2007","journal-title":"Neuroreport"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"109","DOI":"10.1016\/j.toxicon.2013.07.027","article-title":"Botulinum neurotoxin A2 reduces incidence of seizures in mouse models of temporal lobe epilepsy","volume":"74","author":"Kato","year":"2013","journal-title":"Toxicon"},{"key":"ref_54","first-page":"141","article-title":"Epilepsy surgery: Recent advances in brain mapping, neuroimaging and surgical procedures","volume":"59","author":"Dorfer","year":"2015","journal-title":"J. Neurosurg. Sci."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1016\/j.brainres.2015.10.056","article-title":"Intrastriatal injection of botulinum neurotoxin-A is not cytotoxic in rat brain\u2014A histological and stereological analysis","volume":"1630","author":"Mehlan","year":"2016","journal-title":"Brain Res."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1002\/jnr.24110","article-title":"Intrastriatal administration of botulinum neurotoxin A normalizes striatal D2R binding and reduces striatal D1R binding in male hemiparkinsonian rats","volume":"96","author":"Wedekind","year":"2018","journal-title":"J. Neurosci. Res."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1016\/j.neuroscience.2018.01.053","article-title":"Dopamine, Noradrenaline and Serotonin Receptor Densities in the Striatum of Hemiparkinsonian Rats following Botulinum Neurotoxin-A Injection","volume":"374","author":"Mann","year":"2018","journal-title":"Neuroscience"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"461","DOI":"10.1016\/0006-8993(69)90122-X","article-title":"Direct chemical stimulation of dopaminergic mechanisms in the neostriatum of the rat","volume":"14","author":"Ungerstedt","year":"1969","journal-title":"Brain Res."},{"key":"ref_59","doi-asserted-by":"crossref","unstructured":"Pellett, S., Bradshaw, M., Tepp, W.H., Pier, C.L., Whitemarsh, R.C.M., Chen, C., Barbieri, J.T., and Johnson, E.A. (2018). The Light Chain Defines the Duration of Action of Botulinum Toxin Serotype A Subtypes. MBio, 9.","DOI":"10.1128\/mBio.00089-18"},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1016\/j.toxicon.2017.09.006","article-title":"Design of modified botulinum neurotoxin A1 variants with a shorter persistence of paralysis and duration of action","volume":"139","author":"Scheps","year":"2017","journal-title":"Toxicon"},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"345","DOI":"10.3390\/toxins3040345","article-title":"Re-assembled botulinum neurotoxin inhibits CNS functions without systemic toxicity","volume":"3","author":"Ferrari","year":"2011","journal-title":"Toxins"},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"1750","DOI":"10.1021\/bc4003103","article-title":"Synthetic self-assembling clostridial chimera for modulation of sensory functions","volume":"24","author":"Ferrari","year":"2013","journal-title":"Bioconjug. Chem."}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/10\/5\/175\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T15:02:06Z","timestamp":1760194926000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/10\/5\/175"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2018,4,25]]},"references-count":62,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2018,5]]}},"alternative-id":["toxins10050175"],"URL":"https:\/\/doi.org\/10.3390\/toxins10050175","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2018,4,25]]}}}