Used as a pharmaceutical-grade purified low dose preparation, botulinum neurotoxin serotype A (BoNT-A) is employed in various disorders involving motor, autonomic and sensory nerve hyperactivity (Rossetto and Montecucco, 2019; Jankovic, 2017; Anandan and Jankovic, 2021). It poisons the presynaptic nerve terminals for several months firstly by binding to polysialoganglioside and synaptic vesicle 2 (SV2) protein, followed by endocytic entry into recycled synaptic vesicles. Upon vesicle acidification, the toxin translocates its 50 kDa neurotoxic light chain into the presynaptic cytosol, and cleaves synaptosomal-associated protein of 25 kDa (SNAP-25) (Schiavo et al., 1993; Rossetto et al., 2014) with consequent long-term blockage of the Ca2+-triggered release of ACh and other neurotransmitters Gundersen (1980); Pirazzini et al. (2017). In focal dystonias and spasticity, its therapeutic benefits have been commonly attributed to local neuromuscular paralysis of injected muscles, in turn, leading to indirect central plastic changes (Rosales and Dressler, 2010). However, mounting data point to a direct central interaction with sensory and motor systems (Matak and Lacković, 2014; Ramachandran and Yaksh, 2014; Ramirez-Castaneda et al., 2013; Mazzocchio and Caleo, 2015). The BoNT-A may normalise the spastic co-contraction of agonists and antagonists and recover the reciprocal inhibition in uninjected muscles, possibly by influencing the recurrent inhibition at a synapse between motoneuronal collaterals and Renshaw interneurons (Hallett, 2018; Gracies, 2004; Matak et al., 2016; Vinti et al., 2012; Marchand-Pauvert et al., 2013; Aymard et al., 2013; Caleo and Mazzocchio, 2018; Weise et al., 2019). Peripherally injected BoNT-A is axonally transported to spinal cord and brainstem nuclei (Antonucci et al., 2008; Matak et al., 2012; Restani et al., 2012; Koizumi et al., 2014; Caleo et al., 2018). Furthermore, its central antispastic effect has been reported in rat focal muscle hypertonia induced by tetanus toxin (TeNT) (Matak, 2020; Šoštarić et al., 2022), a neurotoxin that blocks the inhibitory transmission (Brooks et al., 1957; Megighian et al., 2021). The BoNT-A central trans-synaptic traffic was found to be necessary for its antispastic effect when local muscular effects of BoNT-A are minimised or start to recover (Matak, 2020; Šoštarić et al., 2022). Other motor consequences of BoNT-A trans-synaptic transport in ventral horn are, up to now, unknown. Spinal premotor inputs regulate the precise activation of different muscle groups during normal locomotion (Laliberte et al., 2019). In dystonia and spasticity, spinal locomotor circuits produce altered patterns of motor pool activation leading to sustained or intermittent muscular hyperactivity (Liu et al., 2015; Bellardita et al., 2017; Pocratsky et al., 2023). Building on our previous research that demonstrated the BoNT-A axonal transport and transcytosis (Caleo et al., 2018; Matak, 2020; Šoštarić et al., 2022), we hypothesised that spinal synapses sensitive to BoNT-A may belong to premotor inputs regulating the precise activation of different muscle groups during locomotion.

To examine this hypothesis, we studied the effect of BoNT-A in the motor system by targeting the bilateral lower leg and hind-paw motor pools in adult rats. We employed direct toxin injections into the sciatic nerves (i.n.) rather than into multiple hindlimb muscles. This enabled a slower-onset toxin effect at the NMJ due to anterograde transport resulting in milder weakness, however with ongoing muscle atrophy, and a central enzymatic action similar to the one seen after i.m. BoNT-A in previous studies (Matak, 2020; Šoštarić et al., 2022). We further characterised the effect of unopposed vs opposed BoNT-A spinal trans-synaptic action on the motor performance by blocking its trans-synaptic transport with intrathecal BoNT-A–neutralising antitoxin. Then, its motor effects were regularly assessed by different behavioural tests up to 56 days after BoNT–A injection. Later, in the same animals we examined BoNT-A central action on TeNT-evoked spasm and exaggerated monosynaptic H reflex and, at the end of experiment, analysed the localization of the toxin enzymatic activity in relation to synaptic markers and known toxin acceptors.