Hebbian associative plasticity has been implied in the formation of the association between sensory and motor representations of actions in the Mirror Neuron System (MNS, Rizzolatti & Craighero, 2004) and, more specifically, in the action-observation network (Heyes & Catmur, 2021). Empirical support for the pivotal role of associative plasticity and sensorimotor learning for MNS development and functioning comes from behavioral, computational, electrophysiological, and non-invasive brain stimulation studies (e.g., Antunes, da Silva, & de Souza, 2018; Brunsdon, Bradford, Smith, & Ferguson, 2020; Catmur, Mars, Rushworth, & Heyes, 2011; Catmur, Walsh, & Heyes, 2007; Hanuschkin, Ganguli, & Hahnloser, 2013; Hou et al., 2017; Zazio, Guidali, Maddaluno, Miniussi, & Bolognini, 2019). Concerning non-invasive brain stimulation, novel insights on the chance of influencing the action-observation network in humans through the induction of visuo-motor associative plasticity have been provided with a cross-modal version of the paired associative stimulation (PAS) protocol (Guidali, Carneiro, & Bolognini, 2020).

The PAS is a dual-target peripheral and central magnetic stimulation protocol shown to affect synaptic plasticity in different sensory and motor systems following Hebbian learning (for reviews, see: Guidali, Roncoroni, & Bolognini, 2021a; 2021b). In the classical PAS protocol, peripheral median nerve stimulations are repeatedly paired with cortical stimuli (delivered using transcranial magnetic stimulation, TMS) over the primary motor or somatosensory cortex (M1, S1, respectively). This kind of stimulation induces neurophysiological changes functionally akin to synaptic effects of long-term potentiation (LTP) or depression (LDP), depending on the interval between the afferent and the magnetic pulse during PAS (Stefan, Kunesch, Cohen, Benecke, & Classen, 2000; Wolters et al., 2005). The recently developed ‘mirror’ version of the PAS (m-PAS) (Guidali et al., 2020) instead pairs TMS pulses over the hand cortical map in M1 with visual stimuli depicting hand actions. The m-PAS was shown to affect cortical excitability as the standard PAS does. However, it can also promote the formation of new visuo-motor associations usually not mapped onto the MNS (i.e., movement of body's parts ipsilateral to the stimulated M1), shaping human motor resonance. This effect is indexed by the emergence, at the end of the m-PAS, of atypical facilitation of cortico-spinal excitability by action observation (i.e., motor resonance, Fadiga, Fogassi, Pavesi, & Rizzolatti, 1995). Notably, the effects of the m-PAS are time-dependent since its modulatory effects rely on the inter-stimulus interval (ISI) between the viewed movement and the cortical pulse and are specific for visual stimuli depicting bodily movements. This evidence suggests that the protocol effectively induces Hebbian-like visuo-motor plasticity within the MNS, exploiting a visual pathway (Guidali et al., 2020).

Beyond the intriguing results of the studies that developed the protocol, which demonstrate the possibility of modulating, at the level of corticospinal excitability, motor resonance with the m-PAS, it is not known the extent of the cortical specificity of such a protocol, nor if this protocol can have behavioral effects attributable to a plastic rewriting of the human action-observation network. To address these issues, the present study aimed at 1) deepening the understanding of the neurophysiological effects of the m-PAS (Experiment 1) and (Experiment 2) assessing its behavioral outcomes, in particular with respect to MNS core functions as the imitation of movements (Experiment 2).

In Experiment 1, the neurophysiological effects of the protocol were deepened investigating the possible contribution of the dominant M1 (i.e., left M1 for right-handed participants) to m-PAS effectiveness. The lateralization of the MNS and, in detail, the contribution of the dominant hemisphere during action observation is still debated (Aziz-Zadeh, Koski, Zaidel, Mazziotta, & Iacoboni, 2006; Caspers, Zilles, Laird, & Eickhoff, 2010; Lange, Pavlidou, & Schnitzler, 2015; Liew et al., 2018; Molnar-Szakacs, Iacoboni, Koski, & Mazziotta, 2005). Somatotopic and mototopic activation of mirror areas during action observation seems confirmed for simple movements, while for complex movements, or goal-directed ones, evidence is more controversial (for reviews, see: Errante & Fogassi, 2021; Kemmerer, 2021). A recent study in monkeys showed that motor resonance during observation of grasping movements does not reflect a hand identity-dependent coding (i.e., lateralized motor resonance effects depending on the viewpoint and identity of the observed effector), leading to bilateral activation of pre-motor and motor cortices (Fiave & Nelissen, 2021). Similarly, in humans, using functional near-infrared spectroscopy, it was shown that observing other people's actions in a fine motor task activated motor regions with mirror properties independently from the observed hand (right or left) used to perform the task (Khaksari et al., 2022). Importantly, the activation of MNS regions during action observation and imitation correlates with participants' handedness. The dominant hemisphere seems to be more activated – and less ‘lateralized’ – than the non-dominant one, as if the observed movement is ‘mirrored’ with the dominant hand independently from the side of the observed one (Aziz-Zadeh, Maeda, Zaidel, Mazziotta, & Iacoboni, 2002; Khaksari et al., 2022; Koski et al., 2002). Considering all this evidence, whether the dominant hemisphere plays a crucial role in the MNS and it is activated during action observation independently from the side of the body observed, we may hypothesize that targeting, during the m-PAS, the left motor cortex – dominant for right-handed people – may induce plasticity within the action-observation network in a domain-general way, potentially affecting motor resonance phenomenon in the non-dominant hemisphere. Conversely, if this version of the protocol proves to be ineffective, it would suggest that the site of TMS during m-PAS administration has to be highly ‘cortical specifical’ to induce plastic effects within the MNS, at least when the visual stimulus conditioned is a simple, lateralized movement as the one exploited in our protocol (i.e., abduction of the index finger). Hence, in this first experiment, besides the standard m-PAS protocol, in which TMS is delivered over the right, non-dominant M1 (ipsilateral to the visual stimulus depicting a right-hand movement), we have tested a second version of the protocol where TMS is delivered over the left, dominant, M1, still during the observation of right-hand movements. In line with our previous study (Guidali et al., 2020), to assess motor resonance before and after the m-PAS, we adopted an action-observation paradigm in which TMS has always been delivered over the right M1 (i.e., over the motor cortex not conditioned in the ‘dominant’ version of the protocol) during the observation of left- and right-hand movements. In this way, the experiment aimed to test the possible modulation of motor resonance by plasticity-induction in the non-dominant right M1. Namely, if the dominant motor system plays a main bilateral control of MNS functioning, we may expect that the administration of the m-PAS over the left M1 (i.e., the dominant hemisphere) would modulate motor resonance also mediated by the non-dominant, contralateral, M1 (i.e., the emergence of motor resonance for the conditioned, ipsilateral movement – Guidali et al., 2020) as its application over the right hemisphere does. Conversely, the absence of changes in motor resonance after left M1 stimulation would prove the hemispheric specificity of such a protocol.

In Experiment 2, we tested the possible behavioral outcomes of the m-PAS, because changing of mirroring mechanism has been proved so far only at a neurophysiological level. Imitating observed actions or movements is a core function of the human MNS (Heyes & Catmur, 2021), and it is subtended by a broad network of cortical areas comprising sensorimotor regions and frontoparietal high-order associative areas (Caspers et al., 2010). Thus, besides effects on motor resonance, exploring possible behavioral outcomes of the m-PAS is crucial to deepen the extent of the plastic effects induced, namely whether the protocol acts only at a neurophysiological (i.e., corticospinal excitability) and/or local level (i.e., within the motor cortex) or it may also modulate high-order cognitive functions subtended to the MNS as, indeed, imitation of actions/movements. To this aim, in the second experiment, we adopted an imitative compatibility task to assess m-PAS influences on automatic imitation. Automatic imitation is a stimulus-response compatibility effect that occurs when the observation of a movement involuntarily facilitates the performance of topographical similar movements and interferes with the performance of topographical dissimilar movements (Heyes, 2011). The difference in the participant's performance (assessed with reaction times – RTs) between imitative/congruent and non-imitative/incongruent observed movements (i.e., imitative compatibility index) is considered a marker of automatic imitation (Cracco & Brass, 2019). This effect has been widely replicated by using different body movements, and it is one of the most used behavioral markers of MNS recruitment (e.g., Brass, Bekkering, & Prinz, 2001; Catmur & Heyes, 2011; Cracco & Brass, 2019; Hétu, Taschereau-Dumouchel, Meziane, Jackson, & Mercier, 2016; Heyes, Bird, Johnson, & Haggard, 2005; Mengotti, Ticini, Waszak, Schütz-Bosbach, & Rumiati, 2013; Quadrelli et al., 2021). We expect that the administration of the m-PAS, through the induction of plasticity within the action-observation network, may influence participants' ability to imitate others' movements. We would also expect this effect, indexed by changes in the imitative compatibility index after m-PAS delivery, to be specific for trials depicting the same visual stimulus conditioned during the protocol (i.e., abduction movement of the right-hand index finger) and an actual movement. Conversely, if a classic behavioral marker of MNS recruitment like automatic imitation is not influenced by the protocol, we may hypothesize that m-PAS effects are detectable only at a neurophysiological level, with no functional effects on human imitation.