Effects of action observation on corticospinal excitability: Muscle specificity, direction, and timing of the mirror response.

Neuropsychologia 64 (2014) 331–348

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Neuropsychologia journal homepage: www.elsevier.com/locate/neuropsychologia

Reviews and perspectives

Effects of action observation on corticospinal excitability: Muscle specificity, direction, and timing of the mirror response Katherine R. Naish a,b,c,n, Carmel Houston-Price d, Andrew J. Bremner e, Nicholas P. Holmes a,b a

School of Psychology and Clinical Language Sciences, University of Reading, Earley Gate, Whiteknights, Reading RG6 6AL, UK Centre for Integrative Neuroscience and Neurodynamics, University of Reading, Earley Gate, Whiteknights, Reading RG6 6AL, UK c Department of Psychology, Neuroscience & Behaviour, McMaster University, 1280 Main Street West, Hamilton, ON, Canada L8S 4L8 d University of Reading Malaysia, Menara Kotaraya, Level 7, Jalan Trus, Johor Bahru, Malaysia 80000 e Department of Psychology, Goldsmiths, University of London, New Cross, London SE14 6NW, UK b

art ic l e i nf o

a b s t r a c t

Article history: Received 3 March 2014 Received in revised form 5 September 2014 Accepted 19 September 2014 Available online 2 October 2014

Many human behaviours and pathologies have been attributed to the putative mirror neuron system, a neural system that is active during both the observation and execution of actions. While there are now a very large number of papers on the mirror neuron system, variations in the methods and analyses employed by researchers mean that the basic characteristics of the mirror response are not clear. This review focuses on three important aspects of the mirror response, as measured by modulations in corticospinal excitability: (1) muscle specificity; (2) direction; and (3) timing of modulation. We focus mainly on electromyographic (EMG) data gathered following single-pulse transcranial magnetic stimulation (TMS), because this method provides precise information regarding these three aspects of the response. Data from paired-pulse TMS paradigms and peripheral nerve stimulation (PNS) are also considered when we discuss the possible mechanisms underlying the mirror response. In this systematic review of the literature, we examine the findings of 85 TMS and PNS studies of the human mirror response, and consider the limitations and advantages of the different methodological approaches these have adopted in relation to discrepancies between their findings. We conclude by proposing a testable model of how action observation modulates corticospinal excitability in humans. Specifically, we propose that action observation elicits an early, non-specific facilitation of corticospinal excitability (at around 90 ms from action onset), followed by a later modulation of activity specific to the muscles involved in the observed action (from around 200 ms). Testing this model will greatly advance our understanding of the mirror mechanism and provide a more stable grounding on which to base inferences about its role in human behaviour. & 2014 Published by Elsevier Ltd.

Keywords: Mirror neuron system Transcranial magnetic stimulation Corticospinal excitability Motor evoked potential Action observation Action execution

Contents 1.

2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332 1.1. Relevance of muscle specificity, direction, and timing of the mirror response. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 1.2. Review method and outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 Muscle specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334 2.1. Barriers to the investigation of muscle specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 2.1.1.Lack of action execution data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 2.1.2.Data obtained from only one muscle or during only one movement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 2.1.3. Sources of EMG signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336 2.1.4. Interindividual variability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 2.2. Overview of specificity findings in the literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 2.3. Studies demonstrating muscle specificity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337

n

Corresponding author at: Department of Psychology, Neuroscience & Behaviour, McMaster University, 1280 Main Street West, Hamilton, ON, Canada L8S 4L8. E-mail addresses: [email protected] (K.R. Naish), [email protected] (C. Houston-Price), [email protected] (A.J. Bremner), [email protected] (N.P. Holmes). http://dx.doi.org/10.1016/j.neuropsychologia.2014.09.034 0028-3932/& 2014 Published by Elsevier Ltd.

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2.4. Studies demonstrating partial- or non-specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Direction of modulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Timing of modulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Mirror response latency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Temporal matching of corticospinal modulation during observation and EMG activity during action execution. . . . . . . . . . . . . . . . . . . 5. A new model of the modulation of corticospinal excitability during action observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Physiological basis of inhibition and excitation comprising the mirror response. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A. Supplementary information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. 4.

“Each time an individual sees an action done by another individual, neurons that represent that action are activated in the observer's premotor cortex. This automatically induced, motor representation of the observed action corresponds to that which is spontaneously generated during active action and whose outcome is known to the acting individual. Thus, the mirror system transforms visual information into knowledge” (Rizzolatti and Craighero, 2004, p. 172).

1. Introduction Around two decades ago, in a laboratory in northern Italy, a macaque monkey became the subject of a lot of excitement in the field of neuroscience. Giuseppe di Pellegrino and colleagues at the University of Parma were using single-unit recording to measure neuronal firing as monkeys performed manual actions such as grasping. The researchers inadvertently discovered that, when a monkey passively observed an experimenter grasping a piece of food, the same neurons in the premotor cortex (PMC) fired as would have fired had the monkey been performing the movement itself (Di Pellegrino et al., 1992; see also Leinonen et al. (1979), for a similar observation). Thus, there appeared to be an internal ‘mirroring’ of the observed action. The neurons that respond in a congruent manner to both action observation and action execution have been called ‘mirror neurons’ (Gallese et al., 1996). Since the discovery of these cells in monkeys, researchers have investigated the existence and characteristics of an analogous mirror neuron system in humans. Due to the invasive nature of single-unit recording, the vast majority of these studies (see Mukamel et al. (2010), for an exception) have used indirect measures such as functional magnetic resonance imaging (fMRI, e.g., Iacoboni et al., 1999), electroencephalography (EEG; e.g., Muthukumaraswamy et al., 2004), and transcranial magnetic stimulation (TMS; e.g., Fadiga et al., 1995) to assess the effects of action observation on motor excitability. Thus, there is very little direct evidence of mirror neurons in the human brain (e.g., highlighted by Dinstein et al. (2008), Hickok (2009, 2014)). There is, however, a wealth of evidence suggesting that the mere observation of movement elicits changes in activity in the motor system; this is taken as evidence of some form of action observation-execution matching system in the human brain. The so-called ‘mirror response’ has been implicated in a wide range of human behaviours and pathologies, from action understanding (e.g., Di Pellegrino et al., 1992) and imitation (e.g., Rizzolatti and Craighero, 2004) to schizophrenia (e.g., Enticott et al., 2008) and autism (e.g., Puzzo et al., 2009; see Hickok (2014), for a more extensive list). One of the primary techniques that have been used to examine the putative human mirror system is TMS, which is used in conjunction with electromyography (EMG) to assess changes in corticospinal excitability during action observation. Specifically, TMS is used to stimulate the primary motor cortex (M1), resulting

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in action potentials along efferent nerves, and producing motorevoked potentials (MEPs) in the muscle or muscles of interest. The amplitude of these MEPs (typically recorded transcutaneously using EMG) gives an indication of the level of excitability in the motor pathways. MEPs are usually recorded as participants observe live or videoed movements, and their amplitudes compared to those elicited during a baseline condition (e.g., during the observation of a static actor or object, a fixation cross, or a blank screen; Baldissera et al., 2001; Hardwick et al., 2012; Alaerts et al., 2010a). An increase in MEP size during action observation compared to baseline is commonly interpreted as evidence of a mirror neuron response, under the rationale that action observation activates mirror neurons, which in turn increase excitability in M1, leading to larger responses in the muscle. The first investigation of the human mirror system was a TMS study by Fadiga et al. (1995), who found larger MEPs in muscles involved in grasping when participants viewed grasping, compared to when they saw a static object or dimming light. Likewise, MEPs in muscles shown to be active during the execution of abstract arm movements (tracing geometric shapes in the air) were facilitated during the observation of these movements. Thus, Fadiga and colleagues provided the first demonstration of muscle-specific mirrorlike activity in humans. Since this first report, numerous papers (see Supplementary Table; Aglioti et al. (2008), Alaerts et al. (2012, 2009d, 2011, 2009a, 2009b, 2009c, 2009d, 2010a, 2010b, 2011, 2012), AzizZadeh et al. (2002), Baldissera et al. (2001), Bianco et al. (2012), Borroni et al. (2011, 2005, 2008), Brighina et al. (2000), Bucchioni et al. (2013), Burgess et al. (2013), Candidi et al. (2014), Catmur et al. (2007, 2011), Cattaneo et al. (2009), Cavallo et al. (2012, 2013a, 2013b), Clark et al. (2003), Désy and Théoret (2007), Donne et al. (2011), Enticott et al. (2008, 2010, 2011, 2012), Fecteau et al. (2005), Fiorio et al. (2010), Fadiga et al. (2005), Funase et al. (2007), Gangitano et al. (2001, 2004), Hardwick et al. (2012), Hill et al. (2013), Hétu et al. (2010), Hogeveen and Obhi (2012), Jola et al. (2012), Jola and Grosbras (2013), Kaneko et al. (2007), Lago et al. (2010), Lago and Fernándezdel-Olmo (2011), Lago-Rodriguez et al. (2013), Leonard and Tremblay (2007), Lepage et al. (2008, 2010), Liepert and Neveling (2009), Liepert et al. (2011), Loporto et al. (2012, 2013), Meister et al. (2012), Molnar-Szakacs et al. (2007), Montagna et al. (2005), Möttönen et al. (2010), Obhi et al. (2011), Ohno et al. (2011), Patuzzo et al. (2003), Puzzo et al. (2009), Ray et al. (2013), Romani et al. (2005), Roosink and Zijdewind (2010), Sakamoto et al. (2012, 2009a, 2009b), Sartori et al. (2011, 2012a, 2012b, 2013a, 2013b), Sartori and Castiello (2013), Senot et al. (2011), Strafella and Paus (2000), Tidoni et al. (2013), Tomeo et al. (2013), Tremblay et al. (2008), Urgesi et al. (2006), van Ulzen et al. (2013), Villiger et al. (2011), and Williams et al. (2012)) have examined changes in corticospinal excitability during action observation. The broad finding that motor activity is modulated during action observation is generally robust; however, a closer look at the literature highlights some differences and inconsistencies between studies. Specifically, there are three aspects of the basic mirror response that are not clear: muscle


specificity, direction, and timing of modulation. These three characteristics are the focus of the current review. 1.1. Relevance of muscle specificity, direction, and timing of the mirror response While many studies have shown that modulation occurs exclusively in the muscles involved in an observed action (e.g., Fadiga et al., 1995), a few have reported modulation of activity in muscles not involved in the movement (e.g., Aglioti et al., 2008; Lago and Fernández-del-Olmo, 2011; Lepage et al., 2010). Whether or not the mirror response is a faithful representation of the observed movement has important implications for both the function and origin of the mirror response. Regarding function, we can take the example of perhaps the most prevalent theory in the field: that the mirror response underlies action understanding (e.g., Di Pellegrino et al., 1992; for extensive discussion and reviews of this theory, see Gallese et al. (2011) and Hickok (2009, 2014)). Although ‘action understanding’ is not very welldefined in this literature (e.g., Hickok, 2009), for an action to be ‘understood’ at any useful level, the response must surely be specific to the observed action. For example, to claim that the motor activation that occurs when we view someone reach towards a cup allows us to infer that the person intends to grasp the cup, the motor response in this instance must be distinct from the response that would occur if we saw the same person pointing towards the cup. The same reasoning can be applied to the theory that mirroring underlies our ability to imitate actions (e.g., Rizzolatti and Craighero, 2004); if mirroring is a key mechanism by which we replicate seen actions, the motor representation evoked for the observation of one action must be distinct from that evoked by other similar movements. Thus, establishing the extent to which the mirror response is muscle-specific is an important step in understanding the function of mirroring. Muscle specificity is also relevant to theories of the origin of the mirror response. Some accounts of the mirror neuron system suggest that the system has evolved to support social behaviour (e.g., Ramachandran, 2000; Rizzolatti and Craighero, 2004). An alternative theory is that mirror neurons acquire their ‘mirror’ properties by means of associative learning. Heyes and colleagues (e.g., Catmur et al., 2007; Cook et al., 2014; Heyes, 2010) have proposed that the co-occurrence of action execution with the observation of one's own or others' actions leads to a strengthening of the synaptic connections between sensory and motor neurons, ultimately leading to motor neurons becoming responsive to both sensory and motor information. According to this view, the mirror response is not strictly tied to the observed action, but can also reflect ‘complementary’ actions that are consistently paired with the execution of a particular action. For example, the sight of somebody stretching out their hand to offer an object to us might activate the motor representation for reaching to grasp the object. Thus, nonspecificity in the mirror response could indicate activation of complementary motor representations. The second aspect of the mirror response that we focus on in this review is the direction of modulation- that is, whether action observation produces an overall increase or decrease in motor activity. Whether the resulting effect (i.e., in the muscle) of action observation is facilitatory or inhibitory is important for inferring the role of the mirror response in behaviour. In particular, the change in excitability at the muscular level determines whether viewing an action leads to overt movement or not, so is relevant to the idea that action observation underlies our ability to imitate others. The majority of reported studies have found that motor pathway activity increases from baseline when an action is observed; however, there are also reports of action observation leading to no modulation (e.g., Ohno et al., 2011) or to decreased

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excitability in the muscle (e.g., Lago and Fernández-del-Olmo, 2011). Importantly, differences in methods of data normalisation and analysis mean that ‘facilitation’ does not mean the same thing from one study to the next. In this review, we examine these discrepancies in relation to between-study differences, including approaches to data normalisation and the selection of baseline conditions. We also consider data from action execution studies, and discuss whether a decrease in activity might reflect the suppression required to prevent overt movement. Finally, the timing of the mirror response is critical for inferring the function and mechanism underlying mirroring. Currently, there is no consensus on precisely when modulation of motor activity occurs relative to the visual presentation of an action. Although we do not need to know the precise timing of mirroring to establish whether behavioural imitation and the mirror response are related, we need this information to infer how much of an action someone needs to see before it is ‘mirrored’, or how soon after action observation imitation should occur. The question of timing is also relevant to the mechanism underlying mirroring. As noted in Section 4, the earliest modulation of motor responses evoked by visual stimuli has been found at around 75 ms. Therefore, we would not expect MEP amplitude to be modulated by action observation earlier than this. If changes in MEP size detected in humans during action observation are the result of direct input from or the activity of mirror neurons, we should see a mirror response present from around 75 ms, or shortly after. However, if modulation is not evident until considerably later, it could imply that the modulation has arisen from mediating factors that are not exclusively driven by sensory inputs. For example, modulation of MEPs during action observation could be mediated by motor imagery (the observer imagining themselves performing the action) which is known to modulate corticospinal excitability in a similar way to action execution. Importantly, a response mediated by slower processes such as motor imagery is not necessarily of lesser interest than a response mediated by mirror neurons. However, the basis of the response would have implications for how the response affects behaviour. For example, if the response is mediated by motor imagery in the observer, we would predict that imagery of a different movement during action observation would produce modulation that reflected the imagined, rather than the observed, action. Alternatively, if the mirror response is indeed mediated by the firing of mirror neurons that discharge specifically to observation and execution of actions, we would expect no interference by the motor imagery, at least in the very earliest stages of mirror neuron processing. The purpose of this review article is not to question the existence of mirror neurons in humans, nor to attempt to infer the exact mechanism behind the mirror response. Rather, our objective is to clarify what we know of the characteristics of the mirror response in humans, to enable us to infer the likely mechanism and function of such a response. To clarify, references to a ‘mirror response’ in this review refer to modulation in the motor system during passive action observation. A relatively slow mirror response could still have functional significance in human behaviour, but suggests that the response is not a straightforward, unmediated transformation of ‘visual information into knowledge’ as it was originally described (Rizzolatti and Craighero, 2004). 1.2. Review method and outline The findings discussed in this review come mainly from studies using single-pulse TMS (spTMS), a method that is well placed to address the issues central to this review for three main reasons. First, TMS has very high temporal precision. From the time at which TMS is triggered, a single pulse takes around 1 ms to be delivered, and the effects of TMS on the muscle are measurable within 10–40 ms. This

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high temporal precision allows us to infer exactly when activity in the pathways to those muscles is modulated relative to action observation, which methods such as fMRI, and to a lesser extent EEG, do not. Second, by measuring the amplitude of MEPs during both action observation and baseline conditions, we can infer whether activity in the muscle increases or decreases from baseline during the observation of movement. As MEPs represent the final output of the motor pathways, the direction of this modulation indicates the resulting effect on excitability: larger MEPs indicating an overall excitatory effect, and smaller MEPs indicating an overall suppression. It should be noted that the terms ‘enhanced’, ‘facilitated’, ‘suppressed’, and ‘inhibited’ in this article are used to refer to an increase or decrease in MEP size; they do not necessarily refer to an excitability change brought about by an active suppressive or facilitatory mechanism. Third, TMS gives us a closer indication than any other method as to whether the responses found correspond to the muscles involved in the observed action. The position of the TMS coil can be optimised for evoking MEPs in specific muscles, while EMG recording can be used to obtain a profile of muscle activity when the movements are actually performed. If action observation is compared to action execution, we can ascertain whether the muscles in which there is observation-related modulation are those activated when the observer himself performs the movement. With regards to the third point, it must be stressed that while TMS can be positioned in order to evoke responses in a specific muscle, this does not mean that responses are produced exclusively in that muscle. It is common for single pulses of TMS to elicit MEPs in more than one muscle simultaneously, due to the size of the stimulation field and the close proximity of the cortical representations of related muscles. Indeed, in most studies recording from more than one muscle, the same location is used to elicit MEPs in all of the muscles of interest. This is not necessarily a limitation of such studies, as researchers tend to measure differences in the amplitudes of MEPs between conditions, rather than the presence or absence of responses. However, it is important to note the limits of what TMS studies can tell us about specificity. Essentially, we can draw a distinction between the ‘muscle specificity’ and the ‘movement specificity’ of modulation. While muscle specificity can be reasonably defined as modulation being present exclusively (or to a significantly greater extent) in muscles involved in the observed movement, this does not necessarily mean that the modulation reflects specifically and only that movement. Related to this, it should be noted that the primary motor cortex is likely organised in terms of movements themselves rather than individual muscles (e.g., Graziano, 2011). Thus, stimulating the cortex in a particular place likely evokes a pattern of modulation related to some aspect of movement (e.g., hand rotation in a particular direction), rather than a specific muscle or muscle synergy. In addition to spTMS, we also consider the smaller number of studies that have used either paired-pulse TMS (ppTMS) or peripheral nerve stimulation (PNS) to examine cortical or corticospinal excitability during action observation. Briefly, ppTMS is used (in the studies discussed in this review) to measure the excitability of connections within the cortex, for example, the connection between the premotor and primary motor cortex (e.g., Strafella and Paus, 2000). PNS is used to stimulate the motor pathway at the peripheral level; for example, it can be applied to the median nerve to evoke a response in hand muscles (e.g., Borroni et al., 2005). The Hoffman (H-) reflex that is produced in the muscle gives an indication of the level of excitability in the pathway between the spinal cord and the muscle (see Palmieri et al. (2004), for more details). In conjunction with data from spTMS, these methods can shed light on the level of the motor pathway at which modulation is occurring. It should be noted here that TMS and PNS studies represent only part of the literature on the human mirror neuron system; fMRI and EEG are also popular tools for studying the effects of action observation. While a complete picture of

the factors that influence the neural effects of action observation would, of course, draw on the literatures relating to these methods as well, this review focuses on studies using TMS, which is the optimal method for addressing the questions asked in this review for the reasons outlined above. The majority of the articles cited in this review were found via a search (conducted in May 2014) of the Thomson Reuters' Web of Science database using the terms ‘action observation’, ‘motor resonance’, ‘mirror neuron’, and ‘mirror system’, followed by a search within those results for articles containing the term ‘transcranial magnetic stimulation’. This initial search produced 261 articles. From these, studies were selected in which TMS was used to assess corticospinal excitability while healthy human participants observed actions. Studies investigating clinical populations were included if the sample included a healthy control group (six studies). We did not include studies in which (a) action observation was combined with imagery or execution and there was no pure observation condition, (b) the stimuli were static images or sounds associated with actions, rather than videoed or live actions (studies in which static images were presented in succession to give the impression of movement, e.g., Cavallo et al., 2013a, were included, however), or (c) the observed effector was being acted upon, rather than being the agent of the perceived action (e.g., a painful stimulus being applied to a hand). A total of 78 papers from the Web of Science results survived these exclusion criteria. A further seven articles known to the authors but which did not arise from the search parameters (Baldissera et al., 2001; Borroni et al., 2008; Brighina et al., 2000; Catmur et al., 2007; Hétu et al., 2010; Ohno et al., 2011; Roosink and Zijdewind, 2010) were also included (see Hedges and Olkin (1985), for a discussion of this inclusion approach). Thus, a total of 85 peer-reviewed TMS and PNS studies were considered in this review (see Supplementary Table). Additionally, a small number of studies reporting data from other methods are cited where they relate directly to an issue raised by discussion of the TMS or PNS data. In Sections 2–4, we discuss evidence pertaining to the three questions about the mirror response raised previously: (1) muscle specificity, (2) direction, and (3) timing. The Supplementary Table provides specific information about the methods and results of each TMS and PNS study (n¼85) cited in this review. In Section 5, we bring together the evidence discussed in the body of the review to propose a model of how corticospinal excitability is influenced by action observation. This model provides testable hypotheses regarding the specificity, direction, and timing of the modulation that occurs when an action is observed. Finally Section 6 provides a summary of our conclusions, and suggestions for how the methods used in this area could be modified or extended to optimally address the remaining unanswered questions about the human mirror response.

2. Muscle specificity If the activation of the motor system during action observation is indeed mirroring the seen action, we would expect the modulation to occur specifically in the pathways innervating the muscles involved in that action. Alternatively, if the mirroring that occurs is more of a general priming of the motor system – and not representative of the specific action observed – we might expect action observation to modulate corticospinal excitability in a manner that is not muscle-specific. According to a seminal report by Gallese et al. (1996), who used single-unit recording to examine the responses of individual cells to action observation, mirror neurons in area F5 of the monkey brain differ in the extent to which they respond exactly to what is seen. In Gallese and colleagues' study, 92 neurons (17% of the 532 F5 neurons that were examined) were found to respond to both action observation and execution, but only 31.5% of these (termed ‘strictly


congruent’ mirror neurons) responded specifically to observation and execution of the exact same movement types (for example, firing in response to execution and observation of a precision grasp using the thumb and index finger, but not to the execution or observation of a whole hand grasp). A smaller proportion (7.6%; ‘non-congruent’ mirror neurons) responded to both action observation and execution, but showed no discernible relationship between the executed and observed movements that they responded to. The majority of mirror neurons (60.9%) were classified as ‘broadly congruent’, as they responded to the observation and execution of actions that were not exactly the same. The broadly congruent mirror neurons fell into three categories, responding to either (1) execution of a specific movement (e.g., precision grip) but observation of different forms of the same action (e.g., precision grip and whole hand prehension), (2) execution of a specific movement but observation of different hand actions (e.g., grasping and object manipulation), or (3) execution of a specific movement but observation of different actions used to obtain a similar, logically-related goal (e.g., grasping with the mouth or hand). As noted in Section 1, the methods used most commonly in human studies allow us to make inferences only about large collections of neurons, so we can only speculate on how the activity of single cells in the human motor system parallels those in the monkey mirror system. However, if the human mirror system is analogous to the monkey mirror neuron system in terms of having both strictly and broadly congruent properties, then we might expect varying degrees of muscle specificity, reflecting strictly and broadly congruent mirror responses. For example, modulation of corticospinal excitability that is not muscle-specific could reflect a response that is only ‘broadly’ mirroring what is being observed, so in some cases observation of a precision grip might elicit a response that mirrors ‘grasping’ in general (i.e., a modulation of MEPs in hand muscles that would be recruited in a whole hand grasp, as well as those recruited more specifically during precision grip). In Fadiga et al.'s (1995) study, TMS was used to elicit MEPs in two intrinsic muscles of the hand (first dorsal interosseous, FDI; opponens

Table 1 Definitions of muscle abbreviations used in the review and the Supplementary Table, with primary function of each muscle. The described muscle functions are based on information provided in Cram's Introduction to Surface Electromyography (Criswell, 2010) and Manual of Nerve Conduction Study and Surface Anatomy for Needle Electromyography (Lee and DeLisa, 2004). Abbreviation Muscle name

Primary function

ADM APB BB

Abductor digiti minimi Abductor pollicis brevis Biceps brachii

ECR

Extensor carpi radialis

ECU

Extensor carpi ulnaris

EDC

FPB GAM

Extensor digitorum communis Extensor indicis proprius Flexor carpi radialis Flexor carpi ulnaris First dorsal interosseous Flexor digitorum superficialis Flexor pollicis brevis Gastrocnemius

Abduction of little finger Abduction of thumb Forearm flexion, supination, and shoulder flexion Wrist extension, abduction, and radial deviation Extension and adduction of wrist Extension of fingers

OP

Opponens pollicis

TA

Tibialis anterior

EIP FCR FCU FDI FDS

Extension of index finger Wrist flexion and radial deviation Flexion and adduction of wrist Abduction of index finger Flexion of wrist and 2nd–5th fingers Thumb flexion and opposition Involved in knee flexion and pointing of the toes Thumb opposition or flexion of thumb metacarpal Dorsiflexion of ankle, and foot inversion

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pollicis, OP; see Table 1 for a full list of muscle abbreviations), and two in the forearm (extensor digitorum communis, EDC; flexor digitorum superficialis, FDS), while participants watched live grasping and arm movements. In a separate condition, recording of muscle activity during the performance of these movements showed that all four of the muscles were active during grasping, while the FDI, EDC, and FDS were also active during the abstract arm movements. Fadiga and colleagues found that, during grasp observation, MEPs in all four muscles were facilitated above a baseline condition (observation of a static hand or a dimming light), while those in the FDI, EDC, and FDS – but not in the OP – were facilitated during the observation of arm movements. This modulation was thus considered muscle-specific, as the same muscles recruited during action execution were modulated during observation. A number of studies published since Fadiga et al.'s (1995) study appear to show similar, muscle-specific effects (e.g., Romani et al., 2005), while a smaller number report seemingly non-specific modulation (Aglioti et al., 2008; Lago and Fernández-del-Olmo, 2011; Lepage et al., 2010). Importantly, there are a large number of studies which, for the reasons described below, preclude the drawing of inferences in relation to muscle specificity.

2.1. Barriers to the investigation of muscle specificity 2.1.1.Lack of action execution data To ascertain whether action observation modulates corticospinal excitability in a muscle-specific manner, we need to know which muscles are recruited during the execution of the movement being viewed. This can be achieved using EMG either alone, to passively record muscle activity during action execution, or in conjunction with TMS, to measure changes in MEP size during action execution. Both of these methods show the relative contribution of different muscles to the movement. Unfortunately, the relatively small number of studies (21 of the 85 in Supplementary Table) that have assessed muscle activity during movement execution means that we cannot always be certain that the modulation reported during observation reflects true muscle specificity. Although action observation and execution data would ideally be collected within the same study (preferably within the same subjects), there are some movements for which an action execution condition is arguably less critical. Discrete, isolated movements of a single effector, such as finger abduction–adduction (e.g., Urgesi et al., 2006) or wrist extension–flexion (e.g., Borroni et al., 2005), involve fewer muscles and have less scope for variability than more complex actions, such as whole-hand grasping. It is therefore easier, in some cases, to infer which muscle or muscles are predominantly active during the movement, and which are not. For example, although modulation during the observation of index finger and little finger abduction has been measured by several of the studies cited in this review (see Supplementary Table), only two of these (Romani et al., 2005; Urgesi et al., 2006) assessed muscle activity during action execution. Romani and colleagues showed that the FDI was more active than the ADM during index finger movement, and that the ADM was more active than the FDI during little finger movement; and Urgesi et al. (2006) somewhat similarly reported that the FDI was more active during index finger movement than little finger movement, while the ADM was more active during little finger movement than index finger movement. Because there are few dimensions on which finger abduction can vary (i.e., only range of motion and speed), it is reasonable to assume that these dissociations in muscle activity likely characterise the index and little finger abduction–adductions shown to participants in other studies (e.g., Aziz-Zadeh et al., 2002; Catmur et al., 2007). The lack of action

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execution data in studies using these types of discrete movements could, therefore, be considered negligible.1 More complex movements that have been examined in action observation studies include reaching and grasping (e.g., Hardwick et al., 2012), pinching (e.g., Loporto et al., 2012), squeezing (e.g., Hogeveen and Obhi, 2012), and lifting (e.g., Hétu et al., 2010) objects, and communicative gestures (e.g., Molnar-Szakacs et al., 2007). Studies have also looked at social actions involving two individuals (e.g., Enticott et al., 2011), actions using tools (e.g., Cattaneo et al., 2013), and whole body movements such as sporting actions (e.g., Aglioti et al., 2008) and dance (e.g., Jola et al., 2012). Using these more complex, goal-directed movements could be argued to increase the ecological validity of the findings, as these movements are commonly encountered in day-to-day life. However, complex movements also involve a larger number of muscles, making it difficult to infer the pattern and time-course of their activation. For example, reaching and grasping involves a large number of muscles of the hand and forearm, the activation of which varies according to the object grasped (e.g., Brochier et al., 2004; Mason et al., 2001). Unless muscle activity is recorded during action execution, one cannot be certain which muscles are more heavily involved during any given grasp. Interestingly, studies that have presented whole-hand grasping movements (as opposed to precision grip using the thumb and index finger) as stimuli sometimes assume the involvement of different muscles. For example, while some studies assume the ADM is involved in whole-hand grasps (Cavallo et al., 2011; Sartori et al., 2011), others treat the ADM as a control site unlikely to be involved in this type of grasp (Enticott et al., 2012; Gangitano et al., 2004). There are data that indicate that the ADM is less involved in reach-to-grasp actions than the FDI (e.g., Hardwick et al., 2012). Given that grasp actions are directed towards differently-shaped and sized objects in each study, it is dangerous to make assumptions about the precise muscles involved for the reasons discussed above. The importance of recording EMG during action execution within a study was demonstrated by the findings and interpretations of Baldissera et al. (2001), and the later clarification of their conclusions (Borroni et al., 2005; Montagna et al., 2005). In Baldissera and colleagues' experiment, muscle responses were recorded from the FDS – a finger flexor muscle – during the observation of a hand extending and flexing the fingers. Activity in the muscle was significantly higher during the observation of finger extension, and lower during the observation of finger flexion. As the modulation was in the direction opposite to that expected intuitively during action execution, the authors concluded that action observation had led to a suppression of activity, and the findings have since been described as an ‘inverted mirror’ effect (e.g., Villiger et al., 2011). After replicating the findings of Baldissera's group, Montagna et al. (2005) sought to explain the suppression effect by examining the pattern of muscle activity that occurred during execution of the action. Unexpectedly, muscle activity in the FDS was greater during hand opening than closing – the opposite to what was originally assumed – indicating that the direction of MEP modulation during action observation did in fact match muscle activity during action execution. As the video stimuli were almost identical in the two studies (Baldissera et al., 2001; Montagna et al., 2005), it is likely that the excitability changes found in both experiments were, in fact, mirroring the course of muscle recruitment during the actors' execution of the movement. Baldissera and Montagna's studies showed modulation of activity in an antagonist muscle during the observation of movement 1 Note that discrete movements of individual digits do recruit multiple muscles (Schieber, 1995); however, it is reasonable to infer that the difference between FDI and ADM involvement will likely be consistently significant for these movements, provided the hand is at rest apart from the finger movement.

(i.e., finger flexor muscle active during finger extension). Indeed, an investigation of muscle activation during movement execution (in monkeys) by Schieber (1995) showed increased activity in muscles that could only serve as antagonist for the movement (indicated by its connecting tendons), as well as in muscles that could not serve as agonists or antagonists (and which presumably play a stabilising role). This highlights, again, that the timing and extent of muscle activity during execution needs to be confirmed by EMG recording, rather than estimated based on knowledge of the primary functions of muscles. 2.1.2.Data obtained from only one muscle or during only one movement In addition to recording muscle activity during action execution, in order to be able to infer whether modulation is muscle-specific a study also needs to fulfil at least one of the following criteria: (1) record from more than one muscle, one of which is involved in the execution of the observed action and one that is not; or (2) assess modulation during the observation of more than one action: one that recruits the muscle to a significantly greater extent than the other. Studies that incorporate both of these criteria – in other words, those that record from two muscles during the observation of two actions, one involving the first muscle and the other involving the second muscle – allow a double dissociation to be demonstrated. For example, in the previously-described study by Romani et al. (2005) and subsequent similar studies, e.g., Catmur et al. (2007, 2011), MEPs were recorded in the ADM and FDI during the observation of abduction–adduction movements of the little finger (greater involvement of the ADM than FDI) and index finger (greater involvement of the FDI than ADM). Due to the known dissociation between these two muscles and their involvements in each movement, we can expect greater modulation of the FDI than ADM during observation of index finger movement, and vice versa during little finger observation. Using two movements and two muscles rules out the possibility that one muscle is simply more excitable than the other, or that one movement evokes motor facilitation more than the other. In studies that record from only one muscle (e.g., Aziz-Zadeh et al., 2002), we do not know whether modulation is specific to the recorded muscle (or muscle synergy) or occurred also in muscles not involved in the action. Likewise, recording activity during only one observation condition (e.g., Ray et al., 2013) fails to establish that modulation arises due to mirroring of the action, rather than from an increase in excitability caused by movement observation more generally. 2.1.3. Sources of EMG signal When surface EMG electrodes are positioned on the skin to record activity from a particular muscle – even when placement is optimal for recording from that muscle – the signal likely arises from more than one muscle (referred to as ‘cross talk’). This was noted, for example, by Weiss and Flanders (2004), who used EMG to assess muscular synergies within the hand, but acknowledged that many of the channels were expected to pick up activity from muscles neighbouring the target muscles. Moreover, in addition to the overlapping EMG signals, there is overlap in the cortical representations of muscles. Stimulation of one part of the cortex can elicit responses in multiple muscles (indeed, the same pulse is usually used to elicit MEPs in all of the muscles examined in studies of this type), and it has been shown that many corticomotor neurons innervate more than one muscle (e.g., McKiernan et al., 1998). These factors mean that inducing and recording MEPs in a muscle cannot provide absolute specificity. The lack of precision provided by EMG is not a problem as long as the muscles recorded from can truly be dissociated. For example, the FDI and ADM are relatively far apart in the hand, so we can be confident that an increase in the FDI is not


contaminated by increases in ADM activity; thus, this is a feasible muscle pair from which to infer specificity. However, muscles in close proximity such as those comprising the thenar eminence would not be dissociable. This point highlights again the importance of establishing the pattern and timing of muscle activity during execution of the movement of interest. All of the studies reviewed in this article used surface EMG (hereafter ‘EMG’) to record muscle activity. Using intramuscular needle or wire electrodes to measure activity would largely negate the problem of cross talk, because electrodes are implanted to record from individual or collections of muscle fibres (see Perry et al. (1981) and Türker (1993), for descriptions and comparisons of surface and intramuscular EMG recording). While intramuscular recording could be used alone as the primary method of muscle activity recording, it could also be used in combination with surface EMG to establish the existence of cross talk (Türker, 1993) and verify optimal surface EMG placement. To our knowledge, no studies of the human mirror system have used wire or needle electrodes for this purpose, so it might be useful for this to be explored in the future. 2.1.4. Interindividual variability Montagna et al. (2005) showed that there are differences between individuals in the timing and amplitude of muscle activation during action execution. Precise measurements of multiple muscles during movement show that different combinations of muscle activation can underlie the same movement both within and between different subjects (Schieber, 1995). This highlights the fact that, ideally, action execution data should come from the subjects who are assessed during action observation. Individual differences in the pattern of muscle activity underlying movements could well explain some of the differences between studies, in all three of the domains (specificity, direction, timing) discussed here. Indeed, in assessing the temporal correspondence between execution and observation, Borroni et al. (2005) found that muscle activity during wrist flexion and extension varied considerably between subjects, but the correspondence between execution and observation within each subject was consistently strong. Thus, the extent to which MEP modulation reflects EMG during execution might be attenuated when the analysed responses are averaged across a group. For example, Ray et al. (2013) found no significant modulation during action observation in their sample (n ¼12) as a whole, but reported MEP facilitation in the individual data of around half of the participants (though it is not clear whether facilitation at the individual subject level was significant). 2.2. Overview of specificity findings in the literature Of the 85 TMS and PNS studies examined in this review, the majority (n¼50) recorded from (a) only one muscle, and involved one or more movement conditions that were not dissociable in terms of the involvement of this muscle (n¼ 29); or (b) more than one muscle, but the relative involvement of these muscles in the various movement conditions was either very similar or unclear (n¼21). Of the remainder (n¼35), six studies recorded from only one muscle but presented different movement conditions that involved this muscle to different extents; nine recorded from more than one muscle during one movement that involved each muscle to a different extent; and 20 recorded from more than one muscle and involved more than one movement in which muscles were involved to different extents (i.e., allowed a double dissociation to be demonstrated). Of these 35 studies, 15 reported action execution data for the same movements observed by participants; while nine did not record muscle activity during action execution within the study, but used a

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simple movement (index or little finger abduction–adduction, or wrist flexion–extension) and recorded from the same muscles as other similar experiments (i.e., ADM and FDI: Romani et al., 2005, Urgesi et al., 2006; ECR and FCR: Borroni et al., 2005). For the reasons discussed above, studies that examined modulation during the observation of a whole hand object-directed grasp but did not report muscle activity during action execution were not considered conclusive in terms of muscle specificity. The following section will therefore summarise the findings, in terms of muscle specificity, of the 24 studies that speak directly to the question of muscle specificity. Of these 24, 16 can be interpreted as showing clear muscle-specific modulation, three studies suggested non-specific effects (Aglioti et al., 2008; Lago and Fernández-del-Olmo, 2011; Lepage et al., 2010), and the remaining five studies reported unclear findings or evidence of partial specificity (Cavallo et al., 2013a; Jola et al., 2012; Jola and Grosbras, 2013; Kaneko et al., 2007; Meister et al., 2012). 2.3. Studies demonstrating muscle specificity In five studies, specificity was indicated by dissociable modulation of MEPs in muscles of the hand during the observation of discrete single finger movements (Group ‘1’ studies in Supplementary Table). As noted earlier, Romani et al. (2005) showed facilitation in the FDI (and not ADM) during index finger movement observation, and in the ADM (and not FDI) during little finger observation. The conclusion of muscle specificity was further corroborated by Romani et al.'s finding of facilitation of MEPs in the EIP (and not in the ADM or FDI) when index finger extension was observed; the EIP is significantly more active during the execution of this movement than the ADM or FDI. Assessing the data in a slightly different way, the four remaining studies in this category reported the differences in MEP size between movements, per muscle condition. All four studies (Catmur et al., 2007, 2011; Loporto et al., 2013; Urgesi et al., 2006) reported that MEPs in the FDI were larger during index finger observation than during little finger observation, and vice versa for the ADM. Muscle-specific modulation during discrete movement observation was also reported by four studies that examined responses to the observation of wrist flexion and extension. Alaerts et al. (2009c) showed that wrist extension was associated with larger MEPs in the ECR (wrist extensor) than in the FCR (wrist flexor), while observation of wrist flexion was associated with larger MEPs in the FCR than ECR. A second study by this group (Alaerts et al., 2009a), which examined wrist extension only, corroborated these results with their finding of ECR (but not FCR) facilitation during the observation of this movement. Borroni et al. (2005, 2008) assessed modulation of the H-reflex (induced by PNS) during the observation of live wrist extension and flexion. Comparing H-reflex modulation in the FCR with the extension–flexion phases during action execution, Borroni and colleagues found that the H-reflex in the FCR was largest during wrist flexion and smallest during extension. Furthermore, assessment of H-reflex in the ECR (Borroni et al., 2005) showed that modulation followed the opposite pattern, with the responses being largest during extension and smallest during flexion observation. These studies show that the pattern of corticospinal modulation – at the spinal as well as the cortical level – matches the muscle recruitment involved in action. Finally, muscle specificity was indicated by seven studies that used relatively complex movement stimuli (Group ‘3’, Supplementary Table). Alaerts et al. (2010a, 2010b) showed in an action execution condition that the OP and ECR were more active than the FCR during grasping of a weight. In line with this, MEPs were larger in the OP and ECR than in the FCR when the same movements were observed (Alaerts et al., 2010a). Furthermore, it was found that MEPs were larger when the observed weight was heavy compared to when it was light, and when the observed action involved high compared to low isometric force. These effects of weight and force – which can be explained by the higher muscle activity associated with increased

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force requirements – were only present in the OP and ECR. These findings suggest muscle specific modulation, which is sensitive to the force requirements of observed actions. Tidoni et al. (2013) also assessed effects of observing an actor lifting heavy versus light objects; however they also manipulated the truthfulness of the action, i.e., the observed movements were either heavy or light, and faked or genuine, actions. An action execution condition showed that the FDI was more active when participants lifted the heavy item, or the light object pretending that it was heavy (truthful or faked heavy), compared to when they lifted the light object or the heavy object pretending it was light (truthful or faked light). MEP modulation during action observation mirrored this: MEPs were larger when the heavy (faked or truthful) lift was observed compared to in the faked and truthful ‘light’ conditions. Interestingly, muscle activity in the FCR followed a different pattern, being highest during the faked light lifting (heavy object lifted as if it was light) than any other condition. As with the FDI, MEP modulation during action observation matched the action execution data; MEPs were larger during the observation of faked light lifting than when the other three movement types were observed. The final four studies that suggest muscle specificity were those reported by Fadiga et al. (1995) and Montagna et al. (2005); both discussed earlier in this section, and Hardwick et al. (2012) and Loporto et al. (2012). Both of the latter studies found that the FDI was recruited to a greater extent than the ADM during execution of the observed movement (grasping and index finger abduction, Hardwick et al., 2012; pinching, Loporto et al., 2012). Demonstrating muscle-specific modulation during observation, both studies reported that MEPs in the FDI, but not the ADM, were facilitated during action observation. 2.4. Studies demonstrating partial- or non-specificity Two of the three studies suggesting non-specificity (Aglioti et al., 2008; Lago and Fernández-del-Olmo, 2011) reported modulation of a hand muscle when a non-hand movement was performed. Aglioti and colleagues found increased excitability in the ADM and flexor carpi ulnaris (FCU) in novices (not experts or players of either sport) when they watched a basketball player throwing a basketball or watched a football player kicking a ball. As we would expect the hand and forearm muscles to be considerably more involved in throwing than kicking, it is surprising that there was no significant difference between the movement conditions. One possible explanation for this finding is that this modulation reflected the whole body movement, rather than the kick specifically. This is feasible as the whole body of the actor was visible in the observed stimuli, so the associated movement of the arm and hand was visible to the participants. In contrast, the ‘expert’ participants (basketball players and expert watchers) examined by Aglioti's group showed larger MEPs during observation of the basketball shot relative to the static player and football kick, with no difference between the latter two conditions. The findings of Aglioti et al. (2008) are similar to those reported by Jola et al. (2012) and Jola and Grosbras (2013), who also assessed the mirror response in groups with varying levels of expertise during the observation of whole body movements. In both of Jola and colleagues' studies, participants watched two types of dance: Bharatanatyam (a type of traditional Indian dance) and ballet. Recording of muscle activity during action execution revealed that the ECR was significantly more involved in ballet than the FDI. During action observation, observers who were experts in ballet showed larger MEPs in the ECR during observation of ballet than observation of Indian dance. In novices and expert Bharatanatyam spectators, however, there were no differences between conditions in ECR (or FDI) modulation. In a later study, Jola and Grosbras (2013) found that the ECR was in fact more active in novices during Indian dance observation than during ballet or acting observation. Thus, similar to Aglioti and colleagues' finding,

modulation appeared to be muscle-specific for the expert groups, but non-specific (or at least, not reflecting the pattern of movement execution) for the non-experts. It could be suggested, based on the findings of these three studies, that the non-specificity in these cases arose from the fact that the participants mirrored the whole body movement, rather than specifically the main effector involved in the action; thus, in Aglioti and colleagues' study seeing the football player's movement towards the ball facilitated the arm and hand muscles to a similar extent as did viewing the throwing action. In Jola and colleagues' study, MEPs in the FDI were modulated to an equal extent to the forearm ECR, because the participants were not focusing specifically on the arm movements. Indeed, in the majority of other studies participants have been presented with stimuli in which only the acting effector (most commonly the hand) is visible. The relative specificity noted in participants who were experts in the actions could reflect more selective attention to the movement most prominent in the movement. To test this hypothesis, a study comparing the muscle specificity of responses when novice and experienced participants view whole-body versus effector-only stimuli would be a very useful addition to the literature. As well as potentially explaining the nonspecificity found by these studies, testing this hypothesis would also be useful for ascertaining whether effects of expertise are driven by differences in visual attention, or more fundamental differences in the neural representations of these actions. Lago and Fernández-del-Olmo (2011) also found that MEPs in a hand muscle (the FDI) were modulated when participants viewed an action performed by the foot (compared to when they viewed a static foot). It is important to note that, in this case, the modulation was a decrease rather than an increase in MEP size. As will be discussed in Section 3, MEP suppression may indicate inhibition of overt movement in the observer. The foot action that was observed in this case was a goal-directed grasp (the foot was seen approaching and contacting the object), and it is possible that this would evoke modulation in the hand due to the same goal being achievable with this effector. Indeed, some work suggests that the mirror response reflects the goal rather than the immediate visual stimulus (e.g., Villiger et al., 2011), so the modulation here might have reflected ‘grasping’ in general rather than being effector-specific. It should be noted that Alaerts et al. (2009c) also presented participants with a foot movement, while MEPs in the arm (ECR, FCR) were recorded. In contrast to Lago and Fernándezdel-Olmo's (2011) findings, no modulation of MEPs was found during the observation of the foot movement. Importantly, however, the foot action presented by Alaerts and colleagues was an intransitive extension–flexion of the foot, so was not goal-directed. This could be interpreted as support for the notion that modulation during the observation of goal-directed actions is not necessarily confined to the effector observed (in this case, the foot), but might also occur in other effectors (i.e., the hand) that could be used to achieve the same goal (i.e., grasping). The third study to find non-specificity was Lepage et al. (2010), who, contrary to other similar studies (e.g., Romani et al., 2005), found no difference in the modulation of the FDI and ADM during index finger abduction. MEPs in both muscles were facilitated above baseline (observation of a static hand or moving dot). Because TMS was delivered considerably earlier in Lepage et al.'s experiment (at 90 ms) than in other studies, the authors proposed that their findings might reflect an early and non-specific modulation of excitability during action observation. Unfortunately, Lepage and colleagues did not compare modulation of the MEPs elicited in the FDI and ADM at any other stimulation times, so they could not establish the duration of the non-specific modulation they observed; however, their hypothesis is supported by the findings of a more recent study by Cavallo et al. (2013a). These researchers assessed MEPs in the FDI and ADM during index and little finger abduction observation but, unlike previous studies of this type, quantified modulation as a ratio of a muscle's


response to index finger observation relative to little finger observation (thus, a ratio of 1 meant no difference in the muscle's response to each movement type). Because MEP size was not calculated relative to a common baseline, it is not clear whether overall excitability was facilitated, suppressed, or unchanged relative to baseline in this study; however it is evident that at two early time points – 100 and 150 ms – modulation was equivalent in the two muscles. At 200, 250, and 300 ms, however, the ‘mirror ratio’ was significantly different for the two muscles. These findings support Lepage et al.'s (2010) proposal of an early, non-specific modulation followed by a later, muscle-specific response. The findings of Meister et al. (2012) also provide tentative support for the suggestion that specificity occurs only after a delay. In this experiment, participants were shown objects either alone or being acted upon, and were required to make semantic, phonological, or visual judgements about the object or action. MEPs were recorded from the APB and FDI, and action execution data showed the APB to be more active than the FDI when the movements were performed. Interestingly, MEPs were significantly smaller in the APB than the FDI during action and object observation. Thus, the relative modulation in each muscle was the reverse of the activation during movement execution. Furthermore, analyses of the effects of muscle, condition (stimulus and judgement), hemisphere stimulated, and time of stimulation (0, 200, 400 ms) revealed a four-way interaction; the APB MEPs elicited in the right hand (left hemisphere stimulation) at 400 ms post movement onset were smaller in the ‘object alone, visual judgment’ condition than in every other condition. Meister and colleagues interpreted this as indicating a muscle-specific (specific to the muscle most active during the movement), time-specific (at 400 ms only) facilitation from baseline (visual judgement of object alone). It is unclear, however, why observation of an object only facilitated MEPs in the phonological and semantic judgement conditions. It could be argued that no ‘mirror’ response was found in this study, because the MEPs elicited during observation of actions towards objects were comparable in size to those elicited when the object alone was observed. The final study that we interpret as finding partial specificity was conducted by Kaneko et al. (2007). In this study, MEPs in the ADM and FDI were assessed during index and little finger abduction and adduction. Consistent with previous research, the FDI was facilitated during index finger abduction observation; however, no modulation was evident in the ADM during little finger movement observation. Their effects were not non-specific, therefore, because modulation during index finger observation was specific to the FDI, but it is unclear why similar modulation was not found in the ADM during little finger observation. It is possible that the failure to detect modulation during observation of little finger movements is due to how modulation was assessed in this study. In several studies discussed in this section, such as Catmur et al. (2011), modulation is considered as muscle-specific if the MEPs in the FDI were larger during index finger than little finger observation, and vice versa for the ADM; it is not known whether MEPs in each condition were facilitated, suppressed, or unchanged from baseline. In Kaneko and colleagues' experiment, MEPs in each muscle and for each movement were compared to baseline, so it is possible that the lack of modulation during little finger observation is actually consistent with other studies that have not compared directly to baseline. Based on the studies discussed in this section, and aspects of the mirror response discussed subsequently in this review, we suggest that the mirror response comprises both a non-specific (at around 90 ms) and subsequent muscle-specific (from around 200 ms) component (see Fig. 1). These timings are based primarily on the reports of Lepage et al. (2010) and Cavallo et al. (2013a), but are supported by data discussed in the subsequent sections of the review. Regarding the non-specific modulation found at later time points (i.e., post 200 ms;

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Fig. 1. Model of how action observation modulates corticospinal excitability (CSE). This model postulates that there is an initial facilitation of corticospinal excitability that occurs from around 90 ms from movement onset. This is followed by a second wave of modulation (from around 200 ms), which is specific to the muscles involved in the perceived action. This size and direction of the second modulation is influenced by factors such as the observer's intentions (which might be influenced by an experimenter's instructions, or the observer's motivation), object affordances, and action correctness. Inhibitory processes prevent this increase in CSE from producing overt imitation of observed movements, and might occur either (or both) in parallel with excitatory processes, or be triggered when the level of excitation reaches a certain threshold (these two possibilities are indicated by the dotted-lined arrows). If inhibition and excitation are competing processes working in parallel, then the resulting level of activity (whether the amplitude of MEPs increases or decreases from baseline) is dependent on whether there is more excitation than inhibition, or vice versa, in the pathways.

Aglioti et al., 2008; Jola et al., 2012; Jola and Grosbras, 2013; Lago and Fernández-del-Olmo, 2011), we speculate that this modulation reflected (a) mirroring of whole body movement (i.e., in Aglioti et al. (2008), Jola et al. (2012) and Jola and Grosbras (2013)), and (b) modulation related to the goal of the action (i.e., grasping, in Lago and Fernández-del-Olmo (2011)) that could have been achieved by movement of the effector in which MEPs were recorded (i.e., the hand).

3. Direction of modulation As can be seen in Supplementary Table, the majority of published findings from TMS studies of the mirror response indicate that observing an action increases activity in motor pathways. In other words, the MEPs produced in the muscle (s) are larger during action observation than in baseline conditions. It is important to note at this juncture that studies vary in their (a) method of data analysis and normalisation, and (b) choice of baseline, which means that the definition of ‘facilitation’ and ‘suppression’ varies between studies. Regarding (a), as noted in our discussion of Kaneko et al.'s (2007) study in Section 2, the way in which data are analysed affects the interpretation of the results. While some studies have drawn conclusions based on differences between conditions, muscles, or participant groups, others base their conclusions on how excitability is modulated relative to a common baseline. In addition, the methods of normalisation vary between studies, with the MEPs entered into the final analyses in some studies being normalised directly to a baseline condition (e.g., percentage change from baseline), and in others being

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normalised to the average of MEPs for that muscle, block, condition, or another subset of the data. While it was beyond the scope of this review to compare and group studies based on their methods of MEP normalisation, these differences should be borne in mind when interpreting the findings of studies. Regarding (b), many studies have used as baseline an image or video of a static hand or actor (e.g., Aglioti et al., 2008), while others use a low level baseline such as observation of a fixation cross (e.g., Bucchioni et al., 2013) or a blank screen (e.g., Ohno et al., 2011), or a condition in which participants have their eyes closed (e.g., Jola et al., 2012). In some reports, conditions are compared only to each other – and not to a common baseline – so, depending on the data provided, it is not always possible to infer whether corticospinal excitability during movement observation is significantly changed from baseline. For the purposes of this review, in cases where the necessary data are provided in the paper but the significance of the difference from baseline not reported, we have estimated whether the difference is significant at a po.05 uncorrected level (see Supplementary Table). Of the 85 studies reviewed here, there were 25 in which the change from baseline, or the data necessary to calculate it, was not reported. Of the 60 studies where change from baseline was reported or accessible, 52 showed facilitation in at least one condition, eight showed no modulation from baseline in one or more conditions, and three showed suppression (in at least one condition relative to baseline). Studies reporting facilitation will not be described in detail in this section, as a number of studies belonging to this category have been described already in this review. It is clear that the finding of MEP facilitation during action observation is considerably more prevalent in the literature than the (subsequently described) finding of suppression or no modulation. The fact that studies showing suppression or no modulation make up a relatively small proportion of the literature does not, however, mean that these studies do not have functional significance. Therefore, it is important to examine the studies whose results differ from the majority, to establish what determines the direction of modulation when an action is viewed. In two of the studies demonstrating suppression (Lago and Fernández-del-Olmo, 2011; Montagna et al., 2005), the suppression was in a muscle that was not an agonist in the movement. In the case of Montagna et al. (2005) study, suppression was found in the FDS (an extrinsic finger flexor muscle) during the observation of finger extension. This demonstrates a lowering of activity in the muscle during the phase of movement where it would serve as an antagonist, while an increase in excitability was found during the flexion phase. In Lago and Fernández-del-Olmo's (2011) study, suppression was found in the hand when the participant viewed an object being grasped using the foot, compared to when a static foot was viewed. Sartori et al.'s (2012a) study showed evidence of suppression in muscles that were involved in the observed action. In their study, participants viewed trials showing a precision grip of a small object, or a whole-hand grasp of a large object, while MEPs were recorded from the ADM and FDI. In previous studies it has been shown that the FDI is activated to a greater extent than the ADM during precision grip, while both muscles are involved to a similar extent in whole-hand grasp. On some trials, the small or large target object was flanked by a large or small (respectively) distractor object (which would warrant a whole hand or precision grasp, respectively). Sartori and colleagues' reported analyses showed that MEPs in the FDI were larger overall for precision grip than whole-hand grasp, while ADM MEPs were larger for the whole-hand grasp than precision grip. Furthermore, modulation in the ADM was influenced by distractor presence. Specifically, MEPs were larger when a precision grip flanked by a larger object was viewed, compared to when a precision grip alone was viewed, whereas MEPs were smaller when a whole-hand grasp was viewed flanked by a small object compared to when the whole-hand grasp alone was

observed. Thus, the incongruent distractor seemed to enhance or attenuate motor excitability depending on whether the muscle was involved in the grasp afforded by the flanking object. These findings indicate muscle-specific modulation. Interestingly, our own calculations based on the means provided by Sartori et al. (2012a) indicate that MEPs were suppressed in some conditions and facilitated in others relative to the low-level baseline (fixation cross). Specifically, MEPs in the ADM were facilitated during observation of the whole-hand grasp, regardless of whether there was a distractor present, and were not modulated from baseline when a precision grip was viewed alongside a large distractor (i.e., when the movement involved minimal ADM activation, but the distractor object primed whole-hand grasping). In contrast, MEPs in the FDI were suppressed in all conditions (precision and whole-hand grasp, regardless of distractor presence), as were MEPs in the ADM during the observation of a precision grip with no distractor present. Sartori and colleagues' data suggest, therefore, that when a whole-hand grasp is observed, MEPs in the ADM were facilitated while those in the FDI were suppressed. When the precision grip was viewed, MEPs in the ADM were suppressed or not modulated, while the FDI was, again, suppressed. To understand these results, it is useful to look at other studies where excitability has been found to be suppressed in one condition relative to another. In a study reported by Villiger et al. (2011), MEPs were recorded from a flexor muscle of the hand (the flexor pollicis brevis; FPB) as participants watched videos of a hand reaching for, grasping, and lifting an apple. MEP modulation was compared between conditions in which the grasp phase of the movement was either visible or occluded by a screen, and in which the apple was present or absent (a mimed grasp). MEPs were significantly smaller in the objectpresent condition than in the object-absent condition. Suppression was also found by Hardwick et al. (2012), who assessed MEPs in the FDI and ADM as participants watched actions (a grasp or finger abduction) with the intention to imitate or to answer a ‘true or false’ question about the action. MEPs in the FDI (the muscle most involved in the action) were suppressed in the observe-to-imitate condition relative to the other condition. As the MEPs were elicited before the participant was required to move, it is possible that the smaller MEPs in the imitation condition indicate the involvement of a suppression mechanism that inhibits explicit action imitation before it is required. Thus, there may have been an initial enhancement of motor pathway activity on observing the actions in both conditions, but if the activation was greater in the imitation condition due to the intention to imitate, this could have triggered neural suppression to prevent the movement being executed until the instruction to imitate was given. If a decrease in corticospinal excitability reflects suppression of motor activity to prevent overt movement in the observer (e.g., as suggested by Baldissera et al. (2001)), this could explain why Sartori et al. (2012a) found suppression in the muscles most active during the actions being observed (e.g., in the FDI during observation of a precision grip). It would also explain why MEPs in a muscle not involved in an action might be suppressed. If this is the case, however, we would expect more studies to have found suppression of MEPs during action observation, as action observation studies tend to require participants to refrain from movement. It is possible that some types of movement or observation environments are more likely than others to elicit action imitation, and that these conditions consequently make a suppression mechanism necessary. It has been shown that viewing an object alone activates brain motor areas associated with actions afforded by the object (e.g., Grèzes et al., 2003), and that viewing an object can enhance MEPs in the muscles that would be involved in acting on it (e.g., grasping; Buccino et al., 2009). Similarly, there are cells in the monkey PMC that fire in response to the presentation of objects, some firing only when a grasp is subsequently viewed (in dorsal PMC; Boussaoud and Wise, 1993), and others firing when the object alone is viewed (F5; Murata et al., 1997). The findings


of Villiger et al. (2011) also indicate that object presence modulates the effect of action observation on corticospinal excitability. It is possible that object affordances increase the drive to mimic observed actions, necessitating a stronger suppression of corticospinal activity to prevent execution. Such findings are compatible with suggestions of an initial increase in excitability that would, without inhibition, produce overt movement imitation. However, other studies that have investigated the effects of observing object-directed actions have found overall increases in corticospinal excitability (e.g., Fadiga et al., 1995; Gangitano et al., 2001), demonstrating that the direction of MEP modulation is not straightforwardly or exclusively determined by object presence. Another factor that might determine the direction of corticospinal modulation is the observer's intention or motivation to move or to imitate the action they are observing. In comparable studies where participants simply watch actions without moving, the instructions given by the experimenter could influence the effects of action observation. It is known that making a conscious effort to inhibit an action involves prefrontal and inferior frontal gyrus activation (e.g., Krams et al., 1998), so an explicit and repeated instruction to refrain from movement would be expected to cause greater inhibitory prefrontal activation than a single instruction at the start of an experiment. Villiger et al. (2011) repeatedly reminded participants to relax, both at the beginning of each block of trials and when muscle activity was detected during trials. It is possible that observing actions always increases the excitability of motor pathways initially but that, in situations where people are consciously refraining from movement, the activity is suppressed to a greater degree, and that this is detected as a decrease in MEP amplitude. Alternatively, a suppression mechanism might come into force when excitability in motor pathways reaches a particular threshold. Those studies that have found a decrease in MEP amplitude may have done so because, without a suppression of activity, the activation would have produced movement. A further possibility is that, rather than reflecting suppression of motor excitability, a decrease in MEP amplitude could simply indicate a lower level of excitation. While spTMS alone cannot distinguish these two possibilities, data obtained from ppTMS using the short intracortical inhibition paradigm have shown increased MEP amplitudes during action observation to be associated with a decrease in the activity of intracortical inhibitory pathways (Patuzzo et al., 2003; Strafella and Paus, 2000). To our knowledge, no action observation study that has found an overall decrease in MEP size has also measured intracortical inhibition, but it would be reasonable to hypothesise that decreased MEP amplitude would be associated with increased inhibition. Action execution data also support the notion of movement suppression. In a go/no-go task in which participants had to perform or withhold a motor response (wrist flexion), the size of MEPs evoked in the muscles involved in the movement was found to increase in the ‘go’ condition but decrease in the ‘no-go’ condition between 70 and 200 ms after the go or no-go instruction (Hoshiyama et al., 1997; Makin et al., 2009). These data show that preparing to execute an action but withholding the response causes a reduction in motor excitability. Similar brain activation occurs during the ‘preparation’ period preceding movement as during movement execution itself (e.g., Krams et al., 1998); thus, the decrease in MEP amplitudes seen when a movement is suppressed could reflect inhibition of the neural activity associated with movement preparation. Furthermore, increases in intracortical inhibition have been demonstrated both when individuals have to inhibit a prepared movement (e.g., Coxon et al., 2006), and just prior to voluntary muscle relaxation (Buccolieri et al., 2004). If modulation during action observation matches that seen during action execution, we would expect increased inhibition when a movement is suppressed.

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To conclude this section, although action observation appears to facilitate corticospinal excitability in the majority of cases, this effect is clearly not as robust as is often assumed. As stated above, 52 studies reported facilitation of MEPs in at least one condition; however, there were conditions within some of these studies where no modulation was present. For example, Enticott et al. (2012) found modulation during grasping observation, but only when the observed grasp was preceded by a negative image, not when it was preceded by a positive image. Kaneko et al. (2007) found MEP facilitation only when participants viewed a hand as if it was their own (kinaesthetic illusion condition) and not when it was viewed in the non-illusion condition (i.e., a condition comparable to the majority of other studies in the field). A lack of modulation was also shown in participants who scored highly (but within the nonclinical range) on a scale of autism traits (Puzzo et al., 2009). While individual explanations can be proposed for each difference in modulation (e.g., that the mirror response is influenced by emotional valence of a stimulus, Enticott et al., 2012), the broad finding here is that the mirror response is not a straightforward increase in motor excitability when an action is viewed; in some cases excitability decreases, and in others there is no modulation at all. We propose that modulation – specifically the second stage of modulation that occurs from around 200 ms from stimulus onset (see Fig. 1) – is influenced by a number of factors. Specifically, we suggest in our model that these factors contribute to the level of excitation that occurs, while the observer's intention (such as an intention to imitate, e.g., Hardwick et al., 2012) affects the level of inhibition that occurs at this stage. The effects of factors such as emotional valence, autism traits, and kinaesthetic illusion need to be explored further to determine whether they are mediated by differences in attention or motivation associated with the conditions or effects specific to the factor.

4. Timing of modulation To determine what is mediating the process that “transforms visual information into knowledge” (Rizzolatti and Craighero, 2004, p. 172), it is important that we know how much time elapses between action observation and the response of the mirror system. Specifically, there are two questions to be asked regarding timing of the mirror response: (1) How long after an action is observed does modulation of corticospinal excitability begin? and (2) How tightly coupled is the time-course of MEP modulation during action observation with the activation of muscles during action execution? Modulation that occurs immediately after an action is perceived or is closely coupled with the timing of muscle activation during execution, is more indicative of an automatic and rapid internal simulation of the action being observed than a delayed modulation would be. Unfortunately, despite the high temporal resolution of TMS compared to other cognitive neuroscience methods, many studies fail to report precise timing information (see ‘Type and time of stimulation’ column in Supplementary Table). Specifically, 34 of the 85 studies identified did not provide details of the times at which stimulation occurred. Of the 51 that did provide timing information, the majority (n¼35) assessed corticospinal excitability more than 1000 ms after observation of the action, while the remainder (n¼16) used time points earlier than 1000 ms. A number of studies elicited MEPs at a range of time points but did not compare these; in these cases we can only infer that modulation (when it occurred) had taken place by the final time point. 4.1. Mirror response latency The time at which we would expect to see a mirror response following action presentation depends on the degree to which an

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action is processed before it is ‘mirrored’. If the response is truly automatic, such that there are no processes occurring between perceiving the action and the modulation of activity in the motor system, the time that elapses before an action begins to be mirrored should simply be the time it takes to perceive the action. The earliest visually-induced modulations of the amplitude of MEPs elicited by TMS over the primary motor cortex occur at around 75 ms (Koch et al., 2006; Makin et al., 2009; O’Shea et al., 2007), so it is unlikely that mirror responses would be seen earlier than this. To our knowledge, the earliest reported modulation of TMSinduced MEPs during action observation occurs at 60–90 ms after movement onset (Lepage et al., 2010). In this study, MEPs were elicited at 10 randomly-selected time points from 0 to 270 ms, during the observation of simple finger abduction or a static hand. Of the five time bins examined (0–30, 60–90, 120–150, 180–210, 240–270 ms), the only time period at which there was significant modulation (an increase in MEP size) was 60–90 ms. In a second experiment, TMS was delivered 90 ms after movement onset to evoke MEPs in the FDI and ADM simultaneously (Lepage et al., 2010). The results of this experiment confirmed those of the first; MEPs elicited at 90 ms were significantly larger in the action observation than in a static control condition. As discussed in Section 2, the data also revealed that the modulation at this time point was not muscle-specific: MEPs in the ADM and FDI were modulated similarly, despite the FDI being significantly more active than the ADM during the performance of index finger abduction (Romani et al., 2005). As discussed, this finding could indicate that there is an early non-specific activation of the motor system when an action is viewed, followed by a later, musclespecific effect of action observation after further processing of the action. As discussed in Section 2, the notion of early non-specific modulation followed by later muscle-specific modulation is supported by the findings of Cavallo et al. (2013a), who found comparable responses in the ADM and FDI to the observation of little and index finger abduction when MEPs were elicited at 100 and 150 ms from movement onset. At 200, 250, and 300 ms, however, the two muscles were modulated differently. Specifically, the ‘preference’ for observation of index finger movement (shown by the ratio of MEP sizes during index finger observation and little finger observation) was significantly higher for the FDI than the ADM at all three of these later time points. Further evidence for separable early and late components to the mirror response comes from a recent study by Candidi et al. (2014), who elicited MEPs in the APB, FDI, and ADM while professional pianists observed a hand performing scales on a keyboard. On some trials, the actor made an error – pressing a key with the thumb (APB involvement) rather than the index finger (FDI involvement), and the participants had to detect whether or not an error was made on each trial. MEPs were larger overall on trials where an error was correctly detected, and this difference was only present in the APB, the muscle considered to control the movement of the thumb that characterised the error. Importantly, the facilitation of MEPs on error trials was present at 300 ms but not 100 ms, and there was a trend at 700 ms from error onset. Candidi and colleagues' findings suggest that the early modulation (facilitation relative to static control condition) was not influenced by whether the movement was ‘correct’ or not, whereas the later modulation at 300 ms was influenced by correctness of the movement. Lepage et al. (2010) highlighted two other studies that support a two-stage model. Roy et al. (2008) used TMS to elicit MEPs in the tongue as participants listened to different speech stimuli. These consisted of frequent words, rare words, and pseudo-words, some of which contain double consonants that require tongue movements to produce (e.g., the Italian double ‘l’), and others that involve very little or no tongue movement (e.g., double ‘b’). TMS was delivered at four time points relative to the start of the double consonant sound within each word: 0, 100, 200 and 300 ms. While there was no difference in

corticospinal excitability between any of the word types when TMS was delivered at 0 ms, at 100 ms MEPs were significantly different between the two types of pseudo-words. At 200 and 300 ms, there was no longer a difference between MEPs evoked as participants listened to the two types of pseudo-words, but differences were found between frequent and rare words, with rare words eliciting larger MEPs than frequent words. Seemingly, the early modulation reflected differentiation between the words based on phonological processing, whereas the later modulation reflected semantic or lexical processing. Another indication that there may be two levels of modulation comes from a study using magnetoencephalography (van Schie et al., 2008), which found increases in activity over sensorimotor areas (C3 and C4) from 83 ms during the observation of hand movements. On each trial, the correct movement was indicated by a visual cue, which specified first, whether the left or right hand should move, and second, whether the hand should move to the left or right side. The observed actor's response was then either correct or incorrect in respect of the two components specified by the cue. At 83 ms, modulation in the observer was influenced by the correctness of the hand that was moved, but not by the correctness of the side to which it moved. In a separate report on the same data set (Koelewijn et al., 2008), it was shown that there was a suppression of beta activity at the onset of the observed movement, followed by a rebound starting around 700 ms after movement onset. Activity in the alpha and beta frequency bands has been found to be inversely related to BOLD signal in sensorimotor cortex during movement execution and imagery (Yuan et al., 2010), so the aforementioned changes in beta are likely to reflect the increased followed by decreased motor activation during movement observation. Both the initial decrease in power and the rebound were modulated by action correctness. Like the results of Candidi et al. (2014), these findings indicate an early mirror response that is not influenced by the correctness of the movement, and a later modulation that differs according to the correctness of the movement (van Schie et al., 2008), reflecting a deeper level of processing than the early response. Interestingly, the so-called ‘beta rebound’ that occurs after both observed (e.g., Koelewijn et al., 2008) and performed (e.g., Jurkiewicz et al., 2006) movements, is thought to represent an active inhibition of motor activity (e.g., Pfurtscheller et al., 2005). These findings therefore support the previously-discussed idea of an early enhancement of motor activity (indicated by early suppression of beta synchronisation, or increased MEP amplitudes in TMS studies), followed by a later modulation that reflects inhibition of the earlier-induced activation (i.e., increased beta power, or decreased MEP amplitudes). 4.2. Temporal matching of corticospinal modulation during observation and EMG activity during action execution Borroni et al. (2005, 2008) showed that modulation of corticospinal excitability during action observation is tightly coupled to the changes in muscle activity seen during action execution. Specifically, these studies showed that the modulation of the H-reflex during the observation of cyclic wrist flexion and extension could be fitted to the same sine wave as that reflecting the cycle of movement (i.e., peak of wave representing maximum flexion). Note that the results of a control condition in which participants observed an oscillating platform following the same movement cycle of the hand produced no discernible pattern of H-reflex modulation. Less straightforward findings were reported by Gangitano et al. (2001), who recorded MEPs at five time points during the observation of a reach-to-grasp movement, and a sixth time point at which no visual stimulus was present. The only significant difference in MEP size was between the sixth time point and all other conditions; no differences were found between the first time point (where the hand was stationary at the start position) and the point at which the fingers were at their maximum grasp aperture. While these direct comparisons of time points did not show


that modulation from baseline was ‘phase-specific’, MEP size and observed finger aperture were found to be positively correlated. Specifically, the greatest increase in MEP amplitude in the FDI was found at the point at which the viewed fingers were maximally open (i.e., the point at which the FDI would be expected to be most active). In a later study, Gangitano et al. (2004) examined again the changes in excitability in the same two muscles at six time points. In addition to the natural grasp movement, participants in this study viewed a reach-to-grasp movement in which the hand did not open until immediately prior to the grasp (i.e., the hand-opening movement was unexpected). While modulation during the natural grasp observation was similar to that found in their 2001 study, the only modulation of MEPs in the FDI during observation of the delayed opening movement was during observation of the static hand (at 400 ms). Thus, observing the grasping hand in the unnatural grasp condition did not modulate excitability relative to viewing the blank screen. Additionally, in contrast to the correlations found in the natural grasp condition, there was no relationship between finger aperture and MEP size when participants observed the delayed opening grasp movement. This suggests that the modulation found by Gangitano et al. (2004) was not an ‘immediate’ mirror response to visual stimuli, but was based on a prediction of the movement about to occur. If the mirror response was indeed an immediate and unmediated transformation of ‘visual information into knowledge’, then we would expect modulation in the FDI to be greatest at the point of maximum finger aperture (plus an additional 75–200 ms for visual processing), regardless of when in the action this point occurred. The fact that the modulation did not reflect what was being perceived at the time of stimulation suggests that the motor effects seen during action observation are based on the observer's prediction of the movement to occur (Gangitano et al., 2004). To summarise our conclusions for this section, the evidence for a close temporal matching of modulation during action observation and action execution is mixed. Studies such as Borroni et al.'s (2005, 2008) indicate a tight coupling of MEP modulation with increases in muscle activity during action execution, and this is supported by the correlations between MEP size and finger aperture found by Gangitano et al. (2001, 2004). However, the size of MEPs in the muscle thought to be more strongly involved in grasping did not differ significantly between time points in either of Gangitano and colleagues' studies. Furthermore, the modulation seen in the delayed hand-opening condition suggests that MEP modulation is dependent on the observer's prediction of the action, rather than what is directly observed. Indeed, it has been claimed numerous times that the mirror neuron system predicts actions or encodes goals. In many mirror neuron studies, experiments are designed so that participants see the same action or image repeatedly within the same block of trials. In these experiments participants are able, after viewing the first few trials, to predict what the action is going to be and approximately when it is going to happen. In such cases, the MEP modulation recorded cannot necessarily be attributed to what the participant is viewing at the time of stimulation or recording. If the mirror system is indeed predicting the outcome of a movement before it has been observed, we have to ask whether this contradicts the fundamental premise of the mechanism, as it was originally described.

5. A new model of the modulation of corticospinal excitability during action observation Based on our review of the literature, we propose a new model of how corticospinal excitability is modulated during action observation (Fig. 1). The model accounts for the majority of TMS findings regarding the effects of action observation, and proposes new hypotheses about the mirror response to be tested in future experiments. It is important to acknowledge again that our review

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focuses primarily on studies using TMS, as this is the only method that addresses the issue of muscle specificity, and is arguably best placed to establish the timing and direction of the mirror response. While methods such as fMRI and EEG are very useful for examining changes in the motor system at the cortical level, they do not provide fine-grained information about muscle specificity, and changes at the spinal and peripheral levels can only be inferred. We propose that, on viewing an action, there is an initial increase in corticospinal excitability, which is based on the most basic level of processing of the stimulus, and which is not muscle-specific. This is estimated to occur as soon as the visual motion associated with an action has been perceived, at around 80–100 ms (as reported by Koelewijn et al. (2008), Lepage et al. (2010), Roy et al. (2008); and van Schie et al. (2008)). This is followed by a later phase of modulation, occurring from around 200 ms (Cavallo et al. (2013a)). This second modulation is muscle-specific (e.g., Romani et al., 2005), and moderated by factors such as action correctness (Candidi et al., 2014), the observer's intention to move (e.g., Hardwick et al., 2012), and the affordances of the observed object or environment (e.g., Villiger et al., 2011). Evidence for an initial phase of muscle non-specific modulation and later muscle-specific modulation of cortical and motor activity is also found in macaque responses to sudden visual and tactile stimulation (Cooke and Graziano, 2003). The model predicts that inhibitory processes prevent the increase in corticospinal excitability from producing overt imitation of observed movements. We propose two alternative ways in which such inhibition might occur. First, inhibitory processes may work in parallel with excitatory processes during action observation, and the resulting modulation of MEP amplitude is dependent on whether there is more excitation than inhibition, or vice versa, in the motor pathways innervating the muscles. A fine balance of inhibition and excitation could explain inter-individual differences (e.g., Montagna et al., 2005; Ray et al., 2013) in modulation. The second – not mutually exclusive – possibility is that an inhibitory mechanism is triggered when the level of excitation reaches a certain threshold. We suggest that, in either scenario, an overall inhibition is likely to be observed in action observation conditions in which suppression of overt movement is required. For example, if participants have been instructed to imitate the movement when they see a cue shortly after observing the movement (e.g., Hardwick et al., 2012), or if some characteristic of the observed scene (such as the object being acted upon) makes the action more imitable, then a suppression mechanism may come into play to override the increased excitability in the motor pathways that could – without suppression – produce overt movement in the observer. The notion of inhibition arising to suppress overt movement is, we believe, supported by physiological and behavioural data arising from studies of action execution. As noted earlier in the review, increases in intracortical inhibition have been found to be associated with suppression of prepared movements (e.g., Coxon et al., 2006) and voluntary muscle relaxation (Buccolieri et al., 2004). In addition, Coxon et al. (2006) found that, in a go/no-go task, participants were less successful in suppressing their responses the closer the instruction to refrain from movement was to movement onset. Associated with this, the size of MEPs in the agonist muscle (involved in executing the required response) increased the closer to movement onset they were elicited, while MEPs in a muscle not involved in the action were relatively unchanged. These results indicate that corticospinal excitability associated with initiating the movement increased as participants prepared to move, even though they knew they might have to suppress the movement. Inhibition of this excitability seemingly occurred to suppress this movement once the participant's intention was determined.

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Behavioural displays of mimicry in naturalistic social settings can also be argued to support our hypothesis. Specifically, we suggest that the inadvertent and apparently unconscious mimicry of body movements that occurs during social interactions (e.g., Chartrand and Bargh, 1999) occurs due to increased corticospinal excitability that is not suppressed due to there being no intention to refrain from movement. This lack of intention is in contrast to typical action observation experimental set-ups in which participants are required to remain static, or in the context of goaldirected actions for which the participants' own intentions and motivations are likely to determine whether they perform the movement or not. Indeed, in studies such as Chartrand and Bargh's (1999) and Hogeveen and Obhi's (2012), where participants mimic the covert body movements of another person, the participants are free to move and have no explicit intentions to not mimic their companion. Importantly, in contrast to purposeful, goal-directed actions, it is likely that the movements mimicked in these studies were not even noticed by the observers at an explicit level, thus making it very unlikely that any intentions would be formed regarding their imitation. In support of this explanation of mimicry, Hogeveen and Obhi (2012) found that participants displaying a relatively high degree of mimicry (imitating the face-touching of a confederate) showed greater motor facilitation during an action observation task than participants who mimicked less. To our knowledge, Hogeveen and Obhi's (2012) study is the only one to examine the relationship between the degree of behavioural mimicry and effects of action observation on corticospinal excitability directly, which is surprising given the mirror system's postulated role in action imitation. This seems to be a gap in the literature which could be easily overcome by assessing both behavioural and physiological effects of action observation in the same participants. A robust association between these factors would provide convincing support for the link between social behaviour and the mirror response (implying that they at least rely on shared mechanisms), and would allow us to explore the proposed role of intention to imitate on mirroring and behaviour. Returning to our two-stage model, we postulate that the level of excitation (which in turn might influence the level of inhibition) is moderated by aspects of the observed action and the observer's state. Specifically, we predict that an observer's motivation towards the observed action (including their motivation to imitate the action, which could be based on object affordances, experimenter instructions, or low-level drives, discussed subsequently) modulates the extent of corticospinal facilitation at the second stage of modulation (from 200 ms; Fig. 1). This is based on the findings of Hardwick et al. (2012), as well as data implying motivational effects on the mirror system shown by fMRI and single-unit recording in humans and monkeys respectively. Specifically, as discussed previously, we propose that the relative suppression found by Hardwick et al. (2012) when participants were preparing to imitate reflected an inhibition of excitability that occurred to prevent overt movement replication. Thus, the intention to move caused a larger degree of excitation initially due to preparation of the movement (as was in fact found by Roosink and Zijdewind (2010)), but this was counteracted by inhibition of excitation to prevent the movement from being carried out immediately. Effects of more low-level motivation were suggested by Cheng et al. (2007), who found greater activation of brain regions considered ‘mirror’ (inferior frontal gyrus and posterior parietal cortex) during observation of grasping food and other objects when participants were hungry compared to when they were satiated. Since this study used fMRI, it is not known whether an effect of motivation on MEPs would have been found; however, this finding implies an initially larger response in the higher motivation condition. This apparent effect of motivation could be

considered consistent with the observation that a larger number of monkeys mirror neurons were found to respond specifically to the observation of eating than were found to be selective for other actions (Fogassi et al., 2005; see also Caggiano et al. (2012)). 5.1. Physiological basis of inhibition and excitation comprising the mirror response Mirror neurons were originally found in the ventral PMC and inferior parietal lobule of the macaque brain (Di Pellegrino et al., 1992; Fogassi et al., 2005), and neurons with ‘mirror’ properties have since been found in other brain regions such as the dorsal PMC and M1 (see Cook et al. (2014), for a review). While it was beyond the scope of the current article to discuss in detail the specific neural underpinnings of the mirror response, we will briefly consider possible mechanisms underlying the inhibition and excitation proposed in our model. Increases and decreases in MEP size as measured using spTMS imply modulation of excitability in the corticospinal tract, but this modulation may arise from excitatory or inhibitory inputs from a number of areas including the PMC. Studies using ppTMS in humans have shown that delivering a conditioning pulse to the PMC (ventral or dorsal) before M1 stimulation enhances the facilitation of MEPs during action observation (Catmur et al., 2011), suggesting that connections from these areas directly play a role in the mirror response. It is possible that corticospinal excitability is also influenced by input from other brain regions during action observation which enhance or attenuate its excitability further. Another possibility is that modulation during action observation arises from direct connections from the PMC to spinal motor neurons (i.e., without M1 involvement). However, it has been noted that the pyramidal tract neurons arising from PMC comprise a small proportion of the cortical input received by the corticospinal tract (Dum and Strick, 1991) compared to input received from M1. In addition, Cerri et al. (2003) showed that stimulation of area F5 (in ventral PMC) in the macaque monkey led to facilitation of M1 but no significant modulation of activity in the muscles. It is likely, then, that modulation of MEPs arises from modulation of the premotor to M1 to spinal motor neurons pathway; however, the effects of action observation could well be mediated by the inputs of other brain regions to these or other cortical or subcortical areas. Suppression of excitability could arise at the cortical level, or further down the corticospinal pathway. It has been shown that preparing an action but then suppressing movement is associated with increased activity in inhibitory circuits between the dorsal PMC and M1 (Koch et al., 2006), so this could also underlie movement suppression when an action is viewed. Additionally, work with monkeys has found neurons in the spinal cord that are excited during action execution but inhibited during observation, leading to the suggestion of a spinal mechanism underlying movement suppression (Kraskov et al., 2009; Stamos et al., 2010; Vigneswaran et al., 2013). Together, these findings suggest that inhibition occurs at both the intracortical and spinal levels during action observation, but further work would be needed to determine the degree to which, and under what circumstances, each occur.

6. Conclusions This review has discussed findings related to fundamental aspects of the mirror neuron response in humans, in an attempt to clarify exactly what we know about changes in corticospinal excitability when actions are observed. The model that we put forward allows the construction of clear hypotheses about the


timing and specificity of corticospinal modulation in action observation studies. Testing these hypotheses will provide a clearer picture of what happens in the motor system when an action is observed, which will in turn allow for more specific and evidencebased predictions of the role of the mirror response in behaviour. As discussed at the beginning of this paper, knowing exactly when and how excitability in the motor system is modulated by action observation is not absolutely necessary for showing an association between the mirror response and any given behaviour. However, it is not possible to infer the nature of such relationships without precise muscle specificity, direction, and timing information. To illustrate this, we can use the example of action understanding. Our review and model suggests that muscle specificity arises from around 200 ms post movement onset, with nonspecific facilitation occurring before this. If subsequent studies confirm our model, it suggests that action understanding (for example, understanding that a person intends to grasp a cup) does not occur immediately when an action is observed. Importantly, modulation occurring after 200 ms appears to be influenced by factors specific to the movement, such as its correctness (e.g., Candidi et al., 2014; Koelewijn et al., 2008) and the goal of the action (e.g., Enticott et al., 2010); thus, the second, muscle-specific phase of modulation appears to be based on a certain level of semantic processing of the action. This makes the theory of the mirror response underlying action understanding rather circular, as it suggests that some level of action understanding must precede mirroring (see Csibra, 2007, for a similar argument). In Enticott et al.s' study (2010), for example, facilitation of corticospinal excitability was found only when a hand was seen grasping a mug, and not when the same hand movement was viewed without the mug present. This difference between these conditions implies that modulation (at 1000–1500 ms) was based on the ‘understanding’ that the hand was going to grasp the mug. Testing our model – specifically, the time at which modulation becomes muscle-specific (i.e., specific to the action observed) – would help us to establish whether the action understanding account is feasible. The existing literature does not, in our view (see also Hickok (2009, 2014)), provide convincing evidence that the mirror response underlies action understanding; however, it also does not necessarily disprove the theory. Tracking the time-course of muscle specificity in the mirror response, along with manipulating contextual information provided within stimuli, could be used to examine the feasibility of the action understanding hypothesis more objectively. In this review, we classified any response of the motor system to action observation as part of a ‘mirror’ response. However, it is debatable whether effects that are not specific to the muscles involved in the observed action should be considered ‘mirror’. Cavallo et al. (2013a), for example, chose not to classify nonspecific effects as mirroring, suggesting that early non-specific facilitation likely reflects general effects of attention or arousal. Although responses of the motor system when an action is merely observed are interesting, we agree that they become less interesting – certainly in terms of behavioural implications – if the responses are not specific to the corticospinal pathways involved in the action being observed. A non-specific response to an action could not explain our ability to understand or imitate that action specifically, because the modulation could represent a range of different actions. For example, if viewing somebody grasping a mug modulates activity in all of the muscles of the hand, then this modulation is not specific to the mug-grasping movement, and therefore cannot be argued to underlie understanding or imitation of the action. Therefore, if action observation elicits both nonspecific and specific modulation, we need to know the timing of these components of the response in order to infer the role of the mirror response in action understanding or imitation.

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Finally, and importantly, this review has highlighted the need for more precise timing information to be reported in studies of the mirror neuron system. The Supplementary Table clearly shows that researchers often provide little or insufficient detail about (1) when TMS is administered, and (2) when changes in corticospinal excitability are evident. This means that information is often lost because researchers either do not set up their experiments in such a way that allows timing to be inferred, or do not deem this information to be important. In order to establish the timing of the mirror response, and to allow researchers to replicate experiments, the timing parameters of all TMS action observation experiments should be reported as standard, regardless of whether they are important to the authors' hypotheses. We believe that meeting these challenges will provide a more solid foundation on which to build and explore theories of the role and function of the mirror response in humans.

Acknowledgements This research was supported in part by a grant awarded to AJB from the European Research Council under the European Community's Seventh Framework Programme (FP7/2007–2013; ERC Grant agreement no. 241242). The authors would also like to thank two anonymous reviewers for their very insightful and useful suggestions, as well as Craig McAllister, Caroline Catmur, and Bhismadev Chakrabarti for their comments on earlier versions of the manuscript.

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Corticospinal excitability modulation during action observation.

Combined action observation and imagery facilitates corticospinal excitability.

Viewing Instructions Accompanying Action Observation Modulate Corticospinal Excitability.

The Effects of Mirror Feedback during Target Directed Movements on Ipsilateral Corticospinal Excitability.

Overt orienting of spatial attention and corticospinal excitability during action observation are unrelated.

Timing and specificity of early changes in motor excitability during movement observation.

The effects of action observation training and mirror therapy on gait and balance in stroke patients.

Preparation and execution of teeth clenching and foot muscle contraction influence on corticospinal hand-muscle excitability.

Phase-dependent modulation of corticospinal excitability during the observation of the initial phase of gait.

Corticospinal excitability modulation to hand muscles during the observation of appropriate versus inappropriate actions.

Age-Related Differences in Corticospinal Excitability during Observation and Motor Imagery of Balance Tasks.

Corticospinal mirror neurons.

Influence of age and posture on spinal and corticospinal excitability.

Response specificity in animal timing.

Muscle Relaxation of the Foot Reduces Corticospinal Excitability of Hand Muscles and Enhances Intracortical Inhibition.

Increasing corticospinal excitability in the antagonist muscle during muscle relaxation with a tracking task.

The effects of expectancy on corticospinal excitability: passively preparing to observe a movement.

Ongoing cumulative effects of single TMS pulses on corticospinal excitability: An intra- and inter-block investigation.

Reversal of Practice-related Effects on Corticospinal Excitability has no Immediate Effect on Behavioral Outcome.

Intensity dependent effects of transcranial direct current stimulation on corticospinal excitability in chronic spinal cord injury.

Effect of tactile stimulation on primary motor cortex excitability during action observation combined with motor imagery.

Symmetric corticospinal excitability and representation of vastus lateralis muscle in right-handed healthy subjects.

Time Course of Corticospinal Excitability and Intracortical Inhibition Just before Muscle Relaxation.

Motor imagery of voluntary muscle relaxation induces temporal reduction of corticospinal excitability.