Participants

The protocols of this study were approved by the University of Nottingham Ethics Committee. A total of 23 adults were recruited from September to November 2012 and gave written informed consent to take part in the study. Researchers had access to personally identifiable participant data during and after data collection. All participants were right-handed, and had normal or corrected-to-normal vision and no history of neurological disorders. Data from four participants who made excessive errors (>30% of trials) or completed only one scanning session were excluded. Nineteen participants were included in the analysis (10 female; median age = 20).

Experimental design

This is an event-related fMRI study with a 2 × 3 factorial design, with two task types (cognitive and spatial) and three demonstration types (Hand, Ghost, and Text). In the cognitive task, images of three different items appeared on the screen simultaneously. Participants were required to select images in an order based on the identity of the items, regardless of their positions. The spatial arrangement of the images varied randomly between the demonstration and execution phases. In order to correctly perform the task, participants had to construct an abstract “cognitive” representation of the identity of the images, rather than a motor or spatial representation. For example, the correct sequence of Fig 1A (Hand) and 1C is flowers → guitar → saw.

In the spatial task, three identical images appeared on the screen, and the spatial arrangement of the items was the critical factor. Participants were required to select the pictures in an order based on their spatial locations, regardless of the content of the pictures. The contents of the pictures changed between the demonstration and execution phases. The sequence shown in Fig 1B (Ghost) is selected in the order bottom-center → top-right → middle-left.

The two tasks were each performed with three different types of demonstration. In a Hand demonstration, a video of a human right hand in first-person perspective operating an fMRI-compatible joystick was presented at the centre of the screen (Fig 1A and 1B, Hand conditions). As the hand moved the joystick toward a particular location, a blue square frame appeared around the image to indicate that the corresponding image was selected. The image then disappeared, signaling that it had been correctly chosen. Thus, in this condition participants learned the sequence rules by observing and copying the actions of another human agent; this condition represents imitation learning.

In a Ghost demonstration, participants saw the same type of sequence of item selection, but the center of the screen was left blank (Fig 1A and 1B, Ghost conditions). As in the Hand demonstrations, a blue square appeared around each image in turn to indicate that the corresponding image was selected; the image then disappeared. Hence, participants learned the sequence rules vicariously, via physical cues (appearance of frame followed by disappearance of the image) rather than social cues (hand movements) provided by an agent, making this condition equivalent to non-social learning.

In a Text demonstration, the correct sequence was shown in English words which vanished one at a time with the same timing as the images disappearing in Hand and Ghost demonstrations (Fig 1A and 1B, Text conditions). Therefore, participants learned the sequence rule by reading text instructions. This condition was included to control for covert speech when learning the sequence order.

In each execution phase, participants performed the sequence rule from the preceding demonstration phase. In this phase, the visual stimuli were carefully controlled, as shown in Fig 1C (this execution phase could have been preceded by any type of demonstration in the cognitive task) and D (which could have been preceded by any type of demonstration in the spatial task). All responses were made using an MRI-compatible joystick; as the participant operated the joystick, a blue square frame appeared around the selected picture, and the corresponding picture vanished, indicating a correct response. This procedure paralleled those witnessed in the Hand and Ghost demonstrations. Custom-written Cogent scripts in Matlab were used to run the experiment.

Participants received feedback on their performance on each trial. If a picture was selected correctly, it disappeared. Therefore, selecting three pictures in a correct sequence led to the vanishing of all pictures. Two types of error could occur: incorrect selection and slow response. When an image was chosen in the incorrect order in a sequence, the word “Error” appeared on the screen until the beginning of the next trial. Also, if the correct response was not made within the allotted time (6 seconds), the word “Error” was shown briefly on the screen before the beginning of the next trial. Participants were instructed to respond as accurately and quickly as possible.

The timings were as follows. The demonstration phase duration was 4 seconds. An interstimulus interval of 1 to 4 seconds was imposed after the demonstration phase. The execution phase duration was 6 seconds. An intertrial interval of 3 or 7 seconds was imposed after the execution phase of one trial and before the demonstration phase of the next trial.

The present study focuses only on the execution phase, in which the visual and motor elements of the task were matched across all 6 conditions. Only the manner in which the participants learnt the sequence in the previous (demonstration) phase differed.

fMRI scanning

Participants became acquainted with the tasks by performing 48 practice trials in the fMRI scanner; during this time, T1 anatomical scans were recorded. Subsequently, two sessions of 48 trials were carried out. Participants took a break between the two sessions. Trials were drawn from the six cells of 2 × 3 factorial design, with 8 trials in each cell. Each session’s trial order was pseudorandomised by permutation of the complete set of possible trials.

fMRI scanning was carried out at the University of Nottingham, in a 3T Phillips scanner (Philips Medical Systems, Best, The Netherlands) with the following settings: double echo imaging, 37 slices per TR (thickness = 3 mm), TR = 2500 ms, TE = 20 and 45 ms, flip angle = 80°, matrix = 64 × 64, field of view = 19.2 cm. 308 images were collected for each session. Double echo imaging was adopted to enhance signal detection [27]. Images of the two sessions were combined by weighted summation according to the signal strength in each brain area [28].

Data analysis

Data and statistical analyses were conducted in the Statistical Parametric Mapping package (SPM12; The Wellcome Centre for Human Neuroimaging, London, UK) implemented in Matlab 2018a (Mathworks, Natick, MA). Preprocessing comprised realignment and unwarping, normalization, and smoothing. For each participant, images were realigned to the first image of session 1 to resolve head movement. The mean image of the 308 realigned images was adopted as an individual parameter for normalization into the standard EPI template. The normalized images were then smoothed with an 8 mm × 8 mm × 8 mm Gaussian filter to resolve neuroanatomical variability between subjects and to increase signal-to-noise ratio. After preprocessing, a registration check was conducted for excessive head movement and to ensure that the preprocessed brains were aligned correctly. Default SPM parameters were used, unless specified otherwise. All 19 participants passed the brain movement check. The registration check showed that the very top part of the brain of one participant had not been recorded; this did not impact on the MNS regions that we focus on, so data from all 19 participants were included.

For first-level analysis, a design matrix modeling demonstration and execution events according to their category was fitted for every participant. Demonstration and execution events have 4-second and 6-second durations, respectively. When errors occurred, both the demonstration and execution event of the trial were modelled in separate “demo-error” and “exe-error” categories. Therefore, there were 14 regressors for each session.

Statistical analysis

Second-level contrasts were computed for the demonstration and execution phases separately; this paper addresses only the execution phase. We focus on the contrast between execution following a Hand demonstration and execution following a Ghost demonstration, because this is most relevant to our hypothesis. Thus, we evaluate and report here the simple effects of Hand-cognitive > Ghost-cognitive and of Hand-spatial > Ghost-spatial. Other simple effects are listed in tables in S1 File. Statistical thresholds were first set to voxelwise p < 0.0001 uncorrected, and we report only clusters that survive a p < 0.05 FWE corrected. The cluster-forming extent threshold k was 10.

Clusters were assigned anatomical labels using the xjView toolbox (https://www.alivelearn.net/xjview).

Cognitive task

Executing a cognitive task sequence after observing a Hand demonstration in contrast to a Ghost demonstration resulted in greater activation in multiple cortical areas, including right calcarine cortex (Fig 2B), left middle occipital region (Fig 2A), bilateral precuneus, right parahippocampus, and left amygdala and hippocampus (Table 1). There was no activity in premotor or parietal cortex for this contrast, even at a lower threshold.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2.

Regions showing greater activity when executing a sequence following a Hand demonstration (red bars) compared to a Ghost demonstration (blue bars) in the cognitive task (panels A and B) and in the spatial task (C). Brackets above the bars indicate which contrasts are significantly different and the focus of the brain plot above. Error bars indicate 95% confidence intervals calculated using within-subjects standard error [29].

https://doi.org/10.1371/journal.pone.0291771.g002

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. Cognitive task, Hand > Ghost: Brain regions, FWE-corrected p values, sizes of clusters (k), T values, and MNI coordinates of clusters showing significant activation following different demonstration types.

https://doi.org/10.1371/journal.pone.0291771.t001

Spatial task

Executing a spatial task sequence after observing a Hand demonstration in contrast to a Ghost demonstration resulted in greater activation in right lingual gyrus (Fig 2C) and left cerebellum (Table 2). There was no activity in premotor or parietal cortex for this contrast, even at a lower threshold.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 2. Spatial task, Hand > Ghost: Brain regions, FWE-corrected p values, sizes of clusters (k), T values, and MNI coordinates of clusters showing significant activation following different demonstration types.

https://doi.org/10.1371/journal.pone.0291771.t002

Clusters with significant activation in other contrasts for the Hand, Ghost, and Text conditions during the execution phase are included in tables in the S1 File. Reaction times are described in a previous publication [20].

The lack of imitation-specific responses in mirror regions

We did not find imitation-specific responses in MNS regions. This contrasts with previous studies which have reported activation in MNS areas during imitation tasks [4, 5, 17, 30]. However, such immediate imitation studies used paradigms in which action observation and execution occurred simultaneously; this generally meant that visual input during imitation conditions was different from that during non-imitation conditions. Consequently, it is difficult to interpret the resulting brain activity as being due strictly to imitation itself.

A stronger test of these hypotheses is to examine delayed imitation, but this has rarely been done. While one previous study using a delayed imitation task reported activity in the MNS [2], it did not directly contrast the imitative versus non-imitative (freely chosen) actions. Another experiment testing delayed hand posture matching [19] did not find strong engagement of mirror systems. Consistent with this latter study, we also found no MNS activity during the execution portion of the task.

The lack of mirror system activation in the execution phase of our study cannot be attributed to a failure to engage these brain regions at all. In the demonstration phase of both the cognitive and spatial tasks with a visible hand, there was activity in MNS areas: in the cognitive task, the Hand > Text contrast revealed an activation difference in right inferior frontal gyrus (IFG). In the spatial task, the Hand > Text contrast showed a difference in left parietal cortex [20]. However, these differences occurred during the demonstration phase only. As such, MNS activity during imitation conditions appears to be restricted to action observation alone, not both action observation and execution, as the MNS hypothesis would predict.

As with some previous neuroimaging studies, the sample size was not large. Yet in both phases of the experiment (demonstration and execution), power was sufficient to detect the differential activation of brain areas in the three conditions. The interpretation of null findings in event-related fMRI is challenging; it could be that MNS areas were active during execution but to a lesser degree than occipital areas. However, even with a lower threshold, we did not find MNS areas to be active during action execution. We maintain that the finding of no differential activation in the MNS during imitative action execution cannot be attributed to insufficient power.

The engagement of occipitotemporal cortex by imitation

In contrast to the lack of activation of MNS regions, we found robust engagement of occipital and occipitotemporal cortex in our tasks. Specifically, when participants executed the cognitive task following a Hand action demonstration (imitation), calcarine sulcus and middle occipital regions were activated. When participants executed the spatial task following a Hand action demonstration, lingual gyrus was activated. These results are again in line with the finding from Makuuchi [19] showing that occipital regions can be engaged when performing an imitative action.

There is evidence from a variety of sources that regions within medial and lateral occipital cortex have an important role in action execution. A careful study from Astafiev and colleagues showed that the extrastriate body area (EBA), in addition to its role in passive observation of bodies and body parts, also responds to self-generated movement [31]. When contrasted under conditions of covert attention, saccades, and pointing actions, EBA showed significantly greater activation for pointing. This demonstrates movement-related modulation of the EBA which cannot be attributed to simple attentional or visual factors of the target stimulus. In addition, a multivoxel pattern analysis study examined the coding of action observation and action performance across the brain [32] and found evidence of cross-modal action representations in occipitotemporal cortex (as well as parietal cortex). These previous results [31, 32] are in agreement with results from a meta-analysis [3] showing that occipitotemporal cortex, along with the MNS, is involved in imitation. Our data corroborate these prior studies’ results. Specifically, our study shows that regions of occipitotemporal cortex have a specific role in the execution of imitative responses following a Hand demonstration—neural activity not seen for identical responses following a non-social Ghost demonstration.

If we seek to understand neural mechanisms of imitative learning, we need to include occipital regions too. EBA and the other parts of occipital cortex highlighted here respond to actions of self and other and to imitation. In particular, the finding that the only brain regions in the cortex which are engaged specifically for imitative actions and not for matched non-imitative actions are located in the medial and lateral occipital cortex suggests that the previous focus on the roles of inferior parietal lobule (IPL) and IFG in imitation has limited our understanding. Further research into the role of occipital cortex in imitation could determine the factors modulating its involvement.

Could the occipital activity in the execution phase be due to “spillover” from the demonstration phase due to the timing of the hemodynamic response function (HRF)? We think this is unlikely for the following reason. For the cognitive task, the occipital areas activated during the demonstration phase for the Hand > Ghost contrast are bilateral occipital gyrus and temporal gyrus (clusters centered on coordinates −54 −74 10 and 56 −70 0). The activated clusters from the execution phase are right calcarine on the medial brain surface (cluster centered on coordinates 14 −50 8) and left middle occipital gyrus (coordinates −32 −82 0), posterior to the cluster from the demonstration phase. For the spatial task, the corresponding areas for the demonstration phase are left occipital gyrus and temporal gyrus (coordinates −50 −70 6) and right occipital gyrus (26 −100 −6), while the execution phase cluster is located in the right lingual gyrus (10 −84 −6), also on the medial brain surface. If the areas activated during the execution phase were simply a result of completion of the HRF initiated due to the demonstration phase stimuli, we would expect them to be the same brain area, rather than different areas.

Grist and mills

Our findings have important implications for the Grist-Mills hypothesis. Heyes claims that social and non-social learning rely on the same basic learning mechanisms (the mill), but that input mechanisms—motivational, perceptual, and attentional processes—may be biased toward information (grist) from social sources [14, 15]. If there is essentially one “mill” that performs all types of learning (whether socially mediated or not), then there should not be detectable brain differences between executing an imitative and a non-imitative action, especially in studies like this one where imitation is delayed and the actions are precisely matched for motor complexity and visual feedback. In fact, our results show that despite the fact that the execution phases across conditions were matched, executing a sequence following a Hand demonstration produces different brain activation patterns from executing a sequence following a Ghost or Text demonstration. That is, social learning involves different neural correlates from non-social learning. This outcome is inconsistent with the core prediction of the Grist-Mills hypothesis, as it strongly indicates that different mechanisms (mills) are involved in learning from social (Hand) and non-social (Ghost) sources.

These activation differences could arise for several reasons. In a recent study of action observation and simultaneous action, participants were instructed to imitate deliberately or to perform a certain action which happened to be imitative [33]. Mid-occipital regions were specifically engaged for intentional imitation, and also showed more activity when participants’ actions matched those observed regardless of instructions. These findings were interpreted in terms of differences in motor planning and attention, with more attention paid when participants were instructed to imitate. However, the results from Astafiev and colleagues [31] suggest that attention alone does not account for different patterns of activity in lateral occipital cortex during motor tasks, and in our study the visual inputs were matched during task execution, which suggests that visual attention should also be matched.

Another possibility might draw on Meltzoff’s original active intermodal mapping (AIM) model, where an “equivalency detector” responds to the correspondence between, for example, the action observed on the face of an adult and the action performed by an infant [34]. A related cognitive framework assumes that social and asocial learning conditions produce distinct cognitive models (or simulations) against which performance during action execution is compared [35]. We might think of occipital and occipitotemporal regions implicated in imitation execution as areas which use the visual input of the demonstrator’s hand actions to generate a model based on what is known about the task (e.g., the content of the demonstration). The model makes predictions about the serial hand actions which the participant will perform, testing these predictions against the motor and proprioceptive feedback from the actual—ongoing—hand actions (for a related model of predictive coding in the MNS, see [36]). However, the differing patterns of activation between the imitation of item-specific sequences in the cognitive task and location-specific sequences in the spatial task suggest that there is no single brain area in which equivalencies are detected or model-based predictions are compared with feedback from either past or present experiences. Rather, multiple areas may perform distinct computations depending on task demands [12, 37].