Neuron
Article
Corticospinal Neurons in Macaque Ventral
Premotor Cortex with Mirror Properties:
A Potential Mechanism for Action Suppression?
Alexander Kraskov,1 Numa Dancause,1 Marsha M. Quallo,1 Samantha Shepherd,1 and Roger N. Lemon1,*
1Sobell Department of Motor Neuroscience and Movement Disorders, UCL Institute of Neurology, London WC1N 3BG, UK
*Correspondence: rlemon@ion.ucl.ac.uk
DOI 10.1016/j.neuron.2009.12.010
SUMMARY
The discovery of ‘‘mirror neurons’’ in area F5 of the
ventral premotor cortex has prompted many theories
as to their possible function. However, the identity of
mirror neurons remains unknown. Here, we investigated whether identified pyramidal tract neurons
(PTNs) in area F5 of two adult macaques exhibited
‘‘mirror-like’’ activity. About half of the 64 PTNs
tested showed significant modulation of their activity
while monkeys observed precision grip of an object
carried out by an experimenter, with somewhat fewer
showing modulation during precision grip without an
object or grasping concealed from the monkey.
Therefore, mirror-like activity can be transmitted
directly to the spinal cord via PTNs. A novel finding
is that many PTNs (17/64) showed complete suppression of discharge during action observation,
while firing actively when the monkey grasped food
rewards. We speculate that this suppression of
PTN discharge might be involved in the inhibition of
self-movement during action observation.
in the inferior parietal lobule (Fogassi et al., 2005; Rozzi et al.,
2008). Their presence may be even more widespread (Cisek
and Kalaska, 2004; Tkach et al., 2007), suggesting that the mirror
neuron system involves a network of areas.
A number of noninvasive studies have demonstrated a similar
mirror neuron system in the human brain (Grafton et al., 1996;
Rizzolatti et al., 1996b), which has been implicated in a variety
of cognitive functions, including understanding the nature and
meaning of actions, a wider role in imitation, speech, and
emotion (Fabbri-Destro and Rizzolatti, 2008; Iacoboni, 2009),
as well as in a spectrum of neurological disorders (Rizzolatti
et al., 2009).
However, despite the diversity of exciting potential roles for
such a system, we still know very little about the identity of the
mirror neurons themselves. Because extracellular recording in
monkey cortex is inherently biased toward large neurons, it is
likely that all mirror neurons are pyramidal neurons rather than
cortical interneurons. However, it is unknown whether mirror
neurons are involved in processing of afferent inputs or represent
outputs to other motor structures. Because PMv contributes to
the corticospinal tract (Dum and Strick, 1991), it is critical to
investigate whether pyramidal tract neurons (PTNs) within area
F5 show mirror-like activity.
INTRODUCTION
RESULTS
The first demonstration of mirror neurons in the macaque brain
(di Pellegrino et al., 1992; Gallese et al., 1996; Rizzolatti et al.,
1996a) showed that they became active both when the monkey
performed a given action and when the monkey observed
a similar action performed by the experimenter. Mirror neurons
were found to constitute a significant fraction (17%) of cells recorded in the rostral division of the ventral premotor cortex
(area F5). Further investigations have demonstrated that mirror
neurons can fire when a grasping action is performed just out
of sight of the monkey (Umiltà et al., 2001) and can differentiate
whether the action is carried out in the peripersonal or extrapersonal space of the monkey (Caggiano et al., 2009). Some F5
mirror neurons are particularly responsive to orofacial movements related to eating and communication (Ferrari et al.,
2003), while others respond to actions performed with tools
(Ferrari et al., 2005) or to sounds characteristic of particular
actions (Keysers et al., 2003; Kohler et al., 2002). Neurons with
‘‘mirror-like’’ activity are not confined to F5 but are also found
This study was carried out in two macaque monkeys trained to
perform both a precision grip task (Baker et al., 1999) and
a tool-use task that involved use of a rake to retrieve food
rewards (Ishibashi et al., 2000; Quallo et al., 2009; Experimental
Procedures). However, this paper reports only studies involving
grasping actions and action observation by these monkeys and
is entirely focused on fully analyzed recordings from 64 identified
PTNs selected for recording on the basis of their antidromic activation from the pyramidal tract (PT). Most of the PTNs (53) were
recorded in a monkey (M43) in which we also made simultaneous
EMG recordings from nine arm, hand, and digit muscles; another
11 PTNs were recorded in the second monkey (M41). All PTNs
were recorded in the rostral division (area F5; Matelli and Luppino, 1996) of the ventral premotor cortex (PMv) in the right hemisphere (Supplemental Data, Figure S1). Area F5 was characterized by brisk neuronal activity for grasp with either the contra- or
ipsilateral hand and by the presence of contralateral digit movements evoked by ICMS at thresholds >15 mA at recording sites.
922 Neuron 64, 922–930, December 24, 2009 ª2009 Elsevier Inc.
Neuron
Mirror PTNs
A
Stimulus
Artifact
−1
Figure 1. Stimulation of the Pyramidal Tract
B
a.u.
a.u.
C
0
1
2
3
ms
a.u.
4
E
L
VI
R
VI
D
a.u.
MLF
IO
ML
PYR
(A) Antidromic volley, recorded during surgery in M41,
from the cortical surface of M1 to stimulation of the ipsilateral PT (bipolar stimulation through two implanted electrodes 5 mm apart in PT, single biphasic shock, 200 mA).
Vertical arrow indicates beginning of antidromic response
(0.7 ms from PT stimulus onset at zero time). Average of
100 sweeps.
(B–D) Short-latency EMG response in contralateral thenar
muscles to PT stimulation (single bipolar shock 250 mA).
These were recorded in M43 awake at the beginning (C),
middle (D), and end (E) of the experimental period. Averages of 50 sweeps.
(E) Transverse section through brainstem of M41, showing
location of tip of posterior PT electrode on right (R) side
and surrounding gliosis. PYR, pyramidal tract; IO, inferior
olive; ML, medial lemniscus; MLF, medial longitudinal
fasciculus; VI, abducens nucleus; XII, hypoglossal nerve.
0
10
XII
1mm
F5 PTNs
Most PTNs (75%) were recorded 1–3 mm from electrode entry
and were probably located on the convexity of the ventral precentral gyrus. The database contained only those PTNs that
satisfied strict criteria for antidromic activation, including the
collision test (see Experimental Procedures), and whose spikes
could be reliably discriminated throughout a range of tests designed to evoke mirror-like activity (Experimental Procedures).
The implanted electrodes used to identify these PTNs were
confirmed to be located in the ipsilateral pyramidal tract (PT)
by a number of electrophysiological and histological tests
(Experimental Procedures, Figure 1).
‘‘Classic’’ Mirror Neurons
The main findings are illustrated by two F5 PTNs (Figure 2) with
completely different patterns of activity. The first PTN, shown
in Figures 2A–2J, showed bursts of activity when the monkey
reached out and used its contralateral hand to grasp a small
piece of food placed in front of it (see photo Figure 2A). Rasters
of discharge for a block of grasp trials (Figure 2B) and averaged
discharge (Figure 2C) were aligned to the cue for the monkey to
begin its reach-to-grasp movement, which was accompanied by
bursts of EMG activity in arm (deltoid), wrist (extensor carpi ulnaris, ECU), and digit (thenar) muscles (Figure 2D). The mirror-like
activity of this PTN is shown in Figures 2F–2I. The experimenter,
sitting directly in front of the monkey, started each trial with her
right hand motionless on a touch-sensitive pad. After a short
baseline period, she released her hand from the pad (magenta
asterisks in Figure 2G) and approached a piece of food previ-
ously placed in an ‘‘action observation area’’
that was in the monkey’s midline and just
beyond its reach. The monkey sat calmly
throughout this process and made no hand or
arm movements, as evidenced by the complete
20
30
40
50
absence of EMG activity in all recorded muscles
ms
during the period of action observation ( 750 to
+750 ms). Figure 2I shows all superimposed
trials of EMG recordings from nine arm, wrist,
and digit muscles; all recordings were essentially flat. As the experimenter’s hand neared the food, the PTN
steadily increased its firing rate, peaking after the experimenter’s
hand touched the food target, and grasped it in a precision grip.
This moment (indicated by the black line at time 0) was detected
by a sensor located within the observation area responding to
a small magnet attached to the tip of the experimenter’s finger
(Experimental Procedures). Note the reproducible pattern of
discharge in the raster plots for the block of action observation
trials. Note also that during action observation, the PTN fired
with maximum rates similar to those observed during the
monkey’s own grasping movement (Figure 2C). Antidromic stimulation and use of the collision test (Baker et al., 1999) confirmed
that the same PTN was recorded throughout the periods of
active grasp (Figure 2E) and action observation (Figure 2J).
Suppression Mirror Neurons
A quite different pattern was found for other PTNs, an example of
which is shown in Figures 2K–2T. In this case, although the PTN
again showed increased bursts of activity as the monkey
reached and grasped the food reward (Figures 2L and 2M), its
background discharge was completely suppressed during
action observation (Figures 2Q and 2R). Once again, this
suppression of activity was highly reproducible from trial to trial
(Figure 2Q). Another example of suppression-type PTNs is
shown in Figure S5.
Population Analysis
Of the 64 PTNs analyzed, 31 (49%) showed statistically significant modulation in their discharge in the 1.5 s period centered
Neuron 64, 922–930, December 24, 2009 ª2009 Elsevier Inc. 923
Neuron
Mirror PTNs
K
P
B
G
L
Q
40 C
H
20
0
−2
−1
0
1
2 s 3 −3
D
−2
−1
0
30 M
20
10
0
1 s 2 −2
−1
I
spikes/s
F
spikes/s
A
0
1
R
2 s 3 −3
N
−2
Biceps
Deltoid
Thenar
Biceps
Biceps
Deltoid
Thenar
ECU
EDC
ECR−L
EDC
−2
−1
E
200
0
−200
1ms
−400
ECU
ECU
ECR−L
ECR−L
FDP
FCU
FCR
0
1
before
2 s 3 −3
PTN E39
−2
J
0
1 s 2 −2
after
−1
O
200
1 s 2
−1
0
1 s 2
FDP
FCU
FCR
Thenar
−1
0
S
Deltoid
Thenar
−1
0
1
before
2 s 3 −3
PTN E45
−2
T
after
0
1ms
−200 1ms
1ms
Figure 2. Examples of Two Different Types of Response in F5 PTNs: One Activated during Action Observation and the Other Suppressed
(A and K) Photo of monkey grasping a piece of food in a precision grip; (F and P) photo of experimenter grasping a piece of food in precision grip; (B and L) raster
plots for two PTNs during self-grasp aligned to cue for onset of reach-to-grasp movement (indicated by black vertical lines); data from ten successive trials are
shown. Note that there were several bursts of activity in both PTNs, associated with initial grasp of reward and then release of food at the mouth, with each burst
associated with peaks of EMG activity in distal muscles, such as thenar (see traces in D and N). (G and Q) raster plots for two PTNs during ten trials of mirror testing
(precision grip with object) aligned to the moment of contact of the experimenter’s hand with the target object (indicated by black vertical lines). Light-blue circles
on each trial indicate beginning of baseline interval for each trial (experimenter’s hand motionless in full view of monkey), and magenta asterisks indicate beginning of experimenter’s movement toward the object. (C, H, M, and R) Average firing rates based on rasters above (spikes/s); (D and N) superimposed records of
EMG activity from three muscles (D: deltoid, ECU, and thenar; N: biceps, ECR-L, and thenar) during the same ten self-grasp trials as in (B) and (L), respectively;
(I and S) superimposed records of EMG activity from all nine muscles during the same ten mirror testing trials shown in (G) and (Q), respectively. Muscles are
divided into three groups of three records. EMG activity from the same trials are in same color; all EMGs are autoscaled to the maximum activity during self-grasp.
Note almost complete absence of EMG activity during mirror testing (I and S). (E, J, O, and T) Antidromic responses from PTNs in response to PT stimulation,
onset of PT stimulus is indicated by arrows, black curves are averages over tens of trials, antidromic spikes had constant latency throughout (facilitation cell
[E and J] 2.4 ms, suppression cell [O and T] 2.8 ms) before (E and O) and after (J and T) mirror testing; red curves show collisions when a spontaneous spike
appeared after the collision interval (indicated by dotted line): the antidromic spike was collided and absent from the record.
around the sensor signal (Experimental Procedures). Of these
PTNs, 14/64 (22%) were of the facilitation type (cf. Figures 2G
and 2H) while 17/64 (27%) showed suppression (cf. Figures 2Q
and 2R). Both types were recorded in both monkeys. For the
924 Neuron 64, 922–930, December 24, 2009 ª2009 Elsevier Inc.
53 PTNs recorded in monkey M43, we analyzed EMG and
excluded a total of 15 PTNs that were recorded in sessions
where there was significant modulation of EMG activity during
action observation (see Experimental Procedures and
Neuron
Mirror PTNs
A
3
a.u.
Population response: Action Observation
Facilitation PTNs
Suppression PTNs
non-mirror PTNs
2
1
0
−1
−2
−3
−2
B
3
−1 .5
−1
− 0.5
0
0.5
1
1.5
s2
Population response: Grasp with contralateral hand
a.u.
Facilitation PTNs
Suppression PTNs
non-mirror PTNs
2.5
2
1.5
1
0.5
0
−0. 5
−1
− 0.5
0
0.5
1
1.5
2
2.5
s 3
Figure 3. Population Summary of Activity in F5 PTNs
(A) Population average of PTN firing rates during action observation of precision grip with an object and (B) for active grasp of a food reward by the monkey
with its contralateral hand. Data plotted separately for mirror PTNs, which
showed facilitation (n = 11, red) and suppression effects (n = 14, blue) during
action observation, or no effect (non-mirror PTNs, n = 23, green). Dotted lines
indicate standard error of the mean. Alignment at time zero in (A) is to the touch
of magnetic sensor by experimenter and in (B) to the cue for the monkey to
begin its reach-to-grasp movement. Note that both facilitation and suppression types of PTN increased their discharge during active grasp, with the
increase beginning shortly after the cue and associated with the first burst of
EMG activity in distal muscles. Firing rate for each PTN is in arbitrary units
(a.u.): the rate was first normalized to unitary standard deviation over 2 to
2.5 s period, and then shifted to have average baseline (between 2 and
1) equal to zero. Data plotted are for PTNs recorded during sessions with
no significant EMG activity during action observation.
Supplemental Data). Of these remaining 48 PTNs, 11 (23%) were
of the facilitation type, while 14 (29%) showed suppression.
The population responses of both types of F5 mirror-like
PTNs during action observation, aligned to the sensor signal,
are presented in Figure 3A. Facilitation (red line) and suppression
(blue) types showed changes in firing rate around the time that
the experimenter began the movement ( 1 s; magenta asterisks
in Figures 2G and 2Q). The facilitation type showed the classical
pattern of mirror-like activity with facilitation during both action
observation (Figure 3A) and grasp (Figure 3B). The suppression
type showed a strong and protracted suppression of discharge
during action observation (Figure 3A), but, like the PTN in Figures
2L and 2M, were facilitated during active grasp (Figure 3B), with
a similar time course to that of the facilitation type of mirror PTN.
Indeed, 14/17 (83%) suppression-type PTNs, like most F5
neurons, showed significantly increased discharge when the
monkey actively grasped the food with the contralateral hand.
Of course, detection of a suppression response during action
observation required the PTN to have some ongoing, background discharge activity, and it is known that PTNs generally
have higher rates than other classes of cortical neuron (Baker
et al., 2001; Merchant et al., 2008). We found that the mean
background firing rate for PTNs showing suppression (14.8 ±
6.0 sp/s) was significantly higher than that of PTNs showing facilitation (6.2 ± 6.1 sp/s; p < 0.001, Mann-Whitney U test). The
average firing rate of F5 PTNs that did not exhibit any mirrorlike activity (Figure 3A, green line) showed only a slight deviation
from baseline throughout whole observation period. During
active grasp (Figure 3B, green), they were activated to a somewhat less marked degree than mirror PTNs.
‘‘Mirror-like’’ Modulation in Different Contexts
Most F5 PTNs exhibited mirror-like modulation of discharge
during observation of grasps carried out in different contexts
by the experimenter (Experimental Procedures). In the example
shown in Figures 4A–4C, the PTN’s ongoing discharge was
completely suppressed when the monkey observed the experimenter performing precision grip of food (Figure 4A), but also
when this same precision grip was carried out without the food
being present (Figure 4B). In other words, this PTN’s discharge
was also suppressed when an ‘‘intransitive’’ action, with no
apparent goal, was carried out by the experimenter. Discharge
suppression also occurred when the experimenter reached
into a small food bowl; her grasp within the bowl was concealed
from the monkey by the bowl’s opaque sides (Figure 4C).
73% of PTNs that were modulated during observation of
precision grip with an object were modulated during precision
grip without an object, and 64% were modulated during concealed grasp. The pie charts in Figures 4D–4F show the proportion of PTNs with mirror-like properties that were detected in
action observation sessions free of EMG contamination. Similar
proportions of PTNs showed significant modulation during
action observation of precision grip with an object (52%;
Figure 4D) and concealed grasp (45%; Figure 4F), but were
less common during precision grip without an object (29%;
Figure 4E). In all three contexts, PTNs showing suppression of
discharge were at least as common as those showing facilitation
of their discharge. Suppression of discharge was also seen in
some of these PTNs during other types of action, such as
advancing of the experimenter’s hand toward a reward and
covering the reward with the outstretched hand, but not grasping
it (Supplemental Data).
Antidromic Latencies of PTNs
Figure 5 shows that F5 PTNs (red bars) had relatively long antidromic latencies (median ADL 2.6 ms, indicated by red arrow)
and clearly belonged to a slowly conducting population
Neuron 64, 922–930, December 24, 2009 ª2009 Elsevier Inc. 925
Neuron
Mirror PTNs
PTN E45
precision grip with object
Populations
D
spikes/s
A
29%
Suppression
25
20
15
10
5
0
Facilitation
23%
precision grip without object
E
12%
n=48
48%
No
Effect
n=52
71%
spikes/s
B
25
20
15
10
5
0
17%
concealed grasp
F
25%
55%
spikes/s
C
25
20
15
10
5
0
-3
n=55
−2
−1
0
1
s 2
20%
Figure 4. PTN Activity during Observation of Grasp in Different Contexts
(A–C) Raster plots and average firing rates during action observation of grasps carried out under three different conditions. The photographs show the actions as
seen by the monkey. In (A), the experimenter performed a precision grip with an object present, in (B), a precision grip without an object being present, and in (C),
the grasp was concealed from the monkey by the opaque walls of the food bowl. Background discharge in this F5 PTN was suppressed in all three conditions.
Rasters and average firing rates aligned to the touch of magnetic sensor by experimenter’s finger tip. Other notations as in Figure 2.
(D–F) Percentage of PTNs recorded in EMG-free sessions (see Experimental Procedures), which exhibited significant ‘‘mirror-like’’ activity (p < 0.05, a MannWhitney U test) during the three grasp conditions. PTNs facilitated or suppressed during action observation in red and blue, respectively. PTNs with no mirror
activity shown in unfilled segment.
significantly slower than the fast-conducting M1 PTNs (open
columns; median ADL 0.9 ms, indicated by black arrow) encountered in the M1 hand area of the same monkeys (Figure S1).
Although the majority of M1 PTNs had ADLs close to 1 ms, we
also recorded some slow M1 PTNs. The distribution of F5
PTNs was not as skewed as in M1 and was more symmetrical
around the median. The subset of F5 PTNs that showed
mirror-like activity is shown with blue bars. Their ADLs were
not significantly different from the subset without mirror-like
activity.
DISCUSSION
We have demonstrated that within area F5 of ventral premotor
cortex mirror-like activity is present in output neurons that
project through the pyramidal tract and in all likelihood to the
spinal cord (Humphrey and Corrie, 1978; Lemon, 2008). Thus,
activity within the mirror-neuron system is communicated
directly to other regions of the motor network, including its
subcortical components, and will probably have functional
consequences for these components. The F5 PTNs we recorded were found in the same region of area F5 as in the original report by Gallese et al. (1996) and were mostly located on
the convexity of the gyrus, as predicted from retrograde labeling
of corticospinal neurons (He et al., 1993). A significant propor-
926 Neuron 64, 922–930, December 24, 2009 ª2009 Elsevier Inc.
tion of these PTNs were activated during observation of
grasping actions in different contexts, including intransitive
precision grip and grasp that was concealed from the monkey.
We do not consider that the mirror-like activity in PTNs that
were facilitated during action observation resulted from covert
movement by the monkey. Our data show unequivocally that
mirror-like activity can be evoked without any concomitant
EMG activity (Figure 2).
A novel finding is the description of F5 PTNs with a steady
resting discharge that was completely suppressed during the
mirror tests. To date, all published records of mirror neurons
are characterized by a very low or absent background discharge
that changes to a brisk increase in firing rate during both action
observation and the monkey’s own grasp (Caggiano et al., 2009;
Fogassi et al., 2005; Gallese et al., 1996; Rozzi et al., 2008).
There have been no published examples of firing suppression
in F5 (but see Supplementary Materials of Caggiano et al.,
2009). The suppression type of PTN was as prevalent in our
sample of F5 PTNs as the facilitation type (56% versus 44%).
Preliminary comparison with recordings of other neurons in the
same monkeys and sessions demonstrates that suppression
of activity may be significantly more common among PTNs in
F5 compared with unidentified neurons in the same area. Interestingly, these suppression PTNs, unlike classical mirror
neurons, provide an unambiguous signal as to whether the
Neuron
Mirror PTNs
50
M1
F5
F5 mirror
40
30
20
10
0
0
2
4
6
8
ms
Figure 5. Antidromic Latencies of F5 and M1 PTNs
Distribution of antidromic latencies (ADLs) for PTNs in F5 (n = 64 in red; mean
ADL 2.8 ± 1.5 ms) and in M1 (n = 100, open columns; mean ADL 1.5 ± 1.2 ms)
and recorded in M41 and M43. The subset of F5 PTNs that showed ‘‘mirrorlike’’ activity is shown in blue. The red vertical arrow indicates median value
of ADLs for F5 PTNs (2.6 ms) and the black arrow for M1 PTNs (0.9 ms). These
two populations were significantly different (p < 1e–6, Mann-Whitney U test).
We did not find any significant difference between ADLs of mirror and nonmirror PTNs.
current state involves action observation (suppressed
discharge) or active grasp (elevated discharge; see Figure 3).
The two classes of facilitation and suppression mirror neurons
may operate independently or be part of the same functional
circuit.
The presence of two different patterns of mirror-like activity in
PTNs provides a possible explanation for contrasting changes in
the excitability of the human motor system during action observation. Thus, facilitation mirror PTNs might explain the ‘‘motor
resonance’’ phenomenon in which the excitability of hand motoneurons in an observer mirrors or resonates with that of the actor
(Cattaneo et al., 2009; Fadiga et al., 1995; Montagna et al., 2005;
Strafella and Paus, 2000). Other studies have demonstrated
dynamic and specific mechanisms that may serve to prevent
such resonance being elaborated into overt movements (Baldissera et al., 2001). We speculate that such changes could result
from a disfacilitation of motoneurons caused by suppression of
activity in PTNs during action observation.
It should be stressed that although we suggest that these
PTNs might be concerned with suppression of self-movement
during action observation, we think of this as a suppression
mechanism that might allow the withholding of a number of
hand actions, rather than suppression of one particular act, for
example the suppression of imitation. Indeed, many F5 PTNs
were suppressed during observation of grasping actions in
a variety of contexts (Figure 4). A role for macaque PTNs in imitation, or its suppression, seems unlikely, since it is known that
adult macaque monkeys have a limited capacity for motor imitation (Ferrari et al., 2006; Visalberghi and Fragaszy, 2002).
In macaques, F5 contributes a relatively small proportion of
the total corticospinal output from the frontal lobe, around 4%
(Dum and Strick, 1991). Most of the F5 PTNs we sampled had
rather slowly conducting axons and are obviously different
from the large, fast PTNs that characterize the M1 output
(Figure 5). Only a minority of F5 PTNs terminate in the lower
cervical motor nuclei innervating digit muscles (He et al.,
1993). In terms of facilitatory actions, it has not yet been shown
that F5 makes any contribution to direct cortico-motoneuronal
excitatory projection (Lemon, 2008; Rathelot and Strick, 2006,
2009) and F5 PTNs exert only weak facilitatory effects on digit
muscles (Boudrias et al., 2009; Cerri et al., 2003). Motor effects
elicited by ICMS, similar to those found at F5 recording sites in
this study, may be largely mediated through cortico-cortical
interactions with M1 (Schmidlin et al., 2008; Shimazu et al.,
2004).
In terms of inhibitory actions, F5 PTNs could exert a net inhibitory effect via segmental inhibitory circuits (Cheney et al., 1985;
Prut and Fetz, 1999) or feedforward inhibition of propriospinal
systems (Isa et al., 2007) at more rostral cervical levels, which
is their principal target (He et al., 1993). They may also be part
of the cortico-cortical circuit that exerts suppression of M1
outputs as part of the active shaping of the hand for grasp
(Prabhu et al., 2009).
We consider it unlikely that the mirror-like activity in PTNs that
were facilitated during action observation resulted from covert
movement on the part of the monkey. This concern was already
recognized by Gallese et al. (1996) but was refuted based on
quiescent EMG activity in hand and mouth muscles during action
observation. However, in that study, EMGs were examined in
separate sessions from those in which the neuronal activity
was recorded. The simultaneous EMG evidence we collected
from M43 supported the conclusion that the activity of the
PTNs evoked during action observation was not due to covert
movement on the part of the monkey (Figure 2). This was further
supported by other lines of evidence that showed that only
a proportion PTNs that were active for the monkey’s own
grasping movement showed mirror-like activity and that it was
possible to record simultaneously from pairs of PTNs, both of
which discharged during active grasp but only one of which
showed mirror properties (Supplemental Data, Figure S4).
Finally, a striking finding of this study is the relatively high
proportion of F5 PTNs tested that showed mirror-like activity. It
is possible that training on the rake task (Ishibashi et al., 2000),
which involved a large amount of interaction with the experimenter and which might have influenced the monkeys’ cognitive
ability to associate observation with action (Iriki and Sakura,
2008), may have influenced this result.
The present findings give new insights into the potential functions of the mirror neuron system. In addition to the wide range of
functions suggested in the literature, including the understanding and imitation of actions carried out by others, and the
prediction of the outcome of such actions (Fabbri-Destro and
Rizzolatti, 2008; Rizzolatti et al., 2009), the mirror neuron system
might also be involved in the inhibition of unwanted self-movement during action observation. This last suggestion will need
to be confirmed by future studies that examine the responses
of F5 PTNs whose discharge is suppressed by action observation, during performance of other tasks requiring the withholding
of a motor response.
Neuron 64, 922–930, December 24, 2009 ª2009 Elsevier Inc. 927
Neuron
Mirror PTNs
EXPERIMENTAL PROCEDURES
All experimental procedures were approved by the Local Ethical Procedures
committee and carried out in accordance with the UK Animals (Scientific
Procedures) Act. Experiments were performed on two adult purpose-bred
Rhesus (M. mulatta) monkeys, (M41, male 8.0 kg and M43, female 5.5 kg).
Behavioral Tasks
Monkeys were trained to perform two active tasks, both with the left hand. The
first was a precision grip task in which the monkey was required to use its index
finger and thumb to squeeze two spring-loaded levers into an electronically
defined target (Baker et al., 1999). The levers were mounted in a manipulandum
positioned just in front of the monkey. Monkeys were trained to move each
lever independently into the target zone and hold it there for around 1 s;
each successful trial was rewarded by the experimenter passing a small
food reward to the monkey (fruit, nuts, or pulses). The second task involved
the monkey using a tool (Ishibashi et al., 2000). A flat white table (62 cm
deep by 68 cm wide) was placed in front of the monkey. The monkey used
a rake to retrieve a small food reward placed by the experimenter on the table
just beyond the monkey’s reach, i.e., in its ‘‘extrapersonal’’ space. Monkeys
were trained on these tasks for several months and reached a steady level
of performance before recording began (M41, 7 months; M43, 9 months).
They were not exposed to the mirror testing protocol until after recording
began.
Mirror Testing
The mirror test procedure comprised of five different tests, each with ten trials.
All five of these tests were carried out in both monkeys, in blocks of ten trials on
the table in front of the monkey. Each trial began with the experimenter’s right
hand resting on a central homepad (Figure S2: HPP). About 1.5 s later, a tone
sounded, which cued the experimenter to release the homepad (Figure S2:
HPR) and begin the specific test. The experimenter wore a glove on the right
hand, and this glove contained a small magnet at the tip of the finger. As the
experimenter approached the action observation area of the table, which
was in the monkey’s midline and just beyond its reach, a magnetic sensor
embedded in the table at the center of this area was activated and generated
a sensor pulse (Figure S2: SP). The timing of the homepad signal and of this
sensor pulse were recorded along with neuronal and EMG data. Tests were
repeated once every 4–5 s.
Mirror Test 1: Precision Grip with Object (see Figure 4A)
A small piece of food was placed above the central sensor. After releasing the
homepad, the experimenter slowly approached the food and grasped it in
a precision grip between the tips of the thumb and index finger. The monkey
was rewarded after every fifth trial.
Mirror Test 2: Precision Grip without Object (see Figure 4B)
After releasing the homepad, the experimenter slowly approached the central
sensor and pantomimed the movement of picking up the food in a precision
grip. The monkey was rewarded after every fifth trial.
Mirror Test 3: Concealed Grasp (see Figure 4C)
A bowl full of small pieces of fruit, which has been used throughout the training
of both monkeys, was positioned above the central sensor. After releasing the
homepad, the experimenter put her right hand into the bowl and retrieved
a piece of food, although the grasp itself was concealed from the monkey
because of the opaque walls of the bowl.
Test 4: Monkey Grasps with Contralateral Hand (see Figure 2A)
The experimenter took a small piece of food and placed her hand on the homepad. As she released the homepad she placed the food reward on the table to
the monkey’s left where it could easily reach and grasp with its left (contralateral) hand. Her release of the homepad cued the monkey’s reach-to-grasp
movement.
Test 5: Monkey Grasps with Ipsilateral Hand
As for test 4, but food placed to right of monkey and grasped with the ipsilateral
hand.
In tests 1–4, the experimenter gently restrained the monkey’s right hand
(ipsilateral to recording); in test 5, the experimenter held the monkey’s left
(contralateral) hand.
928 Neuron 64, 922–930, December 24, 2009 ª2009 Elsevier Inc.
Surgical Preparations and Electrophysiological Recordings
Under deep general anesthesia, a headpiece was surgically implanted to allow
head restraint (Lemon, 1984). During a second surgery (M43 only), we implanted chronic EMG patch electrodes in nine arm, hand, and digit muscles
(Brochier et al., 2004). During a final surgery, a single 20 3 10 mm recording
chamber was mounted so as to give access to the inferior limb of the arcuate
sulcus (for F5 hand area recordings) and to the middle third of the central
sulcus (for M1 hand area recordings) in the right hemisphere (Figure S1 shows
the location for M43). The extent of the craniotomy was based on central and
arcuate sulci locations obtained from previous structural MRI scan (Baker
et al., 1999). During surgery, sulcal locations were measured sterotaxically
through the dura and checked against MRI-derived measurements. During
the same surgery, a pair of fine (240 mm shank diameter) tungsten stimulating
electrodes were chronically implanted in the right medullary pyramid for
subsequent antidromic identification of pyramidal tract neurons (Olivier
et al., 2001). Electrodes had tip impedances of 20–30 kU. The stereotaxic location of the electrodes was anterior +2, lateral 0.5, and posterior 3, lateral 0.5
for anterior and posterior electrodes, respectively. The final depth of each PT
electrode was determined from the lowest threshold point (usually <20 mA) for
activation of the short-latency antidromic volley recorded through the dura
from the ipsilateral M1 (see Figure 1A). Subsequent testing of these electrodes
in the awake monkey confirmed that single PT shocks of 150–200 mA evoked
short-latency EMG responses in intrinsic hand muscles (see Figure 1B; Olivier
et al., 2001), which remained consistent throughout the recording period
(Figures 1B–1D). Finally, histological analysis in M41 confirmed the accurate
location of the electrode tip in the PT (see Figure 1E).
At the end of the experiment in M41, the monkey was killed by an overdose
of pentobarbitone (50 mg kg 1 i.p. Euthanal; Rhone Merieux) and perfused
through the heart. The cortex and brain stem were photographed and removed
for histological analysis. The implanted electrode tips were confirmed to be in
the PT (Figure 1E). M43 is still alive.
Cortical recordings were made using a Thomas Recording 7-channel drive.
The stereotaxic position of the tip of the drive was calculated by triangulation
using fiducial markers on the chamber lid. The drive carried 3–5 glass-insulated platinum electrodes (diameter, 80 mm) with an interelectrode spacing
of 300 mm. The drive was controlled through a networked system that allowed
several experimenters (usually two to three) to advance each electrode independently (Baker et al., 1999).
In M43, EMG was recorded with chronic subcutaneous electrodes from the
following nine muscles: anterior deltoid, biceps brachii, extensor and flexor
carpi ulnaris (ECU, FCU), extensor carpi radialis (ECR-L), flexor carpi radialis
(FCR), flexor digitorum profundus (FDP) extensor digitorum communis
(EDC), and thenar muscles. We used the technique described in Brochier
et al. (2004); correct placement of the EMG electrodes was confirmed by stimulation through the recording leads that were terminated in a multipin socket
on the animal’s back.
Following preamplification (320, Thomas Recording drive), the signals from
each electrode were further amplified (typically 3500 or 31000) and bandpass filtered (0.3–10 kHz). Data were acquired using A/D card (PCI-6071E,
National Instruments) at 25 kHz sampling rate and were recorded together
with EMG activity and experimental time events including home pad and
sensor signals. Wherever possible, recordings were made simultaneously
from several PTNs.
When a full set of data had been recorded for each group of PTNs sampled,
an isolated stimulator (Neurolog NL800 stimulus isolator, Digitimer, UK) was
used to deliver trains of repetitive intracortical microstimulation (ICMS) through
each recording electrode (13 pulses at 333 Hz, intensity typically up to 50–
60 mA, duty cycle 0.5 Hz), in order to characterize the output effects from the
recording site. After the electrodes had been withdrawn, the exposed dura
was treated for 5 min with the antimitotic compound 5-flurouracil, to counteract dural scarring (Spinks et al., 2003), before the chamber was thoroughly
washed with sterile saline and then sealed.
Identification of PTNs
During each recording session, we searched for PTNs by looking for
antidromic responses having an invariant latency (jitter < 0.1 ms) to each PT
shock. A search stimulus of 250–300 mA (biphasic pulse, each phase 0.2 ms)
Neuron
Mirror PTNs
was applied to the PT electrodes. Once a PTN had been identified and wellisolated, its antidromic latency (ADL) was measured from the beginning of
the stimulus artifact to the first negative peak in the action potential. Spontaneous, orthodromic spikes from the PTN were discriminated online using
a software-based double time-amplitude discriminator. Responses evoked
from the PT were then confirmed to be antidromic by using these spontaneous
spikes to collide the antidromic spikes (Baker et al., 1999; Lemon, 1984); see
Figures 2E, 2J, 2O, and 2T). Figure 5 shows the distribution of ADLs for the
PTNs recorded in F5 (red bars; blue bars are PTNs with mirror-like activity).
The distribution is dominated by PTNs with long ADLs (>2 ms) and is significantly different from that for M1 PTNs (open bars) recorded in the same
monkeys.
While we searched for PTNs, the monkey sat quietly and was not performing
a task or being tested for action observation. Thus, our sample is completely
unbiased in terms of the unit’s natural activity which was not tested until stable
recordings had been achieved.
SUPPLEMENTAL DATA
Supplemental Data include details of additional experimental procedures and
control experiments, with five figures, and can be found with this article online
at http://www.cell.com/neuron/supplemental/S0896-6273(09)01000-9.
ACKNOWLEDGMENTS
This work was supported by the Wellcome Trust, Centre for NC3Rs, and the
MRC. We thank Atsushi Iriki, Thomas Brochier, and Marco Davare for advice
and Jim Dick, Victor Baller, Ed Bye, David MacManus, Robin Richards, and
Chris Seers for their technical assistance.
Accepted: December 7, 2009
Published: December 23, 2009
REFERENCES
Analysis of PTNs
PTNs were detected and clustered using modified Wave_clus software (Quiroga et al., 2004). We used an extended set of features that included not only
wavelet coefficients but also first three principal components. Spike shapes
of PTNs obtained after clustering were checked against shapes of spikes
that spontaneously collided antidromic spikes during PT stimulation before
and after mirror testing (see e.g., Figures 2E, 2J, 2O, and 2T). During spike
detection, a very short (200 ms) ‘‘dead’’ time between two consecutive spike
events was used, which allowed detection of different units that fired close
together in time. For bursting units, clusters with minimum 1 ms interspike
interval were accepted; for other units, a minimum interspike interval of 2 ms
was set.
To test whether a cell showed any modulation during action observation, we
compared baseline activity (750 ms before home pad release) and activity
during mirror movement (750 ms before and after the sensor signal) using
a Mann-Whitney U test (p < 0.05).
Only cells that showed statistically significant (p < 0.05) modulation in firing
rate during mirror tests were retained to construct the population averages of
PTNs with mirror-like activity (Figure 3). The time course of each PTN’s
discharge was normalized to unit standard deviation and, for illustration
purposes (Figure 3), shifted to have zero mean value over baseline interval
(here 2 to 1 s).
EMG Analysis
To confirm that mirror activity in PTNs was not due to covert movements, in
monkey M43, we carefully checked data from every session for any signs of
modulation of EMG activity during action observation. We analyzed EMGs
from all nine muscles. EMGs were first low-pass filtered (second order Butterworth, 10 Hz cut off frequency) and the resulting signal was analyzed using the
same time intervals and statistical test as used for analysis of PTN activity. For
each trial, an average baseline EMG activity was estimated as a median over
750 ms interval before the end of the HP signal. It was then compared, using
a Mann-Whitney U test, with the median EMG amplitude estimated over
1.5 s centered on the sensor signal. If the difference was found to be significant, we removed outlier trials from the tails of the distribution. We eliminated
the most distant from the median trial and then repeated the statistical
comparison. This procedure was repeated until the difference was not significant (p > 0.05). Elimination of the trials was performed for each muscle independently. If the difference still remained significant for at least one muscle or
if the total number of remaining trials over all muscles was smaller than seven,
we discarded all PTNs from that recording session from further consideration
(see Figure S3 as an example of a discarded session). After completion of this
analysis in M43, 15 PTNs were excluded. Only six of them showed mirror-like
activity; three PTNs showed facilitation and three suppression. The F5 PTNs
illustrated in the main text (Figures 2 and 4) all showed significant modulation
during action observation that was free of any concomitant EMG activity
(cf. Figure 2I and 2S). The proportion of PTNs with mirror activity in the sample
taken from M43 before and after EMG control was comparable (51% before
and 54% after).
Baker, S.N., Philbin, N., Spinks, R., Pinches, E.M., Wolpert, D.M., MacManus,
D.G., Pauluis, Q., and Lemon, R.N. (1999). Multiple single unit recording in the
cortex of monkeys using independently moveable microelectrodes. J. Neurosci. Methods 94, 5–17.
Baker, S.N., Spinks, R., Jackson, A., and Lemon, R.N. (2001). Synchronization
in monkey motor cortex during a precision grip task. I. Task-dependent modulation in single-unit synchrony. J. Neurophysiol. 85, 869–885.
Baldissera, F., Cavallari, P., Craighero, L., and Fadiga, L. (2001). Modulation of
spinal excitability during observation of hand actions in humans. Eur. J. Neurosci. 13, 190–194.
Boudrias, M.H., McPherson, R.L., Frost, S.B., and Cheney, P.D. (2009). Output
properties and organization of the forelimb representation of motor areas on
the lateral aspect of the hemisphere in rhesus macaques. Cereb. Cortex, in
press. Published online June 26, 2009. 10.1093/cercor/bhp084.
Brochier, T., Spinks, R.L., Umilta, M.A., and Lemon, R.N. (2004). Patterns of
muscle activity underlying object-specific grasp by the macaque monkey. J.
Neurophysiol. 92, 1770–1782.
Caggiano, V., Fogassi, L., Rizzolatti, G., Thier, P., and Casile, A. (2009). Mirror
neurons differentially encode the peripersonal and extrapersonal space of
monkeys. Science 324, 403–406.
Cattaneo, L., Caruana, F., Jezzini, A., and Rizzolatti, G. (2009). Representation
of goal and movements without overt motor behavior in the human motor
cortex: a transcranial magnetic stimulation study. J. Neurosci. 29, 11134–
11138.
Cerri, G., Shimazu, H., Maier, M.A., and Lemon, R.N. (2003). Facilitation from
ventral premotor cortex of primary motor cortex outputs to macaque hand
muscles. J. Neurophysiol. 90, 832–842.
Cheney, P.D., Fetz, E.E., and Palmer, S.S. (1985). Patterns of facilitation and
suppression of antagonist forelimb muscles from motor cortex sites in the
awake monkey. J. Neurophysiol. 53, 805–820.
Cisek, P., and Kalaska, J.F. (2004). Neural correlates of mental rehearsal in
dorsal premotor cortex. Nature 431, 993–996.
di Pellegrino, G., Fadiga, L., Fogassi, L., Gallese, V., and Rizzolatti, G. (1992).
Understanding motor events: a neurophysiological study. Exp. Brain Res. 91,
176–180.
Dum, R.P., and Strick, P.L. (1991). The origin of corticospinal projections from
the premotor areas in the frontal lobe. J. Neurosci. 11, 667–689.
Fabbri-Destro, M., and Rizzolatti, G. (2008). Mirror neurons and mirror systems
in monkeys and humans. Physiology (Bethesda) 23, 171–179.
Fadiga, L., Fogassi, L., Pavesi, G., and Rizzolatti, G. (1995). Motor facilitation
during action observation: a magnetic stimulation study. J. Neurophysiol. 73,
2608–2611.
Ferrari, P.F., Gallese, V., Rizzolatti, G., and Fogassi, L. (2003). Mirror neurons
responding to the observation of ingestive and communicative mouth actions
in the monkey ventral premotor cortex. Eur. J. Neurosci. 17, 1703–1714.
Neuron 64, 922–930, December 24, 2009 ª2009 Elsevier Inc. 929
Neuron
Mirror PTNs
Ferrari, P.F., Rozzi, S., and Fogassi, L. (2005). Mirror neurons responding to
observation of actions made with tools in monkey ventral premotor cortex.
J. Cogn. Neurosci. 17, 212–226.
Ferrari, P.F., Visalberghi, E., Paukner, A., Fogassi, L., Ruggiero, A., and Suomi,
S.J. (2006). Neonatal imitation in rhesus macaques. PLoS Biol. 4, e302.
Fogassi, L., Ferrari, P.F., Gesierich, B., Rozzi, S., Chersi, F., and Rizzolatti, G.
(2005). Parietal lobe: from action organization to intention understanding.
Science 308, 662–667.
Gallese, V., Fadiga, L., Fogassi, L., and Rizzolatti, G. (1996). Action recognition
in the premotor cortex. Brain 119, 593–609.
Grafton, S.T., Arbib, M.A., Fadiga, L., and Rizzolatti, G. (1996). Localization of
grasp representations in humans by positron emission tomography. 2. Observation compared with imagination. Exp. Brain Res. 112, 103–111.
He, S.Q., Dum, R.P., and Strick, P.L. (1993). Topographic organization of
corticospinal projections from the frontal lobe: motor areas on the lateral
surface of the hemisphere. J. Neurosci. 13, 952–980.
Humphrey, D.R., and Corrie, W.S. (1978). Properties of pyramidal tract neuron
system within a functionally defined subregion of primate motor cortex. J.
Neurophysiol. 41, 216–243.
Iacoboni, M. (2009). Imitation, empathy, and mirror neurons. Annu. Rev.
Psychol. 60, 653–670.
Iriki, A., and Sakura, O. (2008). The neuroscience of primate intellectual evolution: natural selection and passive and intentional niche construction. Philos.
Trans. R. Soc. Lond. B Biol. Sci. 363, 2229–2241.
Isa, T., Ohki, Y., Alstermark, B., Pettersson, L.G., and Sasaki, S. (2007). Direct
and indirect cortico-motoneuronal pathways and control of hand/arm movements. Physiology (Bethesda) 22, 145–152.
Ishibashi, H., Hihara, S., and Iriki, A. (2000). Acquisition and development of
monkey tool-use: behavioral and kinematic analyses. Can. J. Physiol. Pharmacol. 78, 958–966.
Keysers, C., Kohler, E., Umiltà, M.A., Nanetti, L., Fogassi, L., and Gallese, V.
(2003). Audiovisual mirror neurons and action recognition. Exp. Brain Res.
153, 628–636.
Kohler, E., Keysers, C., Umiltà, M.A., Fogassi, L., Gallese, V., and Rizzolatti, G.
(2002). Hearing sounds, understanding actions: action representation in mirror
neurons. Science 297, 846–848.
Lemon, R. (1984). Methods for Neuronal Recording in Conscious Animals
(London: John Wiley & Sons).
Lemon, R.N. (2008). Descending pathways in motor control. Annu. Rev.
Neurosci. 31, 195–218.
Matelli, M., and Luppino, G. (1996). Thalamic input to mesial and superior area
6 in the macaque monkey. J. Comp. Neurol. 372, 59–87.
Merchant, H., Naselaris, T., and Georgopoulos, A.P. (2008). Dynamic sculpting
of directional tuning in the primate motor cortex during three-dimensional
reaching. J. Neurosci. 28, 9164–9172.
Montagna, M., Cerri, G., Borroni, P., and Baldissera, F. (2005). Excitability
changes in human corticospinal projections to muscles moving hand and
fingers while viewing a reaching and grasping action. Eur. J. Neurosci. 22,
1513–1520.
Olivier, E., Baker, S.N., Nakajima, K., Brochier, T., and Lemon, R.N. (2001).
Investigation into non-monosynaptic corticospinal excitation of macaque
upper limb single motor units. J. Neurophysiol. 86, 1573–1586.
930 Neuron 64, 922–930, December 24, 2009 ª2009 Elsevier Inc.
Prabhu, G., Shimazu, H., Cerri, G., Brochier, T., Spinks, R.L., Maier, M.A., and
Lemon, R.N. (2009). Modulation of primary motor cortex outputs from ventral
premotor cortex during visually guided grasp in the macaque monkey. J. Physiol. 587, 1057–1069.
Prut, Y., and Fetz, E.E. (1999). Primate spinal interneurons show premovement instructed delay activity. Nature 401, 590–594.
Quallo, M.M., Price, C.J., Ueno, K., Asamizuya, T., Cheng, K., Lemon, R.N.,
and Iriki, A. (2009). Gray and white matter changes associated with tool-use
learning in macaque monkeys. Proc. Natl. Acad. Sci. USA 106, 18379–18384.
Quiroga, R.Q., Nadasdy, Z., and Ben-Shaul, Y. (2004). Unsupervised spike
detection and sorting with wavelets and superparamagnetic clustering. Neural
Comput. 16, 1661–1687.
Rathelot, J.A., and Strick, P.L. (2006). Muscle representation in the macaque
motor cortex: an anatomical perspective. Proc. Natl. Acad. Sci. USA 103,
8257–8262.
Rathelot, J.A., and Strick, P.L. (2009). Subdivisions of primary motor cortex
based on cortico-motoneuronal cells. Proc. Natl. Acad. Sci. USA 106,
918–923.
Rizzolatti, G., Fadiga, L., Gallese, V., and Fogassi, L. (1996a). Premotor cortex
and the recognition of motor actions. Brain Res. Cogn. Brain Res. 3, 131–141.
Rizzolatti, G., Fadiga, L., Matelli, M., Bettinardi, V., Paulesu, E., Perani, D., and
Fazio, F. (1996b). Localization of grasp representations in humans by PET: 1.
Observation versus execution. Exp. Brain Res. 111, 246–252.
Rizzolatti, G., Fabbri-Destro, M., and Cattaneo, L. (2009). Mirror neurons and
their clinical relevance. Nat. Clin. Pract. Neurol. 5, 24–34.
Rozzi, S., Ferrari, P.F., Bonini, L., Rizzolatti, G., and Fogassi, L. (2008).
Functional organization of inferior parietal lobule convexity in the macaque
monkey: electrophysiological characterization of motor, sensory and mirror
responses and their correlation with cytoarchitectonic areas. Eur. J. Neurosci.
28, 1569–1588.
Schmidlin, E., Brochier, T., Maier, M.A., Kirkwood, P.A., and Lemon, R.N.
(2008). Pronounced reduction of digit motor responses evoked from macaque
ventral premotor cortex after reversible inactivation of the primary motor
cortex hand area. J. Neurosci. 28, 5772–5783.
Shimazu, H., Maier, M.A., Cerri, G., Kirkwood, P.A., and Lemon, R.N. (2004).
Macaque ventral premotor cortex exerts powerful facilitation of motor cortex
outputs to upper limb motoneurons. J. Neurosci. 24, 1200–1211.
Spinks, R.L., Baker, S.N., Jackson, A., Khaw, P.T., and Lemon, R.N. (2003).
Problem of dural scarring in recording from awake, behaving monkeys: a solution using 5-fluorouracil. J. Neurophysiol. 90, 1324–1332.
Strafella, A.P., and Paus, T. (2000). Modulation of cortical excitability during
action observation: a transcranial magnetic stimulation study. Neuroreport
11, 2289–2292.
Tkach, D., Reimer, J., and Hatsopoulos, N.G. (2007). Congruent activity during
action and action observation in motor cortex. J. Neurosci. 27, 13241–13250.
Umiltà, M.A., Kohler, E., Gallese, V., Fogassi, L., Fadiga, L., Keysers, C., and
Rizzolatti, G. (2001). I know what you are doing. a neurophysiological study.
Neuron 31, 155–165.
Visalberghi, E., and Fragaszy, D. (2002). ‘‘Do monkeys ape?’’ Ten years after.
In Imitation in Animals and Artefacts, K. Dautenhahn and C. Nehaniv, eds.
(Cambridge: MIT Press).
Neuron, Volume 64
Supplemental Data
Corticospinal Neurons in Macaque Ventral Premotor Cortex with
Mirror Properties: A Potential Mechanism for Action Suppression?
A. Kraskov, N. Dancause, M. Quallo, S. Shepherd, and R.N. Lemon
Control experiments: Mirror‐like responses are not due to covert actions on the part of the monkey Since most area F5 neurons, including PTNs, are movement‐related (Kakei et al., 2001; Kurata, 1993; Kurata and Hoshi, 2002; Rizzolatti et al., 1988; Umilta et al., 2007), it is possible that facilitation of their discharge during action observation was the result of small or covert movements made by the monkey. Overall this seems unlikely, since most of the PTNs sampled showed significant modulation in their activity during the monkey’s own grasping action (53/64; 83%), while only 14 (22%) showed significant excitatory mirror‐like activity. Nevertheless, in a few recording sessions, we noticed sporadic trials in which the experimenter’s actions did indeed prompt the monkey to make small movements and these were accompanied by bursts of EMG activity recorded from contralateral arm and hand muscles; these bursts were of a similar magnitude to those seen during active grasp by the monkey (see Fig. S3). Two further findings reinforced the conclusion that mirror‐like activity in F5 PTNs did not result from the monkey’s own movements. First, in monkey M43, although EMG recording allowed us to exclude possible confounds due to movements of the contralateral hand, we could not exclude PTN activity due to covert movements of the ipsilateral hand, or indeed other body parts, including the mouth, for which no EMG was available. We tested this in a number of ways. For example, a few PTNs were encountered which did not modulate their discharge at all when the monkey grasped with its ipsilateral hand (example PTN shown in Fig. S4A,B). Two out of three PTNs of this kind showed clear ‘mirror‐like’ facilitation of their discharge (see Fig. S4C) which therefore could not have resulted from covert movements of the ipsilateral hand. Second, we recorded a small number of F5 neurons whose activity was related to licking and chewing movements. Covert movements of this kind might be expected to occur during action observation (Cattaneo et al., 2007), when food was being grasped by the experimenter. However, none of these mouth‐related neurons showed significant modulation of discharge during action observation. Finally, because we made simultaneous, multiple‐electrode recordings of PTNs and other unidentified neurons we could check whether ‘mirror‐like’ modulation was due to other non‐specific changes in the monkey’s attention or anticipation. We could demonstrate that while the monkey watched a specific set of actions carried out by the experimenter, some PTNs showed ‘mirror‐like’ activity while others did not. An example is shown in Fig. S4D and E. One PTN showed pronounced mirror‐like facilitation (Fig. S4D), while another, recorded on a separate electrode only a few 100 microns away during the same set of trials, showed no modulation (Fig. S4E). PTNs respond to grasp not kinematics of the approach movement During tests for ‘mirror‐like’ activity, all the actions were carried out slowly and deliberately in front of the monkey. We investigated whether PTNs modulated their discharge while the monkey observed the experimenter approaching and beginning to grasp in the ‘action observation area’ (the ‘pre‐sensor’ period from ‐750 ms to sensor signal) or while the grasp was maintained for a short period, released and the hand withdrawn (the ‘post‐sensor’ period from sensor signal to + 750 ms). In the example shown in Fig. S5, in which the experimenter performed precision grip with an object, the PTN’s discharge was not modulated during the ‘pre‐sensor’ period whereas there was clear suppression of activity in the ‘post‐
sensor’ period. In seven sessions, we tested the effect of the experimenter approximately halving the speed of approach towards the food target. For 6 out of 8 mirror‐like PTNs the time course of its activity was essentially unchanged. One example of such behaviour is shown in Fig. S5. The suppression of PTN discharge during slow and fast approach trials (rank‐ordered for speed in Fig. S5B) were very similar when superimposed (Fig. S5C), suggesting that this PTN was mainly responding to the final grasping action of the experimenter, not to the dynamics of the approaching movement. Mirror like activity for non‐grasp condition In one monkey we performed additional mirror test in which, the experimenter’s hand approached the food reward and covered it with her hand, without grasping it. 25% of PTNs (3/12) that showed suppression of discharge during observation of precision grip also showed suppression during these ‘flat hand’ actions; the other 9 PTNs were unmodulated. Similarly, 33% (3/9) of facilitation PTNs recorded during observation of precision grip were also facilitated during observation of “flat hand” actions; the other 6 PTNs were unmodulated. References Cattaneo, L., Fabbri‐Destro, M., Boria, S., Pieraccini, C., Monti, A., Cossu, G., and Rizzolatti, G. (2007). Impairment of actions chains in autism and its possible role in intention understanding. Proc Natl Acad Sci U S A 104, 17825‐17830. Kakei, S., Hoffman, D.S., and Strick, P.L. (2001). Direction of action is represented in the ventral premotor cortex. Nature Neuroscience 4, 1020‐1025. Kurata, K. (1993). Premotor cortex of monkeys: set‐ and movement‐related activity reflecting amplitude and direction of wrist movements. J Neurophysiol 69, 187‐200. Kurata, K., and Hoshi, E. (2002). Movement‐related neuronal activity reflecting the transformation of coordinates in the ventral premotor cortex of monkeys. J Neurophysiol 88, 3118‐3132. Rizzolatti, G., Camarda, R., Fogassi, L., Gentilucci, M., Luppino, G., and Matelli, M. (1988). Functional organization of inferior area 6 in the macaque monkey. II. Area F5 and the control of distal movements. Exp Brain Res 71, 491‐507. Umilta, M.A., Brochier, T., Spinks, R.L., and Lemon, R.N. (2007). Simultaneous recording of macaque premotor and primary motor cortex neuronal populations reveals different functional contributions to visuomotor grasp. J Neurophysiol 98, 488‐501. Figure S1. Location of PTNs recorded in F5 and M1 for M43. Locations of electrode penetrations in area F5 (ventral premotor cortex) of monkey M43 in which PTNs with mirror activity were encountered either with or without digit rICMS responses are shown by filled and open triangles, respectively. Circles indicate location of PTNs without mirror activity with (filled)/without (open) digit rICMS responses; the latter were obtained with current intensities of 16‐60 µA. F5 PTNS were recorded in 25 separate recording sessions. Open squares indicate locations of PTNs in the hand area of M1 and recorded in the same monkey. ArcS: arcuate sulcus; CS: central sulcus. The PTNs in F5 of monkey M41 were recorded at a similar location in 4 recording sessions. AP, mm
24
23
ArcS
22
21
20
19
18
17
Mirror F5 PTNs, digit ICMS
Mirror F5PTNs, no digit ICMS
16
Non-mirror F5 PTNs, digit ICMS
15
Non-mirror F5 PTNs, no digit ICMS
M1 PTNs
14
13
12
11
10
Ant
9
Med
Lat
8
CS
Post
7
6
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
ML, mm
Figure S2. Time course of mirror tests. Each trial started with home pad press (HPP) by the experimenter, then after approximately 1 s the experimenter released her hand (HPR) and started movement, (shown schematically by red dashed line) towards the magnetic sensor (SP)in the action observation area. When her hand touched the sensor an event signal was generated. HPP
HPR
SP
Experimenter Hand Position
HPP
1s
Figure S3. EMG control. An example of a PTN recorded in an EMG “contaminated” session. All notations are as on Fig. 1. All trials show EMG activity from three example muscles (Biceps, ECR‐L, EDC) are shown. Note the consistent EMG activity during action observation (right panel) just before the sensor touch signal at time zero. Changes in PTN discharge rate overlap with this EMG activity, which was of comparable amplitude to that recorded during active grasp (left panel). 30
spikes/s
spikes/s
30
20
10
0
−2
−2
20
10
−1
−1
0
0
1
2
s
3
0
−3
−2
−1
Biceps
Biceps
ECR−L
ECR−L
EDC
EDC
1
2
s
3
−3
−2
−1
0
1
s
2
0
1
s
2
Figure S4. Control experiments Raster plots and average firing rates of a PTN which was strongly activated when the monkey grasped with the contralateral (A) but not with the ipsilateral hand (B). Alignment is to placement of food reward by the experimenter (black vertical line at time zero) C: The same PTN responded during observation of precision grip with object; rasters aligned to the experimenter touching the object. Note highly significant facilitatory mirror effect.This PTN had an ADL of 0.7 ms. D and E Two PTNs recorded simultaneously during observation of precision grip with object. One PTN (D) was strongly activated during this mirror test (ADL 1.8 ms) while the other (E) was not (ADL 1.2 ms). PTN R02
PTN R09
D
80
40
60
30
spikes/s
spikes/s
A
20
40
10
20
0
−2
−1
0
1
0
−3
2 s 3
−2
−1
0
1 s 2
PTN R10
B
E
80
24
20
40
spikes/s
spikes/s
60
16
12
20
8
0
−2
−1
0
1
2 s 3
C
0
−3
spikes/s
80
60
40
20
0
−3
4
−2
−1
0
1 s 2
−2
−1
0
1 s 2
Figure S5. Demonstration that mirror‐like activity in a PTN is not modified by different hand transport times. A: A raster plot and average firing rate for PTN which showed suppression during mirror testing, (precision grip plus object). B: Raster plot for the same PTN as in A, experimenter varied the hand transport time to the object; trials are ordered according to the duration of the transport time (fast trials first) of the mirror movement from the beginning (magenta asterisk) to the touch of the object (magnetic sensor signal= black vertical line). C: Average firing rates for raster shown in B, the black curve is for all 10 trials, red curve is only for fastest 5 trials, and blue curve PTN E48
A
spikes/s
20
pre post
10
0
B
20
all
fast
slow
spikes/s
C
10
0
−4
−3
−2
−1
0
1 s 2