Movement-related activity during goal-directed hand actions in the monkey ventrolateral prefrontal cortex

Transcription

1 European Journal of Neuroscience, Vol. 42, pp , 2015 doi: /ejn COGNITIVE NEUROSCIENCE Movement-related activity during goal-directed hand actions in the monkey ventrolateral prefrontal cortex Luciano Simone, Stefano Rozzi, Marco Bimbi and Leonardo Fogassi Department of Neuroscience, University of Parma, via Volturno 39, Parma, Italy Keywords: action goal, context, executive, parietal, premotor Edited by John Foxe Received 25 May 2015, revised 6 August 2015, accepted 7 August 2015 Abstract Grasping actions require the integration of two neural processes, one enabling the transformation of object properties into corresponding motor acts, and the other involved in planning and controlling action execution on the basis of contextual information. The first process relies on parieto-premotor circuits, whereas the second is considered to be a prefrontal function. Up to now, the prefrontal cortex has been mainly investigated with conditional visuomotor tasks requiring a learned association between cues and behavioural output. To clarify the functional role of the prefrontal cortex in grasping actions, we recorded the activity of ventrolateral prefrontal (VLPF) neurons while monkeys (Macaca mulatta) performed tasks requiring reaching grasping actions in different contextual conditions (in light and darkness, memory-guided, and in the absence of abstract learned rules). The results showed that the VLPF cortex contains neurons that are active during action execution (movement-related neurons). Some of them showed grip selectivity, and some also responded to object presentation. Most movement-related neurons discharged during action execution both with and without visual feedback, and this discharge typically did not change when the action was performed with object mnemonic information and in the absence of abstract rules. The findings of this study indicate that a population of VLPF neurons play a role in controlling goal-directed grasping actions in several contexts. This control is probably exerted within a wider network, involving parietal and premotor regions, where the role of VLPF movement-related neurons would be that of activating, on the basis of contextual information, the representation of the motor goal of the intended action (taking possession of an object) during action planning and execution. Introduction In primates, interaction with objects of the external world most often occurs through manual interactions. The execution of these actions requires the successful integration of the neural mechanism allowing the transformation of sensory information into the corresponding forelimb movements, with the neural processes being capable, on the basis of behavioural goals, of selecting and controlling the action appropriate for a given context. It is well known that the first mechanism relies on specific parieto-premotor circuits (Rizzolatti & Luppino, 2001), whereas, for the second function, an important role is assigned to the lateral prefrontal cortex (Hoshi et al., 2000; Tanji & Hoshi, 2001). Indeed, it has been shown that the prefrontal cortex takes part in different aspects of executive functions, i.e. the ability to select actions in relation to internal goals through temporal integration of sensory, motor and motivational signals (Tanji & Hoshi, 2008). Interestingly, recent connectional studies in the monkey showed that part of the ventrolateral prefrontal (VLPF) areas 46 and Correspondence: S. Rozzi, as above. stefano.rozzi@unipr.it L.S. and S.R. contributed equally to this work. 12 is strongly linked with the premotor and parietal areas involved in grasping actions (Borra et al., 2011; Gerbella et al., 2013). Previous studies on the monkey prefrontal cortex focused on several aspects of movement planning and the temporal organization of behaviour (Fuster 1997; Saito et al., 2005; Mushiake et al., 2006; Averbeck et al., 2006; Shima et al., 2007; Tanji & Hoshi, 2008; Yamagata et al., 2012; Funahashi & Andreau, 2013). Most of these studies concentrated on the neural activity preceding movement execution; a few of them described movement-related activity (Funahashi et al., 1993; Hoshi et al., 1998), but usually in tasks involving conditional sensorimotor behaviours, i.e. requiring the selection of a movement arbitrarily associated with a sensory signal on the basis of the learned rule. Thus, the role of the VLPF cortex in the guidance and execution of natural object-oriented behaviours remains largely unexplored. The first aim of the present study was to assess whether the VLPF cortex is involved in goal-directed hand grasping actions and whether its neural activity reflects visuomotor transformations and is influenced by visual feedback. Thus, we devised a basic reaching grasping paradigm in which monkeys had to simply observe, or observe and grasp, objects of different size and shape. The actions were performed in the presence or absence of visual control during action execution.

2 Movement-related neurons in prefrontal cortex 2883 The second aim was to verify whether the timing and intensity of VLPF neural activity is influenced when the action is based on object memory. Thus, we used a further task in which monkeys were required to perform the reaching grasping actions under mnemonic guidance. A final aim of this study was to verify whether the activity of VLPF neurons during grasping execution depends on the learned rules of the paradigm. Thus, we also studied the neuronal response by using a naturalistic paradigm. Materials and methods Two female rhesus monkeys (Macaca mulatta), M1 and M2, weighing ~4 kg, aged 4 years and 5 years, respectively, were used in the present experiment. The animal handling, and the surgical and experimental procedures, complied with European guidelines (2010/63/EU) and Italian laws in force on the care and use of laboratory animals, and were approved by the Veterinarian Animal Care and Use Committee of the University of Parma (Prot. 78/12 17/07/2012) and authorized by the Italian Health Ministry (D.M. 294/2012-C, 11/12/2012). Training and surgical procedures Before recordings were made, each monkey was habituated to sit comfortably in a primate chair, to interact with the experimenters, and to become familiar with the experimental setup. At the end of habituation sessions, a head fixation system (Crist Instruments) was implanted. Each monkey was then trained to perform the motor tasks described below, using the hand contralateral to the hemisphere to be recorded. At the end of training, a recording chamber ( mm; Alpha Omega, Nazareth, Israel) was implanted, centred on the VLPF cortex on the basis of a magnetic resonance imaging scan. All surgical procedures were carried out under general anaesthesia (intramuscular ketamine hydrochloride, 5 mg/kg; and intramuscular medetomidine hydrochloride, 0.1 mg/kg), followed by postsurgical antibiotic and pain medications (Fogassi et al., 1996; Rozzi et al., 2006). Experimental apparatus During training and recording sessions, the monkey s head was restrained by fixating the implanted head post to the head positioner of the monkey chair. A box containing three different objects was located in front of the monkey, 22 cm from the monkey s chest. A small door (7 9 7 cm) facing the monkey at eye height allowed, when opened, the objects to be presented one at a time. The objects were a small sphere (diameter, 1 cm), a large cube (side, 2 cm), and a horizontally oriented cylinder (length, 4 cm; diameter, 1.5 cm). The objects were chosen so as to elicit three different types of grip, i.e. precision grip, whole hand prehension, and finger prehension. Two laser spots (instructing cues) of different colours (green and red) could be projected onto the box door or onto the object, depending on the task phases. Eye movements were recorded with an infrared pupil/corneal reflection tracking system (Iscan, Cambridge, MA, USA) positioned above the box. The sampling rate was 120 Hz. Behavioural paradigms Visual and motor conditions VLPF neuronal activity was evaluated by employing a series of motor tasks in which the monkeys were required to execute grasping actions in different conditions. The monkeys first performed a Go/ NoGo task (Fig. 1), consisting of two basic conditions: a Motor condition and a Visual condition. Each trial of both conditions started with the monkey s hand at a starting position. Then, one of the instructing cues was turned on and projected onto the closed box door, starting the trial. In both conditions, the monkey was required to maintain fixation within a fixation window centred on the instructing cue. In the Motor condition, the monkey was required to fixate the green cue for a randomized time interval (from 500 ms to 1100 ms); the box door was then opened and, simultaneously, a light was switched on inside the box, allowing the monkey to see one of the objects. At this time, the green cue was still on, projecting onto the object. During object presentation, the monkey had to maintain fixation. After a randomized time interval of ms, the green cue was turned off (Go signal), instructing the monkey to reach for and grasp the object, and pull it for at least 600 ms. If the monkey performed correctly during a trial, a drop of liquid reward was delivered at the end of it. In the Visual condition, the monkey was instructed by a red cue. Then, the subsequent phases until the turning off of the cue were the same as in the Motor condition. When the red cue was turned off, the monkey was required to fixate the object for 600 ms. After correct completion of the trial, the monkey was rewarded as in the Motor condition. A trial was aborted when the monkey did not maintain fixation until the end of the trial, when it released the hand from the starting position during the Visual condition or, in the Motor condition, before the Go signal, or when it did not reach for and grasp the object with the correct prehension or did not hold it for the required time. Discarded trials were repeated at the end of the sequence in order to collect at least 30 correct trials for condition. The order of presentation of both objects and conditions was randomized. Dark Motor condition In order to evaluate whether the presence or absence of visual control during action execution could affect the neuronal discharge, we employed a further condition (Dark Motor condition; Fig. 1) in a second series of recording sessions. The sequence of events of this condition was identical to that of the Motor condition, except that, when the door opened, the light located inside the box was not turned on, informing the monkey of the type of condition. The presented object was clearly visible, because of the green cue, but, at the Go signal, the laser was turned off, so the monkey had to perform the reaching grasping action in complete darkness. The trials of this condition were carried out randomized, together with those of the previously described conditions, in order to collect at least 30 correct trials for each condition. Blocked Motor condition In order to evaluate the possible modulation of the neuronal discharge when the movement has to be performed under mnemonic guidance, and in the absence of a choice request, we introduced a further control condition (Blocked Motor condition; Fig. 1). Before the beginning of the task, one of the three objects was briefly presented to inform the monkey which object was going to be employed during the block. Then, the monkey had to perform a series of correct trials on the same object (average of 18 trials). In each trial, the monkey was required to fixate the green cue for a randomized period ( ms). Then, the cue was switched off and, simultaneously, the box door was opened. These two events

3 2884 L. Simone et al. A Fig. 1. (A) Temporal sequence of events in the various conditions of the behavioural paradigm. The arrow indicates the time line. The objects used in the tasks are depicted at the bottom. In the Visual and Motor conditions, at the beginning of each trial, one of two instructing cues were turned on, indicating to the monkey which condition to perform. The green cue instructed the monkey to perform a Grasping Pulling action with or without visual control of the hand object interaction (Motor condition and Dark Motor condition); the red cue instructed the monkey to simply fixate the presented object (Visual condition). In the Blocked Motor condition, there was only the green cue. In this condition, the monkey had to grasp the object under mnemonic guidance. (B) Lateral view of the monkey prefrontal cortex, showing a recent parcellation of the VLPF cortex. The map is based on both cytoarchitectonic and connectional studies (Gerbella et al., 2007, 2010, 2013; Borra et al., 2011). The superimposed rectangle indicate the approximate location of the recorded region; the dashed lines indicate the architectonic borders. IA, inferior limb of the arcuate sulcus; P, principal sulcus; SA, superior limb of the arcuate sulcus; 8/FEF, area 8-Frontal Eye Field; 8r, rostral area 8; 12l, lateral area 12; 12r, rostral area 12; 46vc, caudal area 46-ventral; 46vr, rostral area 46-ventral. of two conditions: grasping in light and grasping in dark. Each trial of both conditions started with the monkey s hand at a starting position. In the grasping in light condition, the experimenter presented a piece of food to the monkey, who freely reached for and grasped it, brought it to the mouth, and ate it. In the grasping in dark condition, the monkey was prevented from seeing the scene, and the food was introduced near the monkey in a fixed position, so that it could know the position of the food to be grasped. The order of presentation of the trials belonging to the two conditions was randomized. We collected at least 10 correct trials for each experimental condition. The same paradigm was employed for testing neuronal responses during grasping with the mouth. B constituted the Go signal that instructed the monkey to reach for and grasp the object and pull it for at least 600 ms. Note that in this task, in contrast to the Dark Motor condition, the monkey never saw the object at door opening. Naturalistic paradigm In order to better characterize the properties of the neurons studied in the Motor conditions, we employed a naturalistic paradigm consisting Recording techniques and task event acquisition Neuronal recordings were performed by means of a multi-electrode recording system (AlphaLab Pro; Alpha Omega) with glass-coated microelectrodes (impedance, 0.5 1MΩ) inserted through the intact dura. The microelectrodes were mounted on an electrode holder (Microdriving Terminal; Alpha Omega) allowing electrode displacement, controlled by dedicated software (EPS; Alpha Omega). The Microdriving Terminal holder was directly mounted on the recording chamber. Neuronal activity was filtered, amplified and monitored with a multichannel processor, and sorted with a multi-spike detector (MCP Plus 8 and ASD; Alpha Omega). Further spike sorting was performed with the Off-line Sorter (Plexon, Dallas TX, USA). All conditions of the behavioural and naturalistic paradigms were controlled by means of LABVIEW-based software. In particular, digital output signals determined the onset and offset of laser spots, opening of the door, and reward release. In the behavioural paradigm, contact-detecting electrical circuits provided the digital signals related to monkey hand contact and release of the starting point, beginning and end of object pulling. In the naturalistic paradigm, contact-detecting circuits provided the digital signals related to monkey hand contact and release of the starting point, and of the contact between the monkey s hand/mouth and the food. Analog signals provided information about eye position. Behavioural analysis In the behavioural paradigm, the following digital signals were recorded: (i) onset of the coloured instructing cue; (ii) opening of the door; (iii) offset of the instructing cue (Go/NoGo signal); (iv) monkey release of the starting position; (v) beginning of object pulling; and (vi) reward delivery.

4 Movement-related neurons in prefrontal cortex 2885 These signals were also used to evaluate the behavioural performance of the two monkeys in 1590 correct trials. More specifically, we calculated, in the Motor condition, the average times from the offset of the instructing cue to the release of the starting point position (reaction time) and from the release of the starting point position to the beginning of pulling (reaching grasping time). In order to confirm that different objects elicited different hand configurations, we carried out a kinematics analysis of the performance of M1 during execution of the Motor condition. One hundred and twenty trials, 40 for each object, were video-recorded with a digital video camera at 25 Hz, by the use of markers attached to the thumb and the index finger. The recorded images were sent to a PC for two-dimensional motion analysis of the maximal grip aperture, with home-made software. The values of grip aperture and reaching grasping time for each object were statistically evaluated with a one-way ANOVA (factor Object; P < 0.05). Neuronal analysis The digital signals were also employed to align neuronal activity and to create the response histograms and the data files for statistical analysis. In the Visual and Motor conditions, the neuronal activity was recorded for at least 60 successful trials (30 per condition, 10 for each object). For statistical analysis of the neuronal activity (expressed as mean firing rate in spikes/s), nine epochs were defined, on the basis of the digital signals, as follows: (i) Baseline from 750 ms to 250 ms before the onset of the instructing cue; (ii) Pre-cue 250 ms preceding the onset of the instructing cue; (iii) Cue 250 ms following the onset of the instructing cue; (iv) Prepresentation 500 ms preceding the opening of the box door; (v) Presentation 500 ms following door opening (object presentation); (vi) Set 250 ms before the offset of the instructing cue; (vii) Go/ NoGo, from the offset of the instructing cue to the release of the hand starting position (Motor condition) or 250 ms following the offset of the instructing cue (Visual condition); (viii) Grasping Pulling/Object fixation from 250 ms before to 250 ms after the onset of pulling (Motor condition) or a time period ranging from 250 ms to 500 ms after the offset of the instructing cue (Visual condition); and (ix) Reward 500 ms following reward delivery. Single-neuron responses were statistically evaluated by means of a992anova for repeated measures (factors: Epoch and Condition, P < 0.01) followed by Newman Keuls post hoc tests. As trials were randomized, changes in baseline activity across trials were not expected. Neurons were included in our dataset when there was a significant interaction effect (Condition 9 Epoch, P < 0.01), and the subsequent post hoc test showed a significant difference between one or more epochs and both the baseline and the corresponding epoch(s) of the other condition. The present study was focused on movement-related neurons, defined as those neurons in which the post hoc test revealed that the discharge during the Grasping Pulling epoch of the Motor condition was significantly higher than the baseline of the same condition and the Object fixation epoch of the Visual condition. The details of non-movement-related neurons will be the focus of a subsequent article. In order to test grip selectivity and to better define the timing of activation in the movement epoch, all movement-related neurons were further analysed with a ANOVA for repeated measures (factors: Object Cube, Sphere, and Cylinder; Epochs Grasping and Pulling; P < 0.01) in the Motor condition. The Grasping and Pulling epochs corresponded to 250 ms before and 250 ms after the holding onset, respectively. In order to identify the timing, within movement execution, of the strongest response of each movement-related neuron, we performed a further analysis. The mean firing rate, aligned with the release of the starting position or with the beginning of object pulling, was smoothed with a boxcar filter (sliding window, three bins; step, one bin; bin width, 20 ms). Then, the highest response was identified in three different movement epochs, each of a duration corresponding to half reaching grasping time (HRT), defined on the basis of the behavioural data (see above): (i) Reaching a period of HRT beginning with the release of the starting position; (ii) Actual grasping a period of HRT until the beginning of object pulling; and (iii) Pulling a period corresponding to HRT, starting at the beginning of object pulling. The HRT was used in order to compare three different epochs of the same duration. For each neuron, the epoch containing the highest peak response was assessed. Then, the number of movement-related neurons peaking in each epoch was calculated. All movement-related neurons showing a response during the Presentation epoch were further analysed with a one-way ANOVA (factor: Object; P < 0.01) to test for a possible preference for a specific object (sphere, cube, or cylinder) in the Visual and Motor conditions. A59 2 ANOVA for repeated measures (factors: Epoch and Condition; P < 0.01) was used to evaluate possible differences between the two Motor conditions (Dark Motor condition and Light Motor condition). In this case, the task epochs were the same as used in the ANOVA for repeated measures, except for the Pre-cue, Cue, Pre-presentation and Reward epochs, because these were identical in the two conditions. A792ANOVA for repeated measures (factors: Epoch and Task; P < 0.01) was performed to compare the Motor condition with the Blocked Motor condition. The following epochs were considered in the Motor condition: Baseline, Pre-cue, Cue, Set, Go, Grasping Pulling, and Reward. They were compared with the equivalent epochs of the Blocked Motor condition: Baseline, Pre-cue, Cue, Mnemonic-Set, Go, Grasping Pulling, and Reward. We named Mnemonic-Set the 250-ms epoch preceding the Go signal. Note that the Pre-presentation and Presentation epochs were absent in the Blocked Motor condition. The single-neuron responses of movement-related neurons recorded in the naturalistic paradigm were statistically evaluated with a ANOVA for repeated measures (factors: Epoch and Condition; P < 0.01), followed by Newman Keuls post hoc tests. The epochs, defined on the basis of behavioural events, were as follows: Baseline from 2000 ms to 1500 ms before grasping; Pre-contact 500 ms preceding the hand food contact; and Post-contact 500 ms following the hand food contact. The conditions were as follows: Grasping in light, and Grasping in dark. Population analyses In order to characterize the time course and the discharge rate of different neuronal populations with respect to the main task phases, the neuronal activity of each population was aligned with the main behavioural events (see Behavioural events and data analyses ). The population activity was computed as follows. The mean single-neuron activity over trials, in term of firing rate, was calculated for each 20-ms bin in the different conditions. The average baseline activity was then subtracted from the mean single-neuron

5 2886 L. Simone et al. activity over trials for each bin, and the highest remaining bin was taken to divide spike counts in all bins. In this analysis, 0 represents baseline activity, and 1 represents peak activity. After normalization, the net average discharge frequency of each neuron was used for subsequent statistical analyses. Each neuron contributed one entry to each dataset. The statistical designs adopted were the same as those employed for single-neuron activity (see above). All analyses were performed with a significance criterion of P < Anatomical reconstruction of the neuronal properties As the monkeys were still alive, the recording region was reconstructed on the basis of the location (in stereotaxic coordinates) of the penetrations on the magnetic resonance imaging scans of the brains of both investigated monkeys. Penetration depth, as reported by the protocol, was matched with its location with respect to the sulci. Results Behavioural data During the recording sessions, both monkeys reached a successful criterion of performance of >80% of correct trials (M1, 81%; M2, 82%). The timing of the behavioural performance was different between the two monkeys, and, in particular, the release of the starting position after the Go signal was faster in M1 than in M2 (mean reaction time: ms vs ms). The analysis of the kinematics data of M1 revealed that the maximal grip aperture shown during the grasping of the sphere ( cm) was statistically smaller than that shown during the grasping of the cube ( cm) and that shown during the grasping of the cylinder ( cm). Furthermore, the results of the one-way ANOVA revealed that the durations of the three reaching grasping movements were statistically different. The longest movement duration was found for the sphere (353 7 ms), followed by that for the cube (325 9 ms); the shortest duration was found for the action performed on the cylinder (295 9 ms). These data are in agreement with previous studies showing that different objects elicit differences in movement time and grip aperture (Roy et al., 2000). Properties of movement-related neurons We studied the neuronal activity from the VLPF cortices of the left hemispheres of two monkeys (Figs 1B and 2D) during the execution of the different conditions of the behavioural paradigms described in Materials and methods. The recorded sector covers a large cortical region, including most of the VLPF cortex, excluding its rostralmost sector, and slightly extends into the dorsal prefrontal cortex (M1, mm; M2, mm; Fig. 2). From this cortical sector, we recorded 2391 neurons. Of these, according to the inclusion criteria (see Materials and methods), 442 neurons (18.5%) showed differential discharge between the Motor and Visual conditions in at least one epoch, and were included in the dataset. The most represented class was that of movement-related neurons (124; 5.2% of the recorded neurons; 28% of those included in the dataset), defined as those neurons showing stronger discharge in the Motor condition during the Grasping Pulling Epoch than at baseline and in the Object fixation epoch of the Visual condition (interaction effect of Epoch 9 Condition, followed by Newman Keuls post hoc test, P < 0.01). This section and the following sections will describe in details the neuronal properties of the movement-related neurons; the properties of non-movement-related neurons will be dealt with in a separate article. Forty-one movement-related neurons (33%) discharged exclusively during the Grasping Pulling epoch. Figure 2(A) shows an example of a neuron that was active during the movement phase and did not respond during the other task phases. Eighty-three neurons (67%), in addition to showing movement-related discharge, also showed activity in the preceding task epochs. Of these, 44 showed significant differential discharge between the Motor and the Visual conditions in one (n = 27) or more (n = 17) epochs (Fig. 2C). Note that, in these epochs, the large majority showed a preference for the Motor condition, mainly starting from the Presentation epoch. Only a few neurons showed differential discharge during the epochs preceding object presentation (Cue and Pre-presentation epochs). Interestingly, 10 movement-related neurons had prolonged differential discharge, which included two or three epochs preceding the movement phase. An example of a neuron with a prolonged differential discharge starting from the Presentation epoch of the Motor condition is shown in Fig. 2(B). Table 1 summarizes the number of movement-related neurons that were active in the different epochs. Note that a neuron can have a significant discharge in more than one epoch, so the sum of the responses in the epochs preceding movement is higher than the total number of neurons. Figure 2(D) shows the location of movement-related neurons in the two monkeys. Note that these neurons are almost absent in the rostral-most and caudal-most parts of the recorded region. Grip preference In order to evaluate whether movement-related neurons had a grip preference and whether this preference could occur in a specific movement phase, a ANOVA for repeated measures (objects Sphere, Cube, and Cylinder; epochs Grasping and Pulling) was carried out. The results indicated that 34 movement-related neurons (27%) showed a preference for the type of grip used. In particular, 14 neurons showed a preference for the power grip (cube), 17 neurons showed a preference for the precision grip (sphere), and only three neurons showed a preference for finger prehension (cylinder). Figure 3(A) and (B) shows examples of two neurons, one discharging only when the monkey grasped the cube, and the other discharging equally well for all grip types. Note that at the single-neuron level a subpopulation of neurons showed a grip preference, but the whole population of movement-related neurons did not show a significant difference among the three different grips (see Population analyses ). Concerning the preferred epoch (Grasping vs. Pulling), 26 of the 124 movement-related neurons showed a significant difference between the two epochs (main effect or main effect and interaction, P < 0.01), eight of them discharging more strongly during the Grasping epoch, and 18 during the Pulling epoch. Peak of discharge in different movement phases In order to better characterize the relationship between the maximal neuronal discharge and the different movement phases in each monkey, for each movement-related neuron we identified the maximal peak discharge in three behaviourally defined epochs. The first two epochs corresponded to the period between the release of the starting position and the beginning of object pulling, which was subdivided, on the basis of the behavioural data (see above), into two

6 Movement-related neurons in prefrontal cortex 2887 A C D B Fig. 2. Properties and location of movement-related neurons. (A) A neuron discharging exclusively during the Grasping Pulling epoch. (B) A neuron showing prolonged discharge from object presentation to the Grasping Pulling epoch in the Motor condition, with no response in the Visual condition. The activity is aligned (vertical dashed lines) with the following five behavioural events/epochs: onset of the instructing cues; opening of the door (object presentation); offset of the instructing cues (Go/NoGo signal); beginning of the Object fixation epoch (Visual condition) and beginning of pulling (Motor condition); and reward delivery. Triangles: release of the starting position. The two bars below the histograms indicate the statistical epochs of the Motor (upper) and Visual (lower) conditions, respectively (see Materials and methods). Abscissae: time (s); Ordinates: firing rate (spikes/s). (C) Number of movement-related neurons preferring the Visual condition or the Motor condition during the Cue, Pre-presentation, Presentation, Set and Go epochs. (D) Distribution of penetrations containing movement-related neurons (circles) in the recorded region of the two monkeys (M1 and M2). The dots represent penetrations in which movement-related activity was not found. The position of the sulci is based on the penetration depth (see Materials and methods). IA, inferior limb of the arcuate sulcus; O, orbital reflection; P, principal sulcus. epochs of the same duration (M1, 180 ms; M2, 230 ms). The third epoch, of the same duration as the preceding ones, started with object pulling. These three epochs mostly corresponded to reaching, actual grasping, and pulling, respectively. This analysis allowed us to calculate the number of neurons showing the highest response in the different epochs. The results are shown in Table 2. Note that, in the whole population of movementrelated neurons, as well as in the subpopulation of non-grip-selective

7 2888 L. Simone et al. Table 1. Number of movement-related neurons active in the main task epochs Condition preference Cue Pre-presentation Presentation Set Go/NoGo Grasping Pulling Grip-selective Motor Visual No preference Non-grip-selective Motor Visual No preference Total per column The rightmost column indicates the number of neurons active during the Grasping Pulling epoch, and the other columns refer to the additional responses during other epochs. As a neuron can show a discharge in more than one epoch, the sum of the columns referring to these epochs is higher than the number of movement-related neurons. A B Table 2. Peak of discharge of movement-related neurons: number of movement-related neurons, subdivided on the basis of their grip selectivity, showing the peak discharge in each behaviourally defined movement epoch Epochs Non-grip-selective, no. (%) Grip-selective, no. (%) Reaching 16 (17.8) 9 (26.5) Actual grasping 39 (43.3) 14 (41.2) Pulling 35 (38.9) 11 (32.3) Total 90 (100) 34 (100) C Object preference The results of the ANOVA for repeated measures showed that 43 movement-related neurons were also active during the Presentation epoch, 21 of them having differential activity in this epoch. Nineteen of these 21 neurons discharged more strongly in the Motor condition, whereas only two preferred the Visual condition. The one-way ANOVA (factor: Object) carried out on the 43 movement-related neurons that were active during the Presentation epoch revealed that none of them showed object preference. An example of a neuron discharging during the Presentation and Grasping Pulling epochs, independently of the object observed and grip employed, is shown in Fig. 3(C). Fig. 3. Grip and object preference of movement-related neurons. (A) An example of a movement-related neuron discharging during grasping of the cube (power grip) but not of the cylinder or the sphere (finger prehension and precision grip, respectively). (B) An example of a movement-related neuron discharging equally well during the Grasping Pulling epoch, for all objects. (C) An example of a neuron discharging during the Presentation and Grasping Pulling epochs in the Motor condition, without a preference for the observed object and for the type of grip. In A, B and C (right), rasters and histograms are aligned with the beginning of object pulling; in C (left) they are aligned with the door opening (object presentation). Circles: turning on and off of the green instructing cue. Filled squares: object presentation. Diamonds: beginning of object pulling. Empty squares: reward. The bars under the histograms indicate different epochs used for statistical analyses. Other conventions are as in Fig. 2. neurons, the second and third epochs, corresponding to the actual grasping and pulling phases, respectively, were more strongly represented than the reaching phase (chi-square, P < 0.05). Dark Motor condition vs. Motor condition A subpopulation of movement-related neurons (n = 68) were also tested in the Dark Motor condition, in which the monkeys could see the target object until the Go signal, but, in contrast to the Motor condition, had to reach and grasp the object without visual control (see Materials and methods). The results of the ANOVA for repeated measures, followed by the Newmann Keuls post hoc test, revealed that the discharge of 48 of them (70.6%) was not affected by the presence or absence of visual control during action execution (the post hoc test did not reveal a significant difference between the Grasping Pulling epoch of the Motor condition and the Object fixation epoch of the Visual condition), whereas the remaining 20 (29.4%) showed a significant difference in the Grasping Pulling epoch between darkness and light; in particular, 14 showed significantly stronger discharge during the Motor condition, whereas six showed stronger discharge during the Dark Motor condition. Figure 4(A C) shows examples of the three categories of neurons. Considering the epochs preceding the Grasping Pulling epoch, the ANOVA for repeated measures revealed that three movementrelated neurons showed differential discharge in the Set epoch, two of them discharging more strongly during the Motor condition,

8 Movement-related neurons in prefrontal cortex 2889 A B C Fig. 4. Effect of visual control on the discharge of movement-related neurons during action execution. The three movement-related neurons discharged in the Grasping Pulling epoch both in the Motor condition and in the Dark Motor condition. (A) Movement-related neurons showing the same discharge during the Grasping Pulling epoch in the two motor conditions. (B) Movement-related neurons discharging more strongly during the Grasping Pulling epoch in the Motor condition. (C) Movement-related neurons discharging more strongly during the Grasping Pulling epoch in the Dark Motor condition. Other conventions are as in Fig. 2. whereas eight neurons showed differential discharge at the Go signal, seven of them discharging more strongly during the Motor condition. Note that all these latter neurons had prolonged discharge, and the that differential activity observed during the Go epoch was always congruent with that of the Grasping Pulling epoch. Blocked Motor condition In order to evaluate whether the use of mnemonic information about the target object could influence the discharge of movement-related neurons, we used a further motor condition, run in blocks. In this condition, the monkey could see the object to be grasped only before the first trial of the block, and the task was then carried out in total darkness in all subsequent trials. Note that, in this condition, the door opening coincided with the Go signal (fixation point offset). Thirty movement-related neurons tested in the Visual and Motor conditions were also tested in the Blocked Motor condition. The results of the ANOVA for repeated measures revealed that 28 neurons had a significant main effect of epoch (P < 0.01), and that the discharge in the Grasping Pulling epoch was significantly different from that in the Baseline epoch (Newman Keuls post hoc test, P < 0.01). Nineteen of them had similar discharge in both the Motor condition and the Blocked Motor condition (Fig. 5A1 and A2). The remaining nine neurons had significantly different discharge in the Grasping Pulling epoch. Specifically, eight discharged more strongly during the Motor condition, whereas one preferred the Blocked Motor condition. Six of them also had differential activity in one or more epochs preceding movement. In order to evaluate the impact of the mnemonic load specific to this task, we looked for significant differences between the Mnemonic-Set epoch of the Blocked Motor condition and the Set epoch of the Motor condition. The results of the ANOVA for repeated measures revealed that only one neuron discharged more strongly in the Mnemonic-Set epoch of the Blocked Motor condition, thus almost completely excluding the possibility that the memory of the object can be encoded by these neurons in this condition. Motor responses in the naturalistic paradigm In order to verify whether the movement-related responses also occur when the monkey acts in a more ethological context, without the constraint of behaving in response to abstract cues, we compared the activities of 24 movement-related neurons tested in both Motor conditions, in light and darkness, with those recorded during a naturalistic paradigm, in which the monkeys had to grasp food (with or without visual control) and eat it. The ANOVA for repeated measures revealed that all neurons but one also showed movement-related activity in this paradigm (main effect of epoch, followed by a Newmann Keuls post hoc test, P < 0.01, and/or interaction effect followed by a post hoc test, P < 0.01). Note that, of the 23 neurons that also responded in the naturalistic paradigm, the large majority (78%) did not show differences between light and darkness, either in the Motor conditions or in the naturalistic paradigm. Figure 5(A) shows that the same neuron discharging during reaching and grasping in the Motor conditions (A1) also responded in the naturalistic paradigm (A3). Note that, in the latter, the neuron showed the same discharge profile both when the monkey grasped the target object and when, subsequently, it grasped a piece of food (A4). Interestingly, 19 of the 23 movement-related neurons that were active during hand grasping in the naturalistic paradigm were also tested with the same paradigm performed with the mouth. The ANOVA revealed that eight of them also responded in this condition. Figure 5(B) shows an example of a movement-related neuron discharging during grasping of the small sphere with the hand in the Motor condition (B1), and during grasping of a small piece of food with the hand (B2) and with the mouth (B3) in the naturalistic paradigms. Thus, the neuronal discharge appears to be independent of the effector used to take possession of the target. Population response during the behavioural paradigms Single-neuron recordings revealed the presence of a set of neurons with movement-related responses. In order to evaluate the behaviour and the time course of the activity of the whole population of

9 2890 L. Simone et al. A1 B1 A2 B2 A3 B3 A4 Fig. 5. Effects of different contexts, task rules and effectors on the discharge of movement-related neurons. Left: discharge of a movement-related neuron during grasping in different contexts and under different rules. (A1) Neuronal discharge during Motor conditions in light and dark. The neuron discharge begins in the Presentation epoch, and reaches its maximum in the Grasping Pulling epoch. Note that no significant difference in any epoch was present between the two conditions. (A2) Neuronal discharge in the Blocked Motor condition. The neuron starts firing in the Mnemonic-Set epoch, and reaches its maximal discharge in the Grasping Pulling epoch. (A3) Neuronal discharge in the naturalistic paradigm. The neuron shows a discharge profile similar to that found in the behavioural paradigm. The response begins slightly before the release of the starting position, peaks during hand object interaction, and ends after the accomplishment of grasping. Also in this case, the neuronal discharge during grasping does not differ between the light and dark conditions. (A4) Neuronal discharge in a further naturalistic condition in which the monkey first grasps an object (small sphere), and subsequently grasps a small piece of food. The neuron shows the same discharge during both grasping acts. Right: a movement-related neuron discharging during grasping execution, independently of the employed effector. (B1 B3) Neuronal discharge during grasping of the small sphere with the hand in the Motor condition (B1), and during grasping of a small piece of food with the hand (B2) and with the mouth (B3) in the naturalistic paradigms in light. In the Motor condition, the neuronal discharge starts at the beginning of movement, peaks in the Grasping Pulling epoch, and ends after the accomplishment of grasping. The neuron shows a similar discharge profile in the naturalistic paradigm executed with the hand. The neuronal discharge is slightly weaker when the grasping is performed with the mouth. Conventions are as in Figs 2 and 4. movement-related neurons in the different epochs of the Motor and Visual conditions, we plotted the net normalized mean activity of all neurons aligned with the main behavioural events/task epochs (see Materials and methods). Figure 6(A) shows the time course and the intensity of the response of this population of neurons in the two conditions. In order to compare the intensity of the discharge in the various epochs of the two conditions, we performed a ANOVA for repeated measures (factors: Epoch and Condition), followed by Newman Keuls post hoc tests. This analysis revealed that the population activity was significantly different during the Presentation and Grasping Pulling/Object fixation epochs than at baseline (main

10 Movement-related neurons in prefrontal cortex 2891 A Fig. 6. Temporal profile of the net normalized mean activity and of the net normalized differential activity of the populations of movement-related neurons tested in different experimental conditions. (A) Visual and Motor conditions (n = 124). (B) Motor condition and Dark Motor condition (n = 68). (C) Motor condition and Blocked Motor condition (n = 30). In the upper part of each panel, the lines indicate the population net normalized mean activity in the Visual condition, Motor condition, Dark Motor condition, and Blocked Motor condition. The shaded contours represent their standard errors. In the lower part of each panel, the line represents the differential activity (A, Motor condition minus Visual condition; B, Motor condition minus Dark Motor condition; C, Motor condition minus Blocked Motor condition). Error bars: three standard errors. The activity is aligned (dashed lines) with the main behavioural events/epochs, and the two bars under the graphs indicate the statistical epochs used in the different conditions (see Materials and methods). Abscissae: time (s). B C effect of epoch, P < 0.01, F = 29.94). The results of the post hoc tests on the interaction effect (P < 0.01) indicated that the activity during the Grasping Pulling epoch of the Motor condition was significantly higher than that during the Object fixation epoch of the Visual condition and at baseline. This result was confirmed by the plot of the time course of the differential activity between the Motor condition and Visual condition (Fig. 6A, bottom), which showed that the activity of the population of movement-related neurons started to diverge 180 ms after the Go signal, peaked during the Grasping Pulling epoch, and ended at the end of pulling. In order to evaluate, at the population level, the possible effect of object preference during object presentation and object grasping, we carried out two separate analyses on all movement-related neurons. With respect to grip selectivity, the results of the ANOVA for repeated measures (factors: object Sphere, Cube, and Cylinder; epochs Grasping and Pulling; P < 0.01) revealed that no difference was present for the different grips or between the Grasping and Pulling epochs. Comparing the population activities of the movement-related neurons during the object presentation in the two conditions, the ANOVA for repeated measures (factors: object Sphere, Cube, and Cylinder; conditions Motor and Visual; P < 0.01) revealed no significant differences among objects, whereas a difference emerged between conditions, as the discharge during the Motor condition was higher than that during the Visual condition (main effect, P < 0.01, F = 14.67). These results are in agreement with those obtained at the single-neuron level. To compare the behaviour of movement-related neurons during the execution of the Motor condition and the Dark Motor condition, we plotted the net normalized mean activity of the population of neurons tested in both conditions (Fig. 6B). The results of the ANOVA for repeated measures revealed that the discharge recorded during the Presentation, Set, Go/NoGo signal, Grasping Pulling/Object fixation and Reward epochs were significantly different from those at baseline (main effect of epoch, P < 0.01, F = 66.56), whereas there was no difference in any task phase between the two conditions (absence of a significant interaction effect). To compare the behaviour of movement-related neurons during the execution of the Motor condition and the Blocked Motor condition, we plotted the population analysis of the neurons tested in both conditions (Fig. 6C). The ANOVA for repeated measures revealed a significant main effect of epoch (P < 0.01, F = 44.68), and the post hoc test indicated that only the Grasping Pulling epoch discharges were significantly higher than those at baseline. No difference between the Grasping Pulling epochs of the two conditions was present, indicating that the movement-related discharge was not affected by the fact that the object had to be grasped under mnemonic guidance, and without request of a choice. Interestingly, there was also no difference between the two conditions in the Cue and Set/Mnemonic-Set epochs (absence of an interaction effect).

11 2892 L. Simone et al. Discussion The main finding of the present study is that a sector of the VLPF cortex hosts neurons that are active during the execution of goaldirected reaching grasping actions (movement-related neurons). These neurons were typically activated both with and without visual control of hand object interaction, when the object had to be grasped under mnemonic guidance, and in a naturalistic context in the absence of learned rules. Some movement-related neurons were also active during object presentation, generally discharging more strongly when the object had to be grasped rather than simply observed. Finally, although some movement-related neurons showed a preference for a grip type, none of them showed selectivity during object presentation. Properties of VLPF movement-related neurons This is the first study to report on the presence of neuronal responses during the execution of reaching grasping actions in a wide range of contexts. The presence of neurons showing movement-related activity in the VLPF cortex has been previously described, but in tasks requiring either eye or proximal arm movements (Kubota & Niki, 1971; Niki, 1974a,b; Niki & Watanabe, 1976; Kubota & Funahashi, 1982; Quintana et al., 1988; Funahashi et al., 1989, 1990; Tanila et al., 1992; Boussaoud & Wise, 1993b). In addition, many experiments have mainly focused on the phases preceding movement, related to the attentional mnemonic (Quintana & Fuster, 1992) or perceptual processes (Boussaoud & Wise, 1993a) aimed at selecting behaviour, rather than on the phase of actual execution as such. Where present, the arm movement-related neuronal response was shown to be dependent on specific task rules (Hoshi et al., 1998). In our study, we have demonstrated that movement-related neurons are activated during grasping in different behavioural situations (grasping under visual control, grasping in darkness, memory-guided grasping, and simple grasping of food), indicating that VLPF neuronal activity is not necessarily dependent on the learned relationship between instruction and motor output. It is worth noting that the percentage of movement-related neurons recorded in the present experiment, out of the total number of recorded neurons, is apparently quite small, but it must be considered that, in order to verify the presence of this type of response, we explored a wide prefrontal sector that includes several cytoarchitectonic areas and is partly functionally heterogeneous. This is clear when looking at the maps, which show many penetrations with movement-related properties close to sectors devoid of this type of activity. Interestingly, the sector covered by movement-related neurons appears to overlap with that where Hoshi et al. (1998) recorded neurons controlling forelimb movements used for the achievement of a specific goal. Approximately two-thirds of movement-related neurons also responded during task epochs preceding movement execution. It is interesting to consider these responses in the light of the role usually assigned to sensory signals in the literature on the prefrontal cortex. Indeed, several studies have shown that the VLPF cortex employs information about the visual context to generate goals by forming associations between cues and goals (White & Wise, 1999; Asaad et al., 2000; Miller, 2000; Wallis et al., 2001). Accordingly, lesions including the VLPF cortex lead to impairment of new associations between arbitrary visual cues and forelimb movements (Bussey et al., 2001). In our task, we can identify two phases in which visual information can be used to generate behavioural goals: the instructing cue appearance and the object presentation. Note that, whereas in the Visual condition the cue is enough to define the task goal, because object presentation is irrelevant for accomplishment of the task (to maintain fixation and refrain from acting), in the Motor condition the monkey also needs information about the target object. Our results show that only a few movement-related neurons respond to cue appearance, usually in the absence of a differential discharge between the two conditions. Conversely, one-third of the neurons discharge during object presentation, and, of those that are selective for one of the two conditions in this epoch, almost all prefer the Motor condition. Altogether, these data indicate that the presence of a graspable object, rather than the cue type, is important for triggering some movement-related neurons. This finding is in line with clinical evidence indicating that lesion of the inferior half of the prefrontal cortex in humans can lead to utilization behaviour (Lhermitte et al., 1986), i.e. the compulsory release of grasping movements driven by the presence of an available object. A high percentage of movement-related neurons respond during the Set and/or Go epochs of the Motor condition. This is in agreement with several studies describing the role of the VLPF cortex in movement planning (Quintana & Fuster, 1992; Funahashi et al., 1993; Averbeck et al., 2002; Shima et al., 2007; Yamagata et al., 2012; for review see Tanji et al., 2007), although, in those studies, the percentage of neurons showing set-related activity was higher than in our dataset. This difference can be explained by two factors. First, in our paradigm, during the Set epoch of the Motor condition, all of the information that is necessary for task accomplishment (cue and target object visible) is available, so that the monkey is not required to keep in memory the task rules linked to target location or identity, as is the case in the delayed response tasks. Second, it is possible that the category of movement-related neurons recorded in the present study only partially overlaps with that of neurons modulated in the delay periods. Among the movement-related neurons responding during the Set and Go epochs, many show prolonged differential activity starting from object presentation. This discharge is not affected by the different contextual conditions (Motor condition in light and darkness; Blocked Motor condition), as shown by the population analyses, and could thus represent a type of preparation related to object graspability or the maintenance of action goal representation. This supports the idea that the VLPF cortex could play a role in action planning and execution, extending this role to the case of natural actions (see results for the naturalistic paradigm). Comparison of VLPF movement-related neuronal activity with parieto-premotor grasping-related activity The neuroanatomical literature (Petrides & Pandya, 1999; Borra et al., 2011; Yeterian et al., 2012; Gerbella et al., 2013) indicates that a VLPF cortex sector compatible with that in which we found movement-related neurons (Fig. 2D) is connected with parietal and premotor areas involved in high-order hand motor control. Thus, we could hypothesize that these neurons play a role in a wider network subserving grasping actions. In favour of this hypothesis, our data show that most neurons have a peak discharge in the actual grasping or holding phase, and that, at the population level, the strongest discharge is reached at the beginning of object pulling. Furthermore, one-third of the neurons are grip-selective. Given the above-described anatomical connections and the present functional data, it is important to compare the properties of movement-related neurons with those of parietal and premotor grasping neurons. This comparison suggests similarities in the time course of the discharge, which generally encompasses a motor act (grasping) or