Surgery and Intracortical Microstimulation

All surgeries were conducted in compliance with the standards set forth by the National Institutes of Health and with the approval of the Vanderbilt Animal Care and Use Committee. Each animal was initially anesthetized with ketamine hydrochloride (10–30 mg/kg im), so it could be placed in a stereotaxic frame, and then anesthesia was maintained with 2% isoflurane. The skull was opened to expose parietal and frontal cortex of the left or right cerebral hemispheres. (For clarity and easier comparison across cases, results in all cases are illustrated on left hemispheres.) After the dura was retracted, the motor cortical areas M1, PMD, PMV, and the posterior parietal areas were mapped with electrical stimulation using low impedance (0.5–1 MΩ) microelectrodes. Trains of pulses were generated by a Master 8 stimulator (AMPI; www.ampi.co.il) with biphasic stimulus isolation (Bak Electronics; www.bakelectronicsinc.com). Stimuli consisted of short (60 ms) and long (500 ms) trains of 0.2-ms biphasic pulses delivered at 300 Hz. Current amplitudes were at levels 1–40 μA for M1 and up to 400 μA (in one case 500 μA) for PMC and PPC. Differences in current thresholds for evoking movements and the type of movements evoked allowed us to define the borders that separate M1, PMD, and PMV (see Stepniewska et al. 1993; Preuss et al. 1996). The microelectrode was lowered perpendicular to the cortical surface to a depth of 1.5–1.8 mm for the lowest thresholds of stimulation. Penetration sites were marked on a high-resolution photograph of the exposed cortex. During microstimulation anesthesia was maintained with ketamine hydrochloride (30–50 mg·kg⁻¹·h⁻¹) diluted with physiological saline (1:4) and delivered intravenously with an infusion pump, supplemented every 2–4 h with 0.2–0.5 mg/kg im of xylazine.

Inactivation Procedure

After functional domains in forelimb and face representations within M1, PMD, PMV, and PPC were located by ICMS, we inactivated one of these domains by focal injection of the GABA agonist muscimol (Sigma-Aldrich; www.sigmaaldrich.com) in a concentration 5 μg/1 μl of buffered physiological saline and microstimulated domains in other areas. The effectiveness of muscimol for inactivating a restricted volume of cortex has been previously reported (Kermadi et al. 1997; Schmidlin et al. 2004). The volumes of muscimol injected at each site, the number and position of sites, and the distance between them to cover the desired territory were determined from our preliminary data and published reports (Arikan et al. 2002; Brochier et al. 1999; Kubota 1996; Martin 1991; Schieber 2000; Schieber and Poliakov 1998; Sommer and Tehovnik 1997; Tehovnik and Sommer 1997). Muscimol injections (1–4 μl) in different locations within frontal and posterior parietal regions were guided by the ICMS maps. In a given penetration, 1 μl of the muscimol solution was injected at two different depths (2 × 0.5 μl) at the approximate rate of 0.1 μl/min. Our injection parameters were designed to inactivate a restricted cortical territory (1–2 mm radius around the injection core), which allowed us to target specific functional domains within M1, PMD, PMV, and PPC. Although the effects of a muscimol inactivation can last as long as 24 h (Collins et al. 2005; Martin and Ghez 1993), all data were collected within 4 h after muscimol injections. We retested sites after muscimol injections using the same current thresholds that evoked movements before muscimol treatment. If the site was unresponsive on three retesting attempts over the course of 1–2 min, then we continued testing with incremental increases in current amplitude up to 250 μA for M1 and up to 500 μA for PPC. Sites that remained unresponsive were considered “suppressed.” We occasionally retested such sites at later times throughout the duration of the experiment to ensure that the result was due to muscimol inactivation and not an inadvertent effect of anesthesia.

Microstimulation of the injected region and its surroundings confirmed the extent of effective inactivation. The estimated borders of the inactivation zone were placed between sites that remained responsive and sites that were not (suppressed sites). In addition, the expression of cytochrome oxidase (CO) was reduced after hours of inactivation, and a decrease in histochemical staining of CO was used to estimate size and location of the inactivated domain produced by muscimol injection (Fig. 1). The existence of sites responsive to stimulation identified regions that were clearly outside the inactivation zone. Blocked sites that sometimes occurred outside the estimated inactivation zones suggested a fringe of reduced effectiveness. In most cases the same or very closely situated sites were stimulated before and after muscimol injections. At the end of microstimulation, several sites were marked by electrolytic lesions throughout the depth of the cortex. Although microlesions were readily apparent in histological preparations, there was no evidence of visible damage from the biphasic stimulation.

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Fig. 1.

A cortical section from PMD-M1 border region cut parallel to the surface and stained for cytochrome oxidase (CO) in G 08–01. Note the reduced CO level within the area marked by arrows, and the four electrolytic lesions (open ovals) placed at the functional borders of the reach domain. Muscimol was injected at three sites within the domain (see Fig. 7B).

Analysis of Evoked Movements

Movements evoked by microstimulation were observed by two or three investigators and classified into categories outlined in our previous studies of galagos (e.g., hand-to-mouth, reaching, grasp, or defensive; Stepniewska et al. 2005, 2009a). While one investigator moved and placed the stimulating electrode in various cortical locations, the other investigators observed and classified the movements without direct knowledge about the cortical location of the electrode. There was nearly complete agreement on the type of evoked movements across investigators, and the stimulation was sometimes repeated to establish consistency of the movement. Evoked movements were distinguished from the occasional spontaneous movements that sometimes occur under ketamine anesthesia, by the consistency and the latency of the evoked movement sequence relative to onset of electrical stimulus. Movements were classified during the course of experiment to guide mapping strategy. In addition most movements were recorded on videotape to verify movement classification and to determine complexity, latency, and kinematics of movements. A digital video camera (Sony, DCR-HC65) was positioned in front of the animal, and a mirror next to the animal at a 45° angle to the line of sight of the camera, to provide two nearly orthogonal viewpoints for motion tracking. The camera recorded the forelimb and its reflection in the mirror at 30 frames/s. For each stimulation site, movement sequences were synchronized with the onset of pulse trains, which were visualized by a light-emitting diode (LED) that was captured in the same video recording frame. Thus the start of the stimulation train could be determined to the nearest video frame (33 ms). Movement sequences, especially those evoked from the same site before and after muscimol injection, were traced from video frames, and their trajectories compared.

Organization of Complex Movement Domains in Sensorimotor Regions

The locations of M1, PMD, PMV, and PPC in galagos and New World monkeys are shown in Fig. 2. For all cases these regions were first mapped with long (occasionally also with short) trains of current pulses to establish locations of functional domains within the regions. M1, PMV, and PMD were responsive to short- and long-train stimulation, with PMD evoking movements usually at much higher current intensity (up to 200 μA) than M1 (below 40 μA). Stimulation of PMV evoked movements at slightly higher thresholds than M1. PPC was responsive to long-, but not short-, train stimulation at current intensities of 100–400 μA. For some cases (e.g., OM 08–21, OM 08–19), more complete maps were obtained than for others (e.g., OM 05–38, G 08–01). In all studied species, long-train stimulation evoked similar complex movements from sites in M1, PMC, and PPC regions. Clusters of sites that evoked a particular movement defined behavior-specific domains. Small differences in the relative location of functional domains were noticed between monkeys and galagos (see Kaas et al. 2011), and smaller differences were seen across individuals of the same species.

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Fig. 2.

Schematic illustration of functional domains in frontal and parietal cortex in the left hemisphere of prosimian galago (A), squirrel monkey (B), and owl monkey (C). Territories investigated with microstimulation are depicted in light grey and the approximate locations of functional domains are depicted in color. The black frame corresponds to outlines of schematics in summary Fig. 14. Motor cortical areas: FEF, frontal eye field; M1, primary motor; PMD, dorsal premotor; PMV, ventral premotor; SMA, supplementary motor. Somatosensory cortical areas: 3a, 3b, 1–2. Visual cortical areas: DL, dorsolateral; DM, dorsomedial; FST, fundal superior temporal; IT, inferotemporal; MT, middle temporal; MTc, middle temporal crescent; MST, middle superior temporal; V1, primary visual; V2, secondary visual; V3, third visual. Sulci: CS, central sulcus; FSa, anterior frontal; FSp, posterior frontal; IPS, intraparietal; LS, lateral; STS, superior temporal; PPCr, rostral region of the posterior parietal cortex.

Posterior parietal cortex.

Long-train electrical stimulation (500 ms) evoked movements from domains that paralleled the somatotopic organization of the frontal cortex domains. In galagos only the rostral region of PPC (PPCr) was responsive to ICMS, which includes cortex medial and lateral to the anterior half of intraparietal sulcus (IPS; Fig. 2A). In owl and squirrel monkeys, which do not have an IPS, movements were evoked from a region dorsal to lateral sulcus (LS) and caudal to somatosensory cortex (Fig. 2, ​,BB and ​andC).C). In both galagos and monkeys, most of the responsive PPC region was devoted to forelimb movements, which cluster in cortical domains emphasizing different types of complex movements, similar to those evoked from M1 and PMC.

In galagos, the domain for reaching movements was located most caudal in PPCr, whereas defensive and hand-to-mouth movement domains were in central PPCr. Defensive and hand-to-mouth movement domains were contiguous, with hand-to-mouth movements usually evoked from more rostrolateral sites than defensive movements. We previously reported a small grasping domain in rostral PPC near the border with area 1–2 (see Fig. 6 in Stepniewska et al. 2009a), but it was not identified in the present cases. Eye movements could be evoked from lateral PPC (see Figs. 2–4 in Stepniewska et al. 2009a), but this movement domain was not consistently revealed in previous cases and was not identified in the present cases. Ventral PPC (lateral to the IPS) included a domain for defensive face movements (grimace), sometimes correlated with mouth opening and teeth exposure. Occasionally, defensive face movements were concurrently evoked with forelimb movements.

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Fig. 4.

The effect of inactivation of M1 grasp domain in OM 05–38 (A) and inactivation of M1 hand-to-mouth domain in SM 07–118 (B). In A large circles depict retested sites that were close, but did not overlap the original sites, which are depicted as small dots. Black dots in PMD and PPC depict sites that evoked ear movements. In B note the change in movement sequences evoked from PPC sites (71, 72, 79, 82) during retesting. Arrows from a stimulation sites point to the responses evoked during retesting (shown in squares). For detailed description of results, see the text. Other conventions as in Figs. 2 and ​and33.

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Fig. 6.

Trajectories of lateral defensive movements evoked from site (35) in the M1 forelimb representation in OM 08–19. Trajectories traced from video records show the original movement (“before” muscimol injection) and the movement evoked during retesting (“after” muscimol injection). Black lines show the path of the hand during stimulation, where each dot depicts the tip of digit 3 in successive video frames (33 ms). The end-point of each trajectory is circled for transparency. The forearm is illustrated in a starting position.

Much as in galagos, a mediolateral series of movement domains was revealed in PPC of monkeys, but the movement region was tilted mediocaudally. Thus reach, defense, and grasp domains were organized in PPC in a caudomedial to rostrolateral progression (Gharbawie et al. 2011a; see also cases OM 08–19 and OM 08–21, see Figs. 5 and 11B). A reach domain was identified in caudomedial PPC in all monkeys except monkey SM 07–118 (see Fig. 4B). A defense domain was lateral and slightly rostral to the PPC reach domain. A grasp domain was lateral and slightly rostral to the defense domain and directly caudal to area 1. A hand-to-body domain was not always identified (e.g. OM 08–21, see Fig. 11B), but when present, it was in central PPC near the defense domain (e.g. OM 08–19, see Fig. 5). Face movements (grimace or others) and eye movements were evoked, respectively, from sites in rostrolateral PPC and sites directly medial to the lateral sulcus (Stepniewska et al. 2006a). In squirrel monkeys, evoked face movements were usually accompanied by defensive forelimb movements. Only a few sites evoked face movements alone, and they were typically caudal to the mixed forelimb/face domain (SM 07–118, see Fig. 4B; SM 08–09, see Fig. 7A). Face movement sites were found only in one owl monkey in this study (OM 08–19, see Fig. 5). In this case the rostrolateral PPC region was mapped more extensively than in the other three owl monkeys.

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Fig. 5.

The results of inactivation of the M1 face defense domain in OM 08–19. Note significant changes in movement sequences evoked from site 10 in M1 and sites 79 and 81 in PPC after muscimol injection. Arrows from a stimulation sites point to the responses evoked during retesting (shown in squares). For detailed description of results see the text. Conventions as in Figs. 2–4.

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Fig. 7.

The results of inactivation of the PMD reaching domain in SM 08–09 (A) and PMD-M1 reach domain in G 08–01 (B). In A notable changes in movement sequences occurred during retesting of 3 sites (23, 27, and 30) in M1 and 1 site (104) in PPC. Arrows from a stimulation sites point to the responses evoked during retesting (shown in squares). The effects of inactivation on movement trajectories in SM 08–09 are illustrated in Figs. 8–10. For detailed description of results see the text. Other conventions as in Figs. 2–4.

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Fig. 11.

The effects of inactivation of grasp domain in PMV in OM 08–41 (A) and reach domain in PPC in OM 08–21 (B). In A stimulation of sites 10 and 23 in M1 evoked movements which trajectories are shown in Fig. 12. In B note the change in movement sequences evoked from 4 sites (60, 61, 110, and 116) in PPC and site 38 in M1 during retesting. Arrows from a stimulation sites point to the responses evoked during retesting (shown in squares). For detailed description of results see the text. Conventions as in Figs. 2 and ​and33.

M1 Inactivation

Inactivating parts of M1 abolished movements that were represented in that part of M1, whereas other parts of M1 remained responsive to microstimulation. Moreover, the types of movements blocked in M1 could no longer be evoked from matching domains in PMC or PPC.

The effects of inactivating parts of M1 were studied in three galagos, two owl monkeys, and one squirrel monkey (Table 1). To study the effects of inactivation of the entire M1 forelimb representation, we placed a small piece of gelfoam soaked in muscimol on the cortical surface after it was sized to the M1 forelimb representation as defined by short ICMS mapping (galago G 05–28, Fig. 3A). The estimated region of inactivation (marked in grey in Fig. 3A) possibly extended into parts of the M1 hindlimb and face representations, as well as part of the PMD forelimb representation. Before muscimol treatment, long-train ICMS evoked complex forelimb movements, including defensive, reaching and hand-to-mouth movements, from numerous sites in dorsal PPC. Eight of these sites were retested during M1 inactivation, and none were responsive to microstimulation. In contrast, stimulation sites in ventral PPC evoked face (and ear) movements that were largely unaffected by muscimol injection in the M1 forelimb representation. Nevertheless, higher currents were necessary for evoking movements from some of those sites. For example, site 43 in ventral PPC evoked face grimace and ear movements before muscimol application, but it evoked only ear movement after M1 inactivation, and the current threshold for this movement increased from 100 to 300 μA. Lack of face movements at this site likely reflects the inhibition of a small part of face representation of M1.

Table 1.

Number of cortical sites stimulated after muscimol inactivation of motor domains

Case No.	Inactivated Domain	All	M1	PMD	PMV	PPC
Galago
G 05–28	M1 forelimb	13	—	—	—	8FL + 5F
G 08–01	M1 + PMD reach	19	3FL + 3F	2FL	1FL + 1F	3FL + 6F
G 08–15	M1 reach	41	3HL + 12FL + 3F	6FL + 2T + 1E	2F + 1F/FL	8FL + 2F + 1F/FL
Squirrel monkey
SM 07–118	M1 hand-to-body	37	8FL + 2F	1FL	—	16FL + 5F + 5FL/F
SM 08–09*	PMD reach	50	2HL + 17FL + 2F	2HL + 12FL	2FL	8FL + 3F + 2FL/F
Owl monkey
OM 05–38	M1 grasp	16	3FL + 4F	7FL	1FL + 1F	—
OM 08–19*	M1 face defense	50	5FL + 12F + 2FL/F	—	4F + 1F/T	1HL + 1HL/FL + 18FL + 3F + 3FL/F
OM 08–21	PPC reach	56	19FL + 2F	5FL	3F/FL	1HL + 1HL/FL + 25FL
OM 08–41*	PMV grasp	44	1HL + 20FL + 4F	2FL + 1FL/F	4FL + 5F + 1F/FL + 1F/T	3FL + 2HL

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M1, primary motor cortex; PMV, ventral premotor cortex; PMD, dorsal premotor cortex; PPC, posterior parietal cotex; E, eye movements; F, face movements; FL, forelimb movements; HL, hindlimb movements; T, trunk movements.

^*Cases with illustrated movements trajectories.

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Fig. 3.

Schematic illustration of the effects of inactivating the entire M1 forelimb representation in G 05–28 (A), and the M1 reaching domain in G 08–15 on other functional domains (B). The grey area in A depicts the approximate location of the muscimol application with gelfoam. Arrowheads in B depict the location of 2 muscimol injections. In A and B, dots represent microstimulation sites. Small dots represent sites that were stimulated before muscimol injections. Large dots represent sites that were stimulated before and after muscimol injections. Large dots with a small black dot in the center represent sites that were stimulated after musimol injection, but not before. Areas highlighted in light colors depict the approximate extent of a functional domain or a cluster of sites that evoked similar movements. Thin solid lines mark approximate areal borders and dashed lines mark borders between body representations within M1. Arrows from a stimulation sites point to the responses evoked during retesting (shown in squares). For example, in A stimulation of all sites in ventral PPC evoked concurrent face grimace and ear movements. Inactivation of the M1 forelimb representation suppressed the face movements evoked from PPC, but the ear movements could still be evoked (site 43). In B stimulation of site 61 in PMV evoked grasp followed by hand-to-mouth and mouth opening. Inactivation of the M1 reach domain suppressed the forelimb movements evoked from PMV, but mouth opening was still evoked. Other conventions as in Fig. 2.

More specific changes were observed in the effectiveness of stimulation when particular functional domains or parts of functional domains were inactivated. In galago G 08–15 (Fig. 3B), two small injections of muscimol were placed in the reach domain of M1. Forty one sites in M1, PMD, PMV, and PPC were retested over the course of 3.5 h after muscimol injection. The inactivated region (marked in grey in Fig. 3B) included more than half of the M1 reach domain. Five sites retested within the inactivated region were unresponsive to stimulation. Lateral to the inactivated region, two sites continued to evoke reaching movements, although stimulation thresholds were significantly higher (10 vs. 150 μA). Such results indicate that the effects of muscimol injections in M1 can be very specific, blocking some but not other sites in an individual domain. M1 sites retested caudal and lateral to injected domain were unaffected as they continued to evoke defensive, hand-to-mouth, and grasping/hand-to-mouth movements. Face movements were evoked from all retested sites in lateral M1. However, the movements evoked from these sites were weaker compared with movements evoked from the same sites before inactivation of M1 reach domain. As expected, sites retested in PMV that evoked face movements remained effective. However, one site (61) in PMV where both face and forelimb movements were evoked before inactivation lost the capacity to evoke forelimb movements, but not the face movements. In PMD, five sites for reaching movements were retested. Three of these sites were unresponsive, and the two responsive sites while near the PMD-M1 border evoked reaching movements but at higher current thresholds (20 vs. 150 μA and 40 vs. 200 μA). Another site at the PMD-M1 border evoked hand-to-body movement before muscimol treatment and continued to do so after muscimol treatment. Two sites for trunk and hindlimb movements also remained effective, as did a nearby site for eye movements in the FEF. Thus inactivation of the M1 reach domain either inhibited reaching movements from PMD or increased the threshold for evoking movements. The loss of function was less specific in PPC after inactivation of the M1 reach domain. The four sites retested in the PPC reach domain were unresponsive. However, four nearby sites for defensive forelimb movements were retested and no movements were evoked as well, even with higher current intensity (500 μA), than before inactivation (150–250 μA). Sites retested in ventral PPC remained effective in evoking face movements.

The effects of selective M1 inactivation were further investigated in owl monkey OM 05–38 (Fig. 4A). The M1 grasp domain was inactivated with four small injections of muscimol (black arrowheads in Fig. 4A). Sixteen cortical sites in M1, PMD, and PMV were retested over the course of 70 min after muscimol injection. The inactivated region encompassed more than half of the M1 grasp domain, but sites that used to evoke hand-to-body movement (either alone or concurrent with grasp) were also involved. Grasping movements could still be evoked from a site caudal to the muscimol-injected region, and face movements could be evoked from M1 sites lateral to the same region. The thresholds for both types of movements remained comparable to those before inactivation.

Six sites in PMD that represented grasping, concurrent grasping, and hand-to-body or just hand-to-body movements were unresponsive to microstimulation during retesting. The loss of those movements may be explained by the fact that the region inactivated by muscimol involved sites for grasping as well as for hand-to-body movements and that these behaviors are mediated by M1. Only one site representing hand-to-body movement remained effective, although the movement was much weaker after inactivation. Retesting a site in dorsal PMV for grasping failed to evoke movements. Inactivation did not interfere with face defensive movement evoked from a lateral PMV site, but movement strength was reduced and threshold increased. PPC was not tested after muscimol injections in this monkey. The results indicate that muscimol effectively blocked much of the grasping domain of M1 and that this inhibited sites in premotor cortex (PMV and PMD) for grasping and partially inhibited sites in PMD for hand-to-body movements.

The effects of inactivation of a single functional domain in M1 on the responsiveness to microstimulation of domains in other sensorimotor areas (especially PPC) was also investigated in squirrel monkey SM 07-118. The M1 hand-to-mouth domain was inactivated with four muscimol injections (Fig. 4B). Thirty-seven sites distributed throughout M1, PMD, and PPC were retested over the course of 4 h after muscimol injections. Some dorsal and rostral sites within the hand-to-mouth M1 domain were unaffected, so inactivation was only partial. Another M1 site caudal to the inactivated territory continued to evoke defensive forelimb movements, but two face movement sites lateral to the inactivated region were unresponsive when stimulated with current up to 150 μA even though their movement thresholds before inactivation were 30 and 90 μA. The single site retested in PMD evoked defensive forelimb movement at an unchanged current threshold. Retesting was more comprehensive in PPC with 26 sites. Most hand-to-mouth (or to body) sites in PPC, which were responsive to current amplitudes 150–250 μA, were unresponsive after inactivation of M1 hand-to-mouth domain. Hand-to-mouth sites that included concurrent ear movements (e.g., site 82) or face grimace (e.g., site 79) were devoid of the hand movement during retesting, but the ear and face movements were spared. However, in three other sites hand and face movements were eliminated. Interestingly, two most rostral PPC sites (71 and 72) that evoked hand-to-body movements during retesting evoked defensive forelimb movements. The lateral direction of the defensive movements were almost the diametric opposite of the original hand-to-mouth movement. All other forelimb sites retested in PPC, including reaching and grasping, were suppressed. Other face sites retested in caudal PPC remained responsive, albeit to higher current thresholds. Increased current thresholds likely reflected partial inactivation of face sites in M1. The results demonstrate a more widespread suppression of PPC function than would have been expected from the size and location of the inactivated M1 territory. PMV was not retested after muscimol injection in this case.

The effects of inactivating a large part of the face representation in M1 on the responsiveness of sensorimotor areas was investigated in owl monkey OM 08–19. The M1 face defensive domain was inactivated with one large injection of muscimol (Fig. 5). Fifty sites distributed across M1, PMV, and PPC were retested over the course of 3.5 h after injection. Most of the M1 face representation was inactivated, including a distant site near the PMV border. However, one site on the rostral edge of the inactivated domain remained responsive. A more rostral site at the border with PMV also remained responsive, but its current threshold increased from 20 to 80 μA during retesting. Two M1 sites at the face/forelimb border that evoked concurrent forelimb and face movements were retested as well. The site closest to the inactivation territory (12) was completely suppressed. Another site (10) no longer evoked face movements but continued to evoke forelimb movements (grasp + supination), albeit with current amplitudes that increased from 40 to 200 μA during retesting. Forelimb sites retested in M1 were not suppressed after inactivation of face M1 (except for site 9 at the border with area 3a) and their thresholds remained the same. In addition, the movement trajectories traced from video recordings were conserved for most of such sites during retesting. For example, site 35 evoked defensive forelimb movements that were consistent across trials before and during inactivation. During inactivation only subtle differences were detected in the latter part of the movement, which included a slightly longer trajectory (Fig. 6). A site near the M1 face domain (37) evoked a weak forearm supination followed by hand-to-body movement. During inactivation, stimulation of this site evoked the same movement, albeit with an additional forelimb lift that resulted (as for site 35) in slightly longer trajectory (not illustrated).

Face sites in PMV also required higher current levels during retesting to evoke movements. Two sites near the M1/PMV border were totally suppressed. Also, all face sites in PPC were suppressed during retesting. The most rostral sites in PPC that evoked a facial grimace and concurrent grasping were either suppressed during retesting (e.g., site 72), or devoid of the face movement with forelimb movement remaining the same (e.g., site 79), or turning out to be more complex (e.g., site 81). During retesting, microstimulation of site 81 evoked a grasp followed by a retraction of the hand towards the body. Some forelimb sites in PPC were suppressed during retesting, but hindlimb sites in medial PPC and their thresholds were largely unchanged. PMD was not retested after the M1 face inactivation.

In summary, muscimol injections into forelimb or face domains in M1 effectively inactivated a territory localized near the injection sites, whereas forelimb and face sites more distant from the injection remained functional but in some instances at higher current thresholds. M1 inactivation suppressed the responsiveness of functionally matched domains in PMC, while effecting a more widespread and less specific suppression in PPC that included functionally matched domains and numerous sites in neighboring (unmatched) domains. However, more experiments are needed to confirm the preservation of function for unmatched domains in PMC.

PMD Inactivation

In another set of experiments, we investigated the effects of PMD inactivation on responsiveness in other sensorimotor areas in one squirrel monkey and one galago. Inactivating the PMD reach domain in both cases altered the responses and thresholds at nearby stimulation sites in PMD but had little effect on other frontal cortex sites. Functionally matched sites in PPC were suppressed, and some of the related sites were altered as well.

In squirrel monkey SM 08-09 (Fig. 7A), one muscimol injection was used to inactivate the PMD reach domain. The injection site also included several sites from which forelimb lifting was evoked. Fifty sites were retested over the course of 3 h from muscimol injections. PMD reach domain inactivation did not affect forelimb or hindlimb sites within PMD, including sites at the M1 border that evoked reaching. However, for most of these sites, higher current thresholds were necessary to evoke movements. Forelimb sites retested in M1 (17 sites) and PMV (2 sites), including those representing reaching, were also responsive to stimulation. Sequences of movements evoked from the M1 sites during retesting were mostly unaltered, but movements were truncated in some instances (e.g., sites 7 and 38, Fig. 8, ​,AA and ​andB).B). In other retesting instances, the complexity of forelimb movements evoked from M1 was altered. For example, stimulation of site 23 after inactivation evoked a grasp only, whereas the original movement included grasp followed by hand-to-mouth movement. In contrast, stimulation of site 30 at the forelimb/face border originally evoked hand-to-mouth movement, but the same movement included a grasp component after PMD inactivation. In addition, movement trajectories revealed different end-point positions for the hand before and after inactivation (Fig. 9A), and the end-point positions after inactivation were consistent irrespective of the starting position (Fig. 9A), supporting the previous observations of Graziano et al. (2002a,b). A site in lateral forelimb M1 (27) originally evoked grasping and concurrent hand-to-body movements, but during inactivation it evoked defensive forelimb movements that followed a completely different trajectory (Fig. 9B). Face sites in M1 were not affected by PMD inactivation and their thresholds remained the same.

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Fig. 8.

Trajectories of hand-to mouth (A) and lateral reach movements (B) evoked from sites 7 and 38, respectively, in the M1 forelimb representation of SM 08–09. Trajectories show the original movement and the movement evoked during inactivation of the PMD reach domain. Black dots indicate the positions of the tip of digit 4 and gray dots indicate the position of distal interphalengeal joint in successive video frames. Note the truncated trajectories of both movements evoked after muscimol inactivation. For site 38, trajectories of hand movements reflected in the mirror are also shown. Other conventions as in Fig. 6.

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Fig. 9.

Trajectories of hand-to-mouth (A) and hand-to-body movements (B) evoked from sites 30 and 27, respectively, in M1 forelimb representation of SM 08–09. Trajectories show the original movement and the movement evoked during inactivation of the PMD reach domain. Black dots indicate the positions of the tip of digit 4 and gray dots indicate the position of proximal interphalengeal joint in successive video frames. Note that the final position of the hand in A during retesting was quite consistent irrespective of the starting position. In B the original hand-to-body movement changed to lateral defensive movement during retesting. Conventions as in Figs. 6 and ​and88.

One site retested in PMV during PMD inactivation continued to evoke reaching movements, albeit with current amplitudes that increased from 40 to 300 μA. The movement was also much weaker and truncated (not illustrated). Similarly, another PMV site (86) continued to evoke hand-to-mouth movement with simultaneous mouth opening during PMD inactivation but with current amplitudes that increased from 40 to 60 μA. The trajectory of this movement was also shorter and the final spatial position was much lower than for the original movement (Fig. 10). As for site 30, end-point positions of the original movement evoked from site 86 were consistent regardless of the starting position.

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Fig. 10.

Trajectories of hand-to-mouth movement evoked from site (86) in PMV of SM 08–09. Trajectories show the original movement and the movement evoked during inactivation of the PMD reach domain. As in Fig. 9A final position of the hand in original movement was consistent irrespective of the starting position. Conventions as in Figs. 6 and ​and99.

Forelimb sites retested in PPC (reach and other domains) were suppressed, whereas face sites remained responsive to the original current amplitudes. PPC site 104 originally evoked reaching and concurrent face movements, but PMD inactivation suppressed the capacity to evoke reaching from this site.

The PMD reach domain was also inactivated in galago G 08-01 (Figs. 1 and ​and7B).7B). Three muscimol injections covered caudal PMD and spread into rostral M1 where reaching was also represented. Nineteen sites across PMD, M1, PMV, and PPC were retested over the course of 2 h of muscimol injections. Although fewer cortical sites were retested in this case compared with SM 08–09 (Table 1), the results for both cases were similar. Thus forelimb and face sites in M1 remained responsive after PMD inactivation. Forelimb and face sites in PMV also remained responsive during retesting. All PPC sites that originally evoked reaching were suppressed during retesting but face sites remained responsive.

PMV Inactivation

We investigated the effects of partial PMV inactivation in a single case. Only mild alterations of evoked movements were observed for sites in M1, whereas widespread suppression of movements was found for sites in PPC.

In owl monkey OM 08-41 (Fig. 11), a single muscimol injection inactivated the PMV grasp domain. Forty-four sites distributed across PMV, PPC, PMD, and M1 were retested over the course of 3 h after muscimol injections. A PMV site that was dorsal and caudal to the inactivated territory evoked grasping and concurrent arm lifting, suggesting only partial inactivation of the PMV grasp domain. Although the muscimol injection may have encroached into the face representation, most face sites in PMV were fully responsive during retesting. Other PMV sites were also unaltered. The three sites retested in PMD continued to evoke reaching and defensive forelimb movements albeit with slightly higher current amplitudes.

Only mild effects of PMV inactivation were observed in M1, and these were largely exclusive to M1 grasping sites. Originally, grasping movements were evoked from two clusters of sites in M1. One cluster was in caudocentral aspects of the M1 forelimb representation and may have overlapped area 3a. The second cluster of sites was more lateral, near the face representation. None of the grasping sites retested in caudocentral M1 were suppressed, but two of three lateral grasping sites were unresponsive even with current amplitudes up to 250 μA. Although the type of movements evoked from other forelimb sites in M1 (reaching, defensive, or hand-to-body) was unchanged during retesting, these movements were weaker and in some instances required higher current amplitudes. Tracings of the trajectories of the reaching movements evoked from site 10 near the M1/PMD border showed that movements were shorted during retesting (Fig. 12A). Shorter movement trajectories were also observed during retesting of site 23 in the center of the M1 forelimb representation. More specifically, site 23 originally evoked a grasp with hand-to-mouth movements, which was truncated during retesting as the hand failed to reach the mouth (Fig. 12B). Four face sites were retested in M1. The two sites in rostral M1 near the PMV border were not suppressed, whereas the two sites near border with 3a were unresponsive.

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Fig. 12.

Trajectories of reaching movement evoked from site 10 and hand-to-mouth movement evoked from site 23 in M1 forelimb representation of OM 08–41 (A). Trajectories show the original movement and the movement evoked during inactivation of the PMV grasp domain. Note the truncated trajectories of both movements evoked after muscimol inactivation. In B “+” marks the mouth position. Other conventions as in Figs. 6 and ​and99.

As in other cases with partial inhibition of frontal motor regions, suppression was widespread in PPC. Thus all forelimb sites, representing grasping, but also reaching, and defensive movements were unresponsive. In contrast, hindlimb sites in PPC were not suppressed.

PPC Inactivation

The effects of PPC inactivation on other sensorimotor areas were investigated in owl monkey, OM 08-21. Changes at other sites in PPC were observed, but sites in PMC and M1 were unchanged.

The PPC reach domain was inactivated with two muscimol injections (Fig. 11B). Fifty-six sites distributed across PPC, PMD, PMV, and M1 were retested over the course of 3 h after inactivation. The reach domain was not completely inactivated since reaching movements could still be evoked from at least two sites in rostral aspects of the domain. The other 18 forelimb sites retested in PPC remained responsive during retesting, and their current thresholds were not much different from the original testing results. However, the sequence of the motor responses were altered for some of these sites during retesting, particularly for sites within grasping domain, which is closely associated with the reach domain (Gharbawie et al. 2011a). For example, the grasping movements originally evoked from two lateral sites 60 and 61, changed during retesting such the forelimb was withdrawn immediately following the grasp. In contrast, the grasping followed by forelimb withdrawal originally evoked from sites 110 and 116 changed during retesting such that the evoked movement was reduced to forelimb withdrawal.

Forelimb defensive sites in PMD as well as hand-to-mouth and face sites in PMV were unaltered during retesting and their thresholds remained the same. All M1 sites including grasping, reaching, defensive, hand-to-mouth, and face were also unaltered during retesting. Only 1 site (38) of the 21 sites retested in M1 evoked a different movement sequence during retesting. This forelimb site was near the border of the face representation in M1, and its original forelimb to body movement changed into a grasp followed by hand-to-body movement.

Trajectories of Evoked Movements

We investigated the possibility that sites not suppressed during muscimol inhibition evoked movements that were qualitatively different from the original movements. Thus we traced trajectories from our video records of the original movements and movements evoked during retesting. A video camera positioned in front of the animal allowed us to trace movement trajectories in two dimensions, and the position of the mirror (see materials and methods) facilitated three-dimensional tracing of movement trajectories for some of the recorded movements. The entire video arrangement was consistent for the duration of each stimulation session for each case, so the original and retested trajectories could be reliably compared.

Hand trajectories traced before and after muscimol for eight sites (7 in M1 and 1 in PMV) are shown in Figs. 6, ​,88–10, and ​and12.12. Movements evoked from these sites drove the hand toward various spatial locations. In those examples, sites that evoked the consistent movements before muscimol during retesting produced a movement sequence that was truncated relative to the movements originally evoked. More significant effects were obvious for sites that evoked a completely different sequence of movements after muscimol, which was reflected in the trajectories of the hand (e.g., SM 08–09, site 27, Fig. 9B).

Frame-by-frame comparisons showed that the speeds of the evoked movements were in some instances reduced during retesting after muscimol, even though spatial trajectories may have been comparable to the original movements. Figure 13 shows speed profiles for each stimulation site before (continuous lines) and after (dashed lines) muscimol injections, which followed an approximate Gaussian distribution. Speed profiles recorded for multiple trials from the same site were very similar or even identical before or after muscimol (e.g., SM 08–09, site 27, Fig. 13G). Slightly different speed profiles were obtained for trials before muscimol for which start position of hand differed (SM 08–09, site 86, Figs. 10 and 13F). Overall speed profiles were reduced after muscimol (Fig. 13). Typically greater peak speeds characterized longer movement trajectories (e.g., hand-to-mouth). Thus the truncated movements recorded during retesting had lower peak speeds. For some stimulated sites, such as OM 08–19 site 35 and SM 08–09 site 30, peak speeds after muscimol remained similar (Fig. 13, ​,AA and ​andH).H). For most movements, the peak speed was achieved 100–133 ms after stimulus onset, and this time profile was comparable for movements evoked before and after muscimol injections. Stimulation-evoked movements were initiated during the first two video frames (0–66 ms) from the start of electrical stimulation. This movement initiation profile was generally conserved during retesting. Only in two instances (OM 08–41, site 23 and SM 08–09, site 7; Fig. 13, ​,CC and ​andD)D) was a delay detected and it amounted to a single video frame.

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Fig. 13.

Speed profiles of hand movements trajectories traced from selected sites in M1 and PMV in cases with muscimol inactivation of M1 face domain (A), PMV grasp domain (B and C), and PMD reach domain (D–H). Each graph includes the speed profile of the original movement (solid line) and movement evoked during retesting (dashed line). Black arrowheads indicate the start of microstimulation train. Note the lower peak speeds for most movements evoked after muscimol application.

Connections Between Posterior Parietal and Frontal Motor Regions

The present results support our hypothesis that PPC, PMC, and M1 consist of a number of domains that are hierarchically related and form functionally distinct, mostly segregated parietal-frontal networks. M1 seems to be a critical link in these networks, since the outputs of M1 are essential to the behavior, whereas PPC and PMC mediate complex movements via their connections with M1. Such results are consistent with known patterns of connections between PPC, PMC, and M1.

Our previous connectional studies showed that in galagos and New World monkeys PMC and PPC have numerous reciprocal corticocortical connections, and both regions are also connected with M1 (Fang et al. 2005; Stepniewska et al. 1993, 2006b). Dorsal and ventral divisions of PMC have direct dense and topographical connections with M1. The connections of PPC with M1 are less dense and are limited to the most anterior parts of PPC. However, PPC may communicate with M1 indirectly via PMC. Our recent studies confirmed that the pathways that connect PPC-PMC-M1 are domain specific. By injecting tracers into functional domains in M1-PMC and PPC identified with ICMS in galagos (Stepniewska et al. 2005, 2009a,b) and New World monkeys (Gharbawie et al. 2011a), we determined that domains in the PPCr and frontal motor cortex (M1-PMC), where different classes of complex movements can be evoked, are preferentially interconnected. In line with these findings, stimulation of PPC domains selectively activated matching domains in M1-PMC as shown by our optical imaging studies (Stepniewska et al. 2011).

Our combined neurophysiological and neuroanatomical approach allowed us to identify a number of the parietal-frontal networks that support different actions in both galagos and New World monkeys (Gharbawie et al. 2011a; Stepniewska et al. 2005, 2009a,b). For example, a dorsal parietal-frontal network interconnecting caudomedial PPC with PMD and M1 is involved in reaching, and a more ventral parietal-frontal network interconnecting rostrolateral PPC, PMV, and M1 is involved in grasping. All networks are further characterized by inputs from other cortical and subcortical structures, but M1, PMC, and PPC form the core of these networks. These regions combine visual signals with other sensory and motor signals and build motor commands for reaching, grasping, and other behaviors. Commands from PMC and from PPC (mostly via PMC) are passed onto the motor cortex, which sends long axons down to the brainstem and the spinal cord to synapse directly or indirectly (via interneuron circuitry) onto the motor neurons for movement execution. Thus suppression or alteration of movements evoked from PMC and PPC after muscimol injections in M1 domains and suppression of movements evoked from PPC domains after muscimol injections in PMC are in agreement with the expected roles of well established anatomical connections. Altogether our results suggest a hierarchical relationship of cortical areas and their domains, with M1 domains being central to movement completion (see also Kakei et al. 2001). This hierarchical organization is also in agreement with results of our previous ICMS studies that demonstrated longer latencies for movements evoked from PPC than from M1 and PMC (Stepniewska et al. 2009a).

A main finding of the present study is that inactivations of M1 functional domains suppressed functionally matched domains in PMC and PPC but had much milder effects on the functions of nonmatching domains. This finding provides further evidence for neuronal clusters (domains) in M1, PMC, and PPC that are interconnected in largely segregated networks specialized for specific behaviors. There is considerable evidence for existence of similar, partially independent, parietal-frontal networks involved in specific motor actions in macaques (see Johnson et al. 1996; Luppino et al. 1999; Matelli and Luppino 2001; Rizzolatti et al. 1998; Wise et al. 1997). Physiological studies have shown that different classes of neurons encoding information relevant to reaching, grasping, or other behaviors are common to parietal and frontal regions, which are also linked by reciprocal connections (Battaglia-Mayer et al. 2001; Caminiti et al. 1991, 1999; Hepp-Reymond et al. 1994; Jeannerod et al. 1995; Kalaska 1996; Marconi et al. 2001; Mushiake et al. 1999; Shipp et al. 1998; Rizzolatti et al. 1988; Sakata et al. 1999; Stanton et al. 1995; Tanne-Gariepy et al. 2002). Most recently, connections between parietal and frontal cortical areas representing grasping (Gharbawie et al. 2011b) or defensive behavior (Cooke et al. 2003; Cooke and Graziano 2004; Graziano et al. 2002b), as defined by long-train ICMS, have been described as well.

The segregation of functional parietal-frontal networks, however, is not complete. Our present results show that although inactivation of a particular domain in a sensorimotor area (e.g., M1) does not suppress the movements evoked from adjacent domains within the area, it may alter their trajectories. Thus domains of different classes interact with each other. The interactions between functional motor networks (e.g., reach and grasp networks) are supported by the existence of common inputs from some parietal areas, as well as by intrinsic connections within PPC, PMC, and M1 (Gharbawie et al. 2011b). Such interlinks between networks may represent channels for coordinating different functions of the forelimb (e.g., reach to grasp). We previously suggested that the excitatory connections between functional domains within a region largely terminate onto inhibitory neurons as a way to reduce the probability of generating competing behaviors (Kaas et al. 2011, 2012). Reducing intraregional inhibition by blocking the outputs of any specific domain would be expected to reduce activation thresholds for other domains, but this possibility has not been systematically evaluated. However, our present results provide new evidence for the interaction of the parallel networks in ways that modify the outputs of each other.

Corticospinal Projections

The corticospinal (pyramidal) system is critical for motor functions in primates. About half of the corticospinal fibers arise in M1, but other frontal areas such as PMD and PMV, as well as parietal areas, also contribute axons to the corticospinal tract (Dum and Strick 1991, 1996; Galea and Darian-Smith 1994; He et al. 1993; Nudo and Masterton 1990; Wise 1996; Wu and Kaas 2000). Each population of corticospinal neurons has terminals in the intermediate zone of spinal segments, whereas direct projections onto motoneurons of the ventral horn originate primarily from M1 (Rathelot and Strick 2009). More numerous corticospinal projections from M1 are reflected in its higher excitability and lower stimulation thresholds for evoking movements compared with PMC or PPC (Kuypers 1981; Porter and Lemon 1993). Although the contributions of premotor and parietal areas to corticospinal pathways are less than that of M1, these areas have the potential to influence the generation and control of movements independently, but the extent of this control is not understood. Our results show that direct projections from PMC and PPC to motor neurons in the spinal cord (and brainstem) are not sufficient for evoking complex movements when stimulated, since stimulation of domains in these regions is ineffective during the inactivation of matching M1 domains. Thus corticospinal projections from nonprimary motor areas (including PPC) are not normally of critical importance to complex movements. The cortical connections of PPC with PMC and M1, as well as the PMC connections with M1, appear to be more important for the initiation and guidance of complex motor behaviors.

We thank Mary Feurtado and Laura Trice for technical assistance.

Present address of M. J. Burish: Department of Neurology, University of California, San Francisco, CA 94143.