Functional organization of the insula and inner perisylvian regions

Ahmad Jezzinia,b,1, Fausto Caruanaa,c,1,2, Ivilin Stoianova,d, Vittorio Gallesea,c,2, and Giacomo Rizzolattia,c

aDipartimento di Neuroscienze, Università di Parma, 43100 Parma, Italy; bDepartment of Neurobiology and Behavior, State University of New York, Stony Brook, NY 11794-5230; cBrain Center for Social and Motor Cognition, Italian Institute of Technology, 43100 Parma, Italy; and dDipartimento di Psicologia Generale, Università di Padova, 35131 Padua, Italy

Edited* by Charles Gross, Princeton University, Princeton, NJ, and approved May 3, 2012 (received for review January 5, 2012) In the last few years, the insula has been the focus of many brain- hypothalamus and other subcortical structures was used in the imaging studies, mostly devoted to clarify its role in emotions and past to investigate complex behaviors such as feeding or mating social communication. Physiological data, however, on which one (20, 21). More recently, it was used in the works by Graziano (22) may ground these correlative findings are almost totally lacking. and Graziano et al. (23) to map the global organization of the Here, we investigated the functional properties of the insular cor- motor/premotor complex in the monkey and by neurosurgeons tex in behaving monkeys using intracortical microstimulation. Be- in epileptic patients (6, 7) (detailed information on stimulation havioral responses and heart rate changes were recorded. The parameters, reasons that justify prolonged stimulation, and data results showed that the insula is functionally formed by two main indicating that stimulation parameters similar to our parameters subdivisions: (i) a sensorimotor field occupying the caudal–dorsal activate selective anatomical pathways is discussed in Methods, portion of the insula and appearing as an extension of the parietal SI Methods, and Fig. S1). lobe; and (ii) a mosaic of orofacial motor programs located in the The results of our study can be summarized as follows. The anterior and centroventral insula sector. These programs show insula is functionally constituted of two major subdivisions: a progressive shift from dorsally located nonemotional motor pro- (i) a sensorimotor sector occupying its caudal–dorsal portion and grams (ingestive activity) to ventral ones laden with emotional (ii) an anterior/centroventral sector consisting of a mosaic of and communicative content. The relationship between ingestive orofacial motor programs. These programs show a progressive and other behaviors is discussed in an evolutionary perspective. dorsal to ventral shift from motor programs related to ingestion to motor programs with emotional and communicative content. he insula is one of the parts of the cerebral cortex that is less A preliminary report on the social and emotional aspects of the Tunderstood. Its location, buried in the depth of the Sylvian insula has been published elsewhere (24). fissure, its opercularization, and its high vascularization are some Results of the anatomical features that render it difficult to study. In recent years, brain-imaging studies provided a series of We stimulated 4,771 sites in the insula and surrounding peri- correlative data on its functions. The results of these studies sylvian region in two monkeys (M1 = 2,999 sites; M2 = 1,772 were, however, not crystal clear. According to them, the insula is sites). Responses to ICMS were elicited from 74.9% of the sites involved in many diverse functions. The work by Craig (1), for (74.4% in M1; 75.4% in M2). The elicited behavior was clas- fi fi example, claims that the insula is involved in maternal love, si ed into ve main categories: sensorimotor responses, feeding fi bowel distention, orgasm, sudden insight, and decision making as behavior, disgust-related behavior, af liative behavior, and well as awareness and consciousness (2). The interpretation of movement inhibition. brain-imaging studies would be much more convincing if they Sensorimotor Responses. These responses consisted of elementary could be grounded on a physiological basis. Unfortunately, there movements involving mouth, face, hand, or upper or lower limbs. are only few physiological data, mostly obtained with electrical They were evoked by stimulation of the upper bank of the Syl- stimulation in epileptic patients (3–6). vian fissure and dorsal insula (Fig. 1A and Fig. S2A). Sensori- As far as monkeys are concerned, besides a series of single- motor responses were elicited from 28.4% of the stimulated sites neuron studies focused on the sensory properties of the insula – (25.6% in M1; 31.2% in M2). (7 13), the only systematic data on the insula organization derive Mouth movements were elicited from the outer part of the from electrical stimulation studies carried out in the middle of A A – Sylvian upper bank (Fig. 1 and Fig. S2 , red dots) and con- the last century (14 16). These studies, however, are of rather stituted 6.2% of the sensorimotor responses (3.9% in M1; 8.5% limited use. First, they were carried out in general anesthesia. in M2). The movements consisted of mouth opening or closing. Second, they were often performed after surgical removal of They lasted the whole duration of the stimulation. large portions of the surrounding opercula. Third, the stimula- Face movements were elicited from the middle part of the tion was carried out with surface macroelectrodes, which allow Sylvian upper bank (Fig. 1A and Fig. S2A, green dots). They only an approximate localization of the observed responses. constituted 9.5% of the sensorimotor responses (10.3% in M1; In contrast to the paucity of physiological data, there is an 8.7% in M2). The observed movements were eye blinking or excellent and exhaustive picture of the anatomical organization contraction of the facial musculature. They lasted the whole of the insula of both humans and nonhuman primates (17–19). One of the main conclusions of these studies is that the “plan of

anatomical organization is virtually identical [in humans] to that Author contributions: V.G. and G.R. designed research; A.J. and F.C. performed research; of the macaque monkey” (17). This similarity renders, therefore, A.J., F.C., and I.S. analyzed data; and A.J., F.C., and G.R. wrote the paper. a particularly valuable picture of the functional organization of The authors declare no conflict of interest. the insula in nonhuman primates. *This Direct Submission article had a prearranged editor. In the present study, we investigated the functional properties 1A.J. and F.C. contributed equally to this work. of the monkey insular cortex using behavioral timescale intra- 2To whom correspondence may be addressed. E-mail: [email protected]. or vittorio. cortical microstimulation (ICMS). This technique employs pro- [email protected].

longed electrical trains to reveal the behaviors that are controlled This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. NEUROSCIENCE by the stimulated region. Prolonged electrical stimulation of the 1073/pnas.1200143109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1200143109 PNAS | June 19, 2012 | vol. 109 | no. 25 | 10077–10082 Downloaded by guest on September 26, 2021 Fig. 1. Unfolded view of the lateral sulcus of the left hemisphere of M1 depicting its upper and lower banks and the insula. Each dot indi- cates the entrance point of the electrode. Black arrows indicate the antero-posterior (AP) posi- tion of the central sulcus (C) and the intraparietal sulcus (IP). In all penetrations, several sites were stimulated every 500 mm below the entrance point and above the exit point. (A) Posterior dorsal field. Red dots, mouth movements; yellow dots, hand movements; green dots, face move- ments; gray dots, lower and upper limbs. (B)An- terior field. Red dots, ingestive behavior; blue dots, disgust behavior; green dots, movement in- hibition. (C) Ventral field. Yellow dots, affiliative behavior. (D) Miscellaneous responses. Gray dots, discomfort reactions; purple dots, gaze–trunk contralateral displacement; yellow dots, twitch of the chest; red dots, tremors. (E) Unresponsive sites. (F) Percentage of sites per each category. Blue, sensorimotor sites; green, ingestive sites; yellow, disgust sites; orange, affiliative sites; red, movement inhibition sites; tan, miscellaneous responses sites; gray, unresponsive sites.

duration of the stimulation time. Occasionally, face movements movements of the contralateral wrist, joint finger closure, and were accompanied by upper limb movements. wrist rotation (grasping movements). Loss of muscular tone was Hand movements were elicited from the mediocaudal sector also observed. Occasionally, hand movements were accompanied of the Sylvian upper bank and dorsal insula (Fig. 1A and Fig. by the adduction or abduction of the arm. Goal-related move- S2A, yellow dots). They constituted 66.0% of the sensorimotor ments of the upper limb were also sometimes elicited from the responses (63.2% in M1; 68.8% in M2). The observed move- same region, including hand movements directed to the mouth, ments were flexion or extension of the contralateral fingers, hand movements directed to body parts, compulsive attempts to

10078 | www.pnas.org/cgi/doi/10.1073/pnas.1200143109 Jezzini et al. Downloaded by guest on September 26, 2021 withdraw something from the mouth, and visual inspections of Inhibition of Ongoing Movements. Movement inhibition was ob- the hand triggered by the stimulation onset (Movie S1). served after stimulation of the rostralmost part of the Sylvian Upper and lower limbs movements were elicited mostly from upper bank (Fig. 1B and Fig. S2B, green dots). It was elicited the most caudal part of the upper bank and dorsal insula (Fig. 1A from 3.7% of the stimulated sites (2.7% in M1; 4.8% in M2). and Fig. S2A, gray dots). They constituted 18.3% of the senso- ICMS of these sites produced an immediate inhibition of any rimotor responses (22.6% in M1; 13.9% in M2). ICMS of these arm movement performed by the monkey, including food sites elicited nonspecific movements of abduction or adduction grasping movements and movements bringing food to the mouth. of the contralateral hindlimbs and forelimbs. During the whole stimulation time, the arm remained still where The analysis of the heart instantaneous frequency (IF) (Fig. it was located at the stimulation onset. S3) showed that stimulation of this region did not evoke any The analysis of the IF (Fig. S3) showed that ICMS applied to fi signi cant effect to the heart rate frequency but a slight brady- the movement inhibition sites evokes a clear bradycardic effect − cardic effect (peak deviation per behavioral outcome = 3.5%). (peak deviation per behavioral outcome = −8.5%).

Ingestive Behavior. Ingestive behavior was evoked from the rostral Miscellaneous Responses. In addition to the responses already sector of the Sylvian upper bank and dorsal insula. It consisted of described, other types of reactions were evoked from the ventral chewing and mouthing, and it was occasionally followed by fi D B B insula and the lower bank of the Sylvian ssure (Fig. 1 and Fig. swallowing (Fig. 1 , Fig. S2 , red dots, and Movie S2). The S2D). They included (i) signs of discomfort reactions, (ii) con- evoked movements were very similar to the movements observed tralateral orienting of the gaze or the trunk, (iii) twitches of chest during spontaneous oroalimentary behavior. It was elicited from muscles, and (iv) tremors. Globally, these reactions were elicited 12.7% of all stimulated sites (7.8% in M1; 17.6% in M2). from 24.4% of the stimulated sites (32.7% in M1; 16% in M2). Stimulations applied during spontaneous chewing changed the Reactions interpreted as caused by discomfort were elicited masticatory rhythm into the rhythm triggered by ICMS. Fur- from the ventral insula. They were evoked from 9.3% of the thermore, ICMS could evoke licking movements that, in some stimulated sites (12.4% in M1; 6.1% in M2) (Fig. 1D and Fig. cases, were context-dependent. Thus, when ICMS was applied D while the monkey was still, the stimulation evoked a repetitive S2 , gray dots). ICMS of these sites produced compulsive re- protrusion of the tongue constantly directed to the same di- petitive movements of the hands or feet and postural adjust- rection. However, when the left or right part of the monkey lips ments. The intensity of the discomfort reactions ranged from was wetted with juice, the evoked tongue protrusion was directed small postural adjustments to a clear psychomotor agitation. In to that part (Movie S3). some cases, ICMS elicited a facial grimace of distress; the gri- The analysis of the IF (Fig. S3) showed that ICMS applied to mace was also often accompanied by psychomotor agitation. the ingestive field evoked a slight bradycardic effect (peak de- Contralateral orienting of gaze and trunk was elicited from the viation per behavioral outcome = −7.7%). lower bank of the Sylvian fissure (Fig. 1D and Fig. S2D, purple dots). It consisted of a rotation of the trunk to the contralateral Disgust and Retching. ICMS of the region ventral to the ingestive space and simultaneously, a shift of gaze in the same direction. field evoked disgust behavior (Fig. 1B and Fig. S2B, blue dots). It This response was elicited from 2.3% of the stimulated sites was elicited from 2.3% of the stimulated sites (2.8% in M1; 1.9% (1.2% in M1; 3.3% in M2). in M2). Twitches of chest muscles were also elicited from the ventral The disgust behavior most commonly observed was a typical insula and Sylvian lower bank (Fig. 1D and Fig. S2D, yellow facial expression characterized by the curling of the upper lip and dots). They were elicited from 3.8% of the stimulated sites (5.7% the wrinkling of the nose. ICMS of the same sites could also in M1; 1.8% in M2). Often, these movements were followed by determine food refusal. In particular, when stimulation was ap- general psychomotor agitation. plied while the monkey was bringing food to the mouth, the Tremors were mostly evoked from the caudal insula (Fig. 1D monkey immediately threw it away; when stimulation was ap- and Fig. S2D, red dots). They were elicited from 9.1% of the plied with the monkey chewing the food already in its mouth, the stimulated sites (13.3% in M1; 4.8% in M2). Tremors were dif- monkey spat it (Movie S4) (24). Occasionally, the stimulation of fused to the limbs or even the whole body. There was no evi- the same sites could evoke retching (Movie S5). dence of a somatotopic arrangement. The effect did not last The analysis of the IF (Fig. S3) showed that ICMS applied to beyond stimulation time. Interestingly, during ICMS-evoked the disgust field evoked a strong bradycardic effect (peak de- tremors, the monkey did not show any sign of discomfort or viation per behavioral outcome = −17.9%). other emotional facial expressions. Affiliative Behavior. Affiliative behavior was evoked by stimulating The analysis of the IF (Fig. S3) showed different types of heart the middle part of the ventral insula (Fig. 1C and Fig. S2C, rate modulation in different behaviors. Discomfort reactions and yellow dots). It was elicited from 3.4% of the stimulated sites twitch of the chest sites showed a biphasic pattern (i.e., a strong (2.8% in M1; 4.1% in M2). Its typical response was lip smacking bradycardic phase quickly replaced by a tachycardic phase; (that is, a repetitive up–down movement of the jaw with the lips −9.4%). The contralateral displacement of gaze and trunk sites also repetitively opening and closing) (Movie S6). The affiliative evoked a bradycardic effect (−8.6%). Twitches sites evoked behavior occurred only when ICMS was applied in the presence a very strong bradycardic effect (−18.7%). Tremors-evoking sites of eye contact between the monkey and the experimenter. It was elicited a tachycardic effect (8.4%). lacking in the absence of it. When stimulation was applied with the monkey exhibiting another emotional expressions (i.e., Unresponsive Sites. The stimulation of 25.1% (25.6% in M1; threat), ICMS interrupted this spontaneous behavior and 24.5% in M2) of the sites did not elicit any detectable behavioral replaced it with affiliative behavior. The original behavior was response. Although unresponsive sites were found in the entire resumed again after the end of the stimulation (24). stimulated region, the vast majority of them were concentrated The analysis of the IF (Fig. S3) showed that ICMS applied to in the lower Sylvian bank and the ventral insula (Fig. 1E and Fig. affiliative sites evokes a slight bradycardic effect (peak deviation S2E). The analysis of the IF showed that ICMS applied to the

per behavioral outcome = −5.1%) starting during the stimula- unresponsive sites often evoked a very slight bradycardic effect NEUROSCIENCE tion and lasting a few seconds after its offset. (peak deviation per behavioral outcome = −5.4%).

Jezzini et al. PNAS | June 19, 2012 | vol. 109 | no. 25 | 10079 Downloaded by guest on September 26, 2021 Discussion providing information on food consistency and texture, may The aim of the present study was to assess the functional orga- control and drive chewing, swallowing, and more generally, in- nization of the insula and the surrounding opercular regions. The gestive behaviors. In conclusion, our data suggest that the main main results of our study can be summarized as follows. (i) There role of this part of anterior insula sector is integrating somato- is a clear functional separation between a large sensorimotor sensory and gustatory information to achieve an effective in- field located in the dorsocaudal portion of the insula and the rest gestive behavior. Note that this sector of the insula is disgranular, of it. (ii) The anterior and centroventral insula consist of a mo- a structure consistent with a motor function. Finally, our pro- saic of different motor programs, all of them related to specific posal is in accord with clinical cases where oroalimentary orofacial motor behaviors. (iii) In the anterior and centroventral movements have been described during epileptic seizures origi- insula, there is a progressive dorsoventral shift from motor nating in this part of the insula (4). programs without emotional content to motor programs with fi such a content. Disgust-Related Field. Ventral to the ingestive eld, electrical stimulation elicited disgust-related responses (24). These Sensorimotor Dorsal Field. The dorsocaudal part of insula and the responses were characterized by the grimace typical of disgust adjacent Sylvian upper bank are somatotopically organized. and occasionally followed by retching. More complex behaviors, Mouth movements are represented dorsally, face movements are such as the refusal of food intake or food spitting from the in an intermediate position, and hand movements are repre- mouth, were also observed. sented ventrally. The hand representation occupies the dorsal Unlike the stimulation of the ingestive field, which determined part of the insula for most of its extension. Caudally, there is only slight modification of the heart rate, stimulation of this field a small representation of upper and lower limbs. showed potent bradycardic effects accompanying the motor ex- Our data are based on electrical stimulation, which obviously pression of disgust. These data are in agreement with previous stresses the motor aspect of an area. There is no doubt, however, findings showing that disgust, unlike other negative emotions, is that the sensorimotor field is essentially a sensory field. This typically associated with a decrease in heart rate (29). In line with finding is shown (besides the simplicity of the observed move- this finding is also the clinical data in the work by Catenoix et al. ments elicited by ICMS) by its cytoarchitectonic granular structure (30), which found that the occurrence of ictal vomiting corre- and single neuron studies showing the presence of somatosensory lated in time with a discharge affecting exclusively the anterior responses in this field (12, 13). part of the insula. Considering the lack of agreement on the extension and par- Note the continuity between two ingestive-related behaviors: cellation of area SII, it is difficult to match the part of our sen- a positive one, located dorsally, and a negative one, located sorimotor field located in the Sylvian upper bank with SII or ventrally. It is worth stressing that IF data show that the ventrally some parts of it. One possibility is that the map that we just located ICMS-evoked motor behavior is associated with a strong described is independent of SII. In favor of this interpretation is emotional component. the cytoarchitectonic data in the work by Roberts and Akert (25), which localized SII in a more caudal position with respect Affiliative Field. The stimulation of the most ventral part of the to our sensorimotor field (ref. 26, figure 10). Furthermore, the midposterior insula evoked communicative responses. These somatotopic organization of the sensorimotor field described responses mostly consisted of lip smacking (an affiliative ges- here is distributed along a dorsoventral axis rather than along ture). They were evoked only if there was eye contact between a rostrocaudal one as in SII. the experimenter and the monkey. The stimulation of the same According to the work by Schneider et al. (12), sensory in- site, in the absence of eye contact, did not produce any overt formation coming from a region approximately corresponding to behavior. When ICMS was applied during a spontaneous gri- our sensorimotor field and information coming from SII (27) link mace expressing threat, the monkey’s behavior changed, primary somatosensory centers to the ventromedial limbic switching from an aggressive to an affiliative behavior, and it regions, thus playing a crucial role in somatosensory learning. In resumed its previous behavior at the end of the stimulation. agreement with this interpretation are the connectivity data Macaques are social animals. A very important element in showing that the posterior part of the insula is connected with SII social interactions among them is eye contact. Direct eye contact complex and the inferior parietal lobule but not the rostral insula starts the interaction between two individuals and is considered (18). Eberstaller, cited in the work by Cunningham (28), wrote an aggressive message during conflict situations. The necessity of that “the anterior insula is connected entirely with the frontal eye contact to evoke lip smacking suggests that this sector of the lobe, whilst the posterior insula is exclusively connected with the insula plays an important role in activating the motor programs parietal and temporal lobes” (28). necessary to establish the social hierarchic positions and ranks Finally, the absence of clear modification of the heart rate within a given social group. Note that, unlike the other behaviors after stimulation of this field is consistent with a sensorimotor elicited by ICMS, affiliative behavior required, in addition to interpretation of its functional role. electrical stimulation, a specific social element involving another individual. This finding suggests that the electrical stimulation, in Ingestive Behavior. Ingestive behavior was evoked from the an- addition to triggering a specific motor behavior, also modified terior sector of the dorsal insula and the adjacent part of upper the mood of the stimulated monkey, rendering it clear to Sylvian bank. The elicited motor acts included chewing, the observer. mouthing, and swallowing. Anatomically, this region occupies As far as the heart rate is concerned, our data showed a slight the orbitofrontal cortex of the Sylvian upper bank and the dorsal bradycardic effect. This finding is in line with the observation disgranular insula shown in the work by Roberts and Akert (25). that, in humans, affiliative behavior is accompanied with an in- This region, from which we evoked ingestive motor acts, is crease of the parasympathetic tone. classically considered a gustatory cortex. Indeed, taste (7, 8) and some specific aspects of somatosensory modalities (9, 10) are Miscellaneous Responses. A series of repetitive and stereotyped represented. However, the percentage of neurons responsive to responses, rather difficult to decipher in terms of their behavioral such stimuli is rather low. In fact, less than 10% of the recorded meaning, were also observed in the ventral insula and adjacent neurons are responsive to taste stimuli, whereas about 20% lower bank of the Sylvian sulcus. As far as the ventral insula is of neurons respond to specific somatosensory stimuli such as concerned, these responses consisted of repetitive movements of texture, viscosity, etc. It is plausible that these last neurons, the hands or feet, possibly caused by unpleasant sensations. The

10080 | www.pnas.org/cgi/doi/10.1073/pnas.1200143109 Jezzini et al. Downloaded by guest on September 26, 2021 intensity of these putative discomfort reactions ranged from functional fields in the human insula: the sensorimotor, the small postural adjustments to a clear psychomotor agitation. In socioemotional, the olfactory–gustatory, and finally, the cogni- some cases, ICMS elicited a facial grimace of distress. It is im- tive fields. A conjunction analysis across these domains revealed portant to stress that these results are not in contrast with the that, aside from the sensorimotor field, all of the others share finding that affiliative behavior was elicited in a partially over- activations in a sector of the anterior dorsal insula. lapping region (SI Methods shows statistical evidence). In fact, The sensorimotor field described in the work by Kurth et al. the affiliative behavior also includes a submission component (36) is located in the dorsal–posterior part of the insula. This and therefore, a stressful and anxiety-related feeling. field corresponds, for its functional properties and location, to In cases in which the discomfort was more evident, the motor our sensorimotor field. As discussed above, this field is related to responses were accompanied by biphasic cardiac responses, the elaboration of sensory information somehow similar to the suggesting a dual activation of the sympathetic–parasympathetic information carried out in SII and adjacent areas, and it is not system. The work by Paton et al. (31) reported that, unlike the related to emotions. classic inverse relation between sympathetic and para- We already discussed the sensory properties of insular in- sympathetic activity, coactivation of the two vegetative systems gestive field. Our conclusion was that the sensory responses of occurs during the nociception. Thus, the heart pattern produced the neurons in this field represent only one, albeit a very im- by ICMS supports our interpretation of the presence of noci- portant, functional aspect of it. However, its fundamental func- ceptive responses in the ventral insula. tion is of using this sensory information for organizing ingestive Stimulation of the lower bank of the Sylvian sulcus evoked actions. The similar anatomical location of the olfactory–gusta- body rotation to the contralateral side, which was generally ac- tory field in humans and monkeys suggests similar ingestive companied by gaze shifting to that same side. It is interesting to functions in both species. note that the work by Ferrier (32) already showed that the In strict agreement with the present data, the part of the insula electrical stimulation of the outer portion of the temporal related to emotional behaviors is located in humans in the ven- operculum elicits deviation of eyes and neck contralaterally. tral part of the insula. As stated in the work by Kurth et al. (36), Clinical observations report that, during epileptic seizures in- “after testing for specific effects evoked by emotional processing, volving the posterior inferior quadrant of the insula and only the anterior–ventral insula and a small cluster on the central extending to the superior temporal gyrus, head rotation is fre- region remained significant” (36). These locations are in full quently observed (4). Taken together, these data suggest that the accord with our data, which also showed two ventral fields re- contralateral orienting behavior could be related to the excita- lated to emotion: a rostral one related to aversive food behavior tion of auditory fields. (disgust) and a central one related to affiliative behavior. A field described in humans that we were unable to find in the Functional Organization of the Insula. The conventional wisdom is monkey is the so-called cognitive field. This field is functionally that insula is a center related to emotions. More specifically, it very heterogeneous, being activated by language processing (e.g., would constitute an intermediate station between the neocortical lexical decision or semantic judgment), overt speech, working association areas and the core structures mediating emotions, memory, and finally, episodic and short-term memory retrieval. such as the hypothalamus, the periacqueductal gray, and the It is hard to believe that all these many and diverse cognitive centromedial nuclei of the amygdala (33). The present data functions are clustered in a small insular sector. It is much more offer a more complex and articulated picture of the insula plausible that these activations occurred as a consequence of functional organization. a common factor. This factor is, most likely, internal verbal- Insula is formed by two separate sectors: a dorsal–caudal ization, obviously necessary for some verbal tasks and un- sector and the anterior one that ventrally extends into caudal derpinning the other cognitive functions mentioned above. direction. The dorsal–caudal sector is functionally similar to the Note that the notion that a sector of the anterior insula is somatosensory areas located in the parietal lobe. Its connections a language center is supported by a vast amount of clinical data. are almost exclusively with the posterior part of this lobe. In The work by Ojemann and Whitaker (37), for example, by using contrast, the anterior and central insula consists of a mosaic of electrical stimulation of the human insula concluded that the motor programs mostly related to mouth and facial movements. rostral insula is an extension of the language center situated in Some of these programs are related with the emotional core the frontal lobe. Consistent with this proposal is the data structures and therefore, are endowed with an emotional va- reported in the work by Vignolo et al. (38), which described cases lence. Thus, the anterior insula contains mouth programs related of global aphasia with lesions centered on the insula. Finally, to ingestive behavior, negative (aversive) food responses in- there is evidence coming from single cases showing aphasia after cluding disgust and retching, affiliative gestures mostly expressed left anterior insular infarction (39). by mouth and face movements, and intermixed, aversive responses triggered by unpleasant and painful sensations. Some Evolutionary Considerations. On the basis of the data of the As far as the organization of the motor mosaic is concerned, present study, it is interesting to discuss some ethological hy- there is a clear dorsal-to-ventral trend from nonemotionally re- potheses concerning the evolution of communication in pri- lated motor programs (ingestive field) to motor programs with mates. According to the work by Van Hoff (40), many of the emotional valence (disgust and affiliative fields). In agreement most common communicative gestures in monkeys, such as lip with these functional data are the connections of the ingestive smacking or lips protruding from faces, are ritualizations of in- insula. These connections are with the frontal lobe (18) and in- gestive actions that monkeys use for affiliative purposes (41). clude the masticatory field (34) located in the ventral premotor Similarly, the work by Redican (42) stressed the similarity be- cortex and the part of F5 that contains ingestive neurons (35). tween lip smacking and other facial communicative gestures and According to the work by Mesulam and Mufson (17), the ingestive cyclicities. The presence of an ingestive center in the human insula has “a plan of anatomical organization virtually insula, shown in the present study, and its proximity with identical to that of the macaque monkey” (17). Do the functional the affiliative region in the insula provide support to these data support this statement? A recent metaanalysis by Kurth ethological proposals. et al. (36), based on a large number of functional MRI studies, Another consequence of the notion that the more advanced provided a comprehensive correlative functional picture of hu- functions of the insula derived from mouth ingestive motor

man data to which the present findings can be compared. programs is the possible link between ingestive behavior and NEUROSCIENCE According to the work by Kurth et al. (36), there are four distinct speech. As suggested in the work by MacNeilage (43), a species-

Jezzini et al. PNAS | June 19, 2012 | vol. 109 | no. 25 | 10081 Downloaded by guest on September 26, 2021 specific organization property of speech is a continual mouth arm and fixed to the monkey head-holder. The electrodes were inserted open–close alternation, the two phases of which are subject to through the dura that was left intact and moved by a hydraulic microma- continual articulatory modulation. The cycle constitutes the syl- nipulator. Neuronal activity was amplified (Bak Electronics) and monitored lable, and the open–closed phases are segments, vowels, and on an oscilloscope. The position of the microelectrodes in the deep cortical consonants, respectively. It is plausible that this communication regions was monitored by means of ultrasound technique (Logiq 400CL related frames evolved when the ingestive-related cyclicities of ProSeries; General Electric Medical System). When the target region was μ mandibular oscillations (associated to mastication, sucking ,and reached, ICMS was applied every 500 m from the upper bank to the lower licking) assumed communicative significance (see lip smacking bank of the lateral sulcus. ICMS was applied by means of a Biphasic Pulse Generator (BAK) connected to an isolation unit (Stimulus Isolator; WPI). and other communicative gestures of nonhuman primates). Fi- Stimulation was triggered by a hand-held button and consisted of a train of nally, the proximity of the insular primitive speech center with μ fi 200- s biphasic pulses with cathodal pulse leading. Trains were delivered at the premotor areas controlling ne mouth and orolaringeal 50 Hz with an intensity of 4 mA for 3 s. The behavioral responses were in- movements (area 44 and ventral premotor cortex) might have – cluded in the dataset only when two observers recognized the elicited be- allowed the transformation of simple open close mouth move- havior, and this behavior could be evoked in more than 50% of ICMS trials. ments into the motorically complex activity necessary for speech. For each site, the first ICMS was delivered after a period of 60 s, during which time the monkey was quiet and its heart rate was around 120 bpm. Methods Electrocardiogram (EKG) traces (SI Methods) were recorded in each site The experiments were carried out on two behaving macaque monkeys during the first stimulation and the next 10 s after stimulation. After the first (Macaca mulatta). Before the experiments, the monkeys were operated stimulation, additional ICMS was applied at least five times. All experiments under general anesthesia, and a head-holder and two recording chambers were videotaped. were implanted (SI Methods). ICMS were performed through low-impedance (<200 KΩ) tungsten ACKNOWLEDGMENTS. We thank Giuseppe Luppino, Stefano Rozzi, Marzio microelectrodes with epoxylite insulation (FHC). Penetrations were made Gerbella, and Elena Borra for technical support and histological analysis. This perpendicularly to the lateral sulcus, and they were spaced at 1- (M1) and study was supported by the Italian Ministero Istruzione Università Ricerca 2-mm (M2) intervals in the rostrocaudal axis and 0.5- (M1) and 1-mm (M2) (G.R.), European Union Contract 027017 (Neuroprobes), and Rete Tecnolog- intervals in the mediolateral axis. A microdrive was attached to a stereotaxic ica Multidisciplinare-Italian Instituite of Technology.

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