The results indicated that activity of some neurons was significantly correlated with one or more of social behaviors. Activity of leaving- and approaching-related neurons changed in response to leaving and approaching behaviors of recording and/or partner monkeys, respectively.
Journal of military pharmaco-medicine 7-2013 STUDY ON NEURONAL ACTIVITIES OF ANTERIOR CINGULATE DURING SOCIAL BEHAVIOR IN MONKEY Can Van Mao*; Etsuro Hori**; Hisao Nishijo** summary Previous studies indicated that the anterior cingulate cortex (ACC) is one of the critical brain areas for social behaviors, and that the effects of PCP might be mediated through the ACC However, few studies investigated neural correlates with social behaviors in the ACC To investigate this issue, we recorded ACC neuronal activity from one of the monkeys while these monkeys socially interacted The results indicated that activity of some neurons was significantly correlated with one or more of social behaviors Activity of leaving- and approaching-related neurons changed in response to leaving and approaching behaviors of recording and/or partner monkeys, respectively Activity of the communication-related neurons changed while the monkeys engaged themselves in a series of social behaviors such as grooming together, lip smacking, facing, etc These results provide neurophysiological bases for ACC involvement in psychiatric disorders with social disturbance such as schizophrenia, austism * Key word: Neural mechanisms; Social interaction Introduction Disturbances in social skills (e.g avoiding social contact, neglecting a surrounding environment, social isolation) are pervasive aspects of many diseases such as: schizophrenia, autism, bipolar disorder However, neural mechanisms of these social deficits in these diseases are still unclear The anterior cngulate cortex (ACC) is one of the pivotal components in the brain network [6], and implicated in cognition, decision making, emotion and social behaviors [9] The previous behavioral studies reported that lesions of the ACC induced deficits in social behaviors in monkeys Post-mortem and clinicopathological studies using individuals with schizophrenia indicated deficits in the ACC; decreased gray matter volume, and reduced neuronal and glial density, reduction of neuronal soma size and cluster of neurons Neuroimaging studies also showed the same morphological changes in the ACC of schizophrenic and bipolar disorder patients Recently, fMRI studies also revealed functional deficits in the ACC in autism patients [1] Previous neurophysiological studies reported that activity of ACC neurons are related to working memory, emotion, error detection, reward prediction [7]… However, there have been no studies to investigate relationships between neuronal activity and social behaviors in the ACC The aim of the present study was to: Investigate activity of monkey ACC neurons during social behaviors, and neural correlates with social interaction were analyzed Materials and methods Subjects Three monkeys (two Macaca fuscata; two female, one male) weighting from to kg were used The animals were fed in the same condition as in the behavioral experiment The ACC neurons were recorded from of these monkeys, and one monkey was used as a partner in a social interaction task All monkeys were treated in strict compliance with the United States Public Health Service Policy on Human Care and Use of Laboratory Animals, and with the Guidelines for the Care and Use of Laboratory Animals at University of Toyama Social interaction task Two monkeys (one recording monkey and other partner monkey) were individually put into the side cages of the same linked cages To prevent direct contact between the recording and partner monkeys so that wiring for neurophysiological recording was not Journal of military pharmaco-medicine 7-2013 disconnected by the partner monkey, only one mesh partition of the side cage for the recording monkey was removed to start social behavioral testing The social behavioral categories were listed in table Communication was defined as a series of the social behaviors, including grooming together, lip smacking, threatening, fighting, and moving around that occurred after they faced each other Neuronal activity and social behaviors were recorded simultaneously The social behaviors were analyzed online by the same computer and software used in the behavioral experiment This computer analyzed data from the CCD camera, emitted TTL signals when one of the behaviors occurred, and sent them to the computer for neuronal recording Social behaviors were also analyzed offline by visual inspection Table 1: Categories and definition of monkeys' social behaviors analyzed in the neurophysiological experiment B e h a v io r s B e h a v io r s - - Approaching 2: The partner monkey approached to the recording monkey - Leaving 2: Partner monkey left from the recording monkey - Grooming 2: The partner monkey groomed by themselves (self-grooming of the partner monkey) - Moving around 2: The partner monkey moved around Approaching 1: The recording monkey approached to the partner monkey - Leaving 1: The recording monkey left from the partner monkey - Grooming 1: The recording monkey groomed by themselves (self-grooming of the recording monkey) - Moving around 1: The recording monkey moved around - Contact: Both monkeys sat close together (distance between the monkeys was less than 10 cm) without social behaviors - Communication: Both the monkeys displayed a series of the social behaviors including grooming together, lip smacking, threatening, fighting, and moving around that occurred after they faced each other - Proximity: Distance between the monkeys was 10 - 60 cm Surgery and training After acclimation of the monkeys, a head-restraining device (a U-shaped resinoid plate) was surgically attached to the skull under aseptic conditions The subject was anesthetized with a combination of medetomizine hydrochloride (0.5 mg/kg, i.m.) and ketamine hydrochloride (5 mg/kg, i.m.) The plate was anchored with dental acrylic to tungsten bolts inserted in the skull During the surgery, heart and respiratory functions and rectal temperature were monitored (LifeScope14, Nihon Kohden, Tokyo, Japan) A blanket heater was used to keep body temperature at 36 ± 0.5°C Antibiotics were administrated topically and systemically for week to prevent infection After one month when monkeys recovered from surgery completely, MRI or X-ray of the heads of the monkeys were taken to locate X-Y coordinates of the ACC Then, the monkeys were trained to sit on a monkey chair with the head painlessly fixed on the stereotaxic apparatus using the head-restraining device * Electrode implantation: The recording electrode assembly consisted of tetrodes (tungsten wire, 20 μm in Journal of military pharmaco-medicine 7-2013 diameter; impedance, 200 - 400 kΩ at kHz) (California Fine Wire, Grover Beach, CA) encased individually in a set of stainless steel guide tubes (33 gauge; Small Parts, Miami, FL) and a microdrive which consisted of a screw coupled with a molded nut attached to the guide tubes A full turn of the screw advanced the electrodes by approximately 400 µm After training for the monkeys, recording electrode assemblies were stereotaxically implanted above the ACC under anesthesia The recording assemblies were covered by a thin film of white petrolatum, and were fixed by dental cement Neurophysiological recording Five days after implantation of the tetrodes, the head of monkey was fixed painlessly on the stereotaxic apparatus on the monkey chair Then, the implanted tetrodes were lowered into the ACC, using the microdrive while neuronal activity was monitored on an oscilloscope The tetrodes were lowered in each step of 20 µm with a pause of between the steps The maximum number of steps within day was 16 (i.e., 320 µm) to minimize damages in the brain Neuronal activity was passed through a high input impedance preamplifier, amplified, and monitored on the oscilloscope Only neuronal activities with signal-to-noise greater than 2.5:1.0 were used When such neuronal activity was detected, the monkey was moved to the linked cage If the neuronal activity was still present for more than 30 in the cage, it was judged to be stable and suitable for recording The analog signals of neuronal activities, triggers for behavioral events emitted from the computer for behavioral analysis, and video signals from another CCD camera were digitized and stored in a computer via a Multichannel Acquisition Processor (MAP, Plexon Inc., Dallas TX) system The amplified neuronal signals were digitized at a 40 kHz sampling rate, and 1.0-msec waveforms that crossed an experimenter-defined threshold were stored on a computer hard disk for off-line spike sorting The digitized neuronal activity was isolated into single units by their waveform components using the Offline Sorter program (Plexon Inc.) Superimposed wave forms of the isolated units were drawn to check their consistency throughout the recording sessions, and then were transferred to the NeuroExplorer program (Nex Technology, Littleton, MA) for further analysis Typically, one to two single units were isolated by means of off-line cluster analysis from channels (wires) of one tetrode Spike sorting was performed with the Offline Sorter program (Plexon Inc.) The waveforms in the tetrode were concatenated (end-to-end) to make one quad-length waveform, and a principal component analysis was performed based on the concatenated data points Therefore, all the principal components were calculated based on the total data derived from the tetrode Each cluster was then checked manually to ensure that the cluster boundaries were well separated and the waveform shapes were consistent with the action potentials For each isolated cluster, an interspike interval histogram was constructed and an absolute refractory period of at least 1.0 msec was used to exclude suspected multiple units Finally, superimposed waveforms of the isolated units were drawn to check the consistency of the waveforms Data analysis For a 2.0 sec period before and after the behavioral onsets, the mean neuronal firing rate was calculated every 100 msec All data are expressed as means ± SEM Significant excitatory or inhibitory responses to each stimulus were defined by a Wilcoxon signed rank (WSR) test (p < 0.05) of neuronal activity between the pre and post sec around the behavioral onsets Response magnitudes were defined as follows; mean firing rates during sec after behavioral onsets minus mean firing rates during sec before behavioral onsets Journal of military pharmaco-medicine 7-2013 Results General A total of 86 neurons were recorded from the ACC of the monkeys Of these, 11 neurons responded to social behaviors (leaving, approaching, contact, communication), while no neurons responded to non-social behaviors (self-grooming, moving around) Figure 1: An example of neuronal activity of an ACC neuron A Waveforms Ch Ch Ch Ch 50 µV 100 µsec B C Autocorrelogram Cluster cutting PC PC 40 20 20 Time (msec) 40 Figure shows an example of raw records of an ACC neuron Typical waveforms of one ACC neurons are shown in Fig 1A, which were simultaneously recorded from the same tetrode (Ch 1-4) Figure 1B shows the results of spike sorting by off-line cluster cutting of the neural activity shown in Fig 1A Each dot represents one spike, and clusters of dots encircled by dotted lines was easily recognized Figure 1C indicates an autocorrelogram of the neuron shown in Fig 1B The autocorrelogram indicated that a refractory period of the neuron was - msec, which indicated that these spikes were recorded from a single neuron Responses to social behaviors The responses of the 11 neurons related to social behaviors fell into categories: leaving-related (n = 7), approaching-related (n = 1), and communication-related neurons (n = 4) Activity of neuron was related to both leaving and approaching Activity of the leaving- Journal of military pharmaco-medicine 7-2013 and approaching-related neurons changed in response to leaving and approaching behaviors of the recording and/or partner monkeys, respectively Activity of the communication-related neurons changed while the monkeys engaged in a series of mutual social behaviors such as grooming together, lip smacking, facing, etc Of the leaving-related neurons, neurons responded when the recording monkey left from the partner monkey (Leaving 1) (3, excitatory; 1, inhibitory), neurons responded when the partner monkey left from the recording monkey (Leaving 2) (all, excitatory) Responses of these neurons to leaving behaviors of the recording monkey (Leaving 1) were -4 -2 sec -2 -4 -2 sec D Approaching2 B Leaving2 -4 E Grooming1 C Approaching1 A Leaving1 sec G Proximity -4 -2 sec H Contact -4 -2 sec F Grooming2 -4 -2 sec K Communication -4 -2 sec -4 -2 sec -4 -2 sec illustrated in Fig Figure 2: Responses of neuron to leaving behaviors of the recording monkey The activity of the neuron was specifically inhibited in response to Leaving1 (A), but not to Leaving (B) However, the neuron did not respond to approaching behaviors of the recording monkey (Approaching 1, C) nor to those of the partner monkey (Approaching 2, Journal of military pharmaco-medicine 7-2013 Contact Proximity Grooming2 Grooming1 Communication 0.0 Approaching2 Leaving2 1.6 Leaving1 A Approaching1 D) The neuron also did not respond to grooming behaviors of the recording monkey (Grooming 1, E) nor to those of the partner monkey (Grooming 2, F) Furthermore, the activity of the neuron did not change when both the monkeys were located within a distance of 60 cm (Proximity, G), when one of the monkeys touched the other monkey (Contact, H), nor when both the monkeys displayed a series of the social behaviors (Communication, K) The response magnitudes of the neuron to various behaviors were compared in Fig 4A Communication -3.2 -4.8 B Contact Proximity Grooming1 Grooming2 0.0 Approaching2 2.0 Leaving2 4.0 Approaching1 6.0 Leaving1 Change in firing rate (spikes/sec) -1.6 -2.0 Figure 4: The response magnitudes of the neuron to various behaviors All of the communication-related neurons displayed excitatory responses Figure illustrates an example of the communication-related neurons Activity of the neuron increased specifically in response to Communication (K) The response magnitudes of the neuron to various behaviors were summarized in Fig 4B The activity of the neuron significantly increased in response to communication Journal of military pharmaco-medicine 7-2013 F Sagittal igure 5: Coronal Electrode Recordin g sites of ACC neurons F igure shows the ACC recording ACC sites of the ACC neurons Most ACC neurons were recorded from the rostral part of the ACC anterior (pregenual) to the genu of the corpus callosum These areas correspond to the areas 24a, 24b, 24c, and area 32 A B Discussion Characteristics of the ACC neurons Activity of some ACC neurons was correlated with the specific behaviors of the recording monkeys (i.e approaching, leaving and communication) These activities were not ascribed to general locomotion since activity of the neurons was selective to approaching or leaving, but not both behaviors Furthermore, movement direction and social relations are important factors to characterize approaching, and leaving behaviors; approaching and leaving are to move toward and away from the other monkey, respectively This selectivity to movement directions further supports the idea that these neuronal activities were related to social behaviors, but not to general locomotion On the other hand, it has been reported that the cingulate motor areas (CMA) are located in the mid part of the cingulate cortex and involved in the cognitive control of voluntary motor behaviors [10], and CMA neurons became active during various voluntary actions, and responded to external targets for selecting an appropriate action It is noted that the ACC neurons in the present study were different from those in the CMA; the ACC neurons were located in the gyrus of the rostral part of the ACC, while the CMA neurons were located in the cingulate sulcus posterior to the rostral part of the ACC Activity of some ACC neurons in the cingulate gyrus was correlated with specific social behaviors of the partner monkeys, but not all social behaviors Previous neurophysiological studies reported that visually-responsive neurons to various non-social and emotional (rewarding and aversive) stimuli were located in the rostral part of the monkey ACC [7] However, these neurons were mainly located in the cingulate sulcus [7], and few neurons in the gyrus of the rostral part of the ACC responded to those stimuli Furthermore, the ACC neurons did not respond to non-social behaviors such as self-grooming in the Journal of military pharmaco-medicine 7-2013 present study These results suggest that these activity changes could not be ascribed to non-specific arousal responses, and the rostral and gyral part of the ACC might be related specifically to social behaviors Functional topography in the ACC The neurons related to social behaviors were found in the rostral part of the ACC This part of the ACC has intimate anatomical connections with the emotion-and social cognition-related areas such as the amygdala in monkeys [8] Findings from human fMRI and neurophysiological studies suggest that the ACC could be divided into the subdivisions; emotional tasks activated the rostral part of the ACC (affective division), while the cognitive tasks activated the caudal part of the ACC (cognitive division) [3] Rodent lesion and pharmacological studies also support the divisions of the ACC [5] The recording area in the present study corresponded to the affective part of the human ACC The present results are consistent with the above previous human studies on the ACC Although there have been no studies that made selective lesions in the rostral part of the ACC in monkeys, lesions that involved at least the rostral part of the monkey ACC induced deficits in social and emotional behaviors Further studies performing selective lesions within the ACC are required to investigate functional differentiation in the ACC The role of ACC in social interaction Recent human studies using noninvasive imaging techniques suggest that the medial prefrontal cortex including the ACC is involved in social cognition and social behaviors [9] The ACC, especially its rostral and gyral part was activated during various social cognition tasks including the prisoner’s dilemma tasks, social judgments, and mentalizing Furthermore, activity of the medial prefrontal cortex including the rostral ACC increased in response to social gaze shifts compared with unsocial gaze shifts [2] These results suggest that the rostral part of the ACC is critical for normal social behaviors Consistent with these imaging studies, previous lesion studies indicated that the ACC, especially its gyral part, was critical for normal patterns of social interaction in monkeys [9]; ACC lesions reduced socially interactive behaviors, time spent in proximity with other individuals, and interest in social stimuli such as other conspecifics In humans, damages of the prefrontal cortex including the ACC induced changes in face expression identification and social behaviors, and disturbed performance in a theory of mind task [4] These studies were consistent with the present results indicating that the neurons related to social behaviors were located at least in the rostral and gyral part of the ACC Conclusions The neurophysiological results indicated that the neurons correlated with social behaviors were recorded from the rostral part of the anterior cingulate cortex, which is one of the critical brain areas for social cognition and most reactive to NMDA antagonists such as PCP The present results provide neuroscientific bases for cingulate involvement in social behaviors and schizophrenia References Borgwardt SJ, McGuire P, Fusar-Poli P, Radue EW, Riecher-Rössler A Anterior cingulate pathology in the prodromal stage of schizophrenia Neuroimage 2008, 39, pp.553-554 Bristow D, Rees G, Frith CD Social interaction modifies neural response to gaze shifts Soc Cogn Affect Neurosci 2007, 2, pp.52-61 Davis KD, Taylor KS, Hutchison WD, Dostrovsky JO, McAndrews MP, Richter EO, Lozano AM Human anterior cingulate cortex neurons encode cognitive and emotional demands J Neurosci 2005, 25, pp.8402-8406 Journal of military pharmaco-medicine 7-2013 Hornak J, Bramham J, Rolls ET, Morris RG, O'Doherty J, Bullock PR, Polkey CE Changes in emotion after circumscribed surgical lesions of the orbitofrontal and cingulate cortices Brain 2003, 126, pp.1691-1712 Malin EL, Ibrahim DY, Tu JW, McGaugh JL Involvement of the rostral anterior cingulate cortex in consolidation of inhibitory avoidance memory: interaction with the basolateral amygdala Neurobiol Learn Mem 2007, 87, pp.295-302 Margulies DS, Kelly AM, Uddin LQ, Biswal BB, Castellanos FX, Milham MP Mapping the functional connectivity of anterior cingulate cortex Neuroimage 2007, 37, pp.579-588 Matsumoto M, Matsumoto K, Abe H, Tanaka K Medial prefrontal cell activity signaling prediction errors of action values Nature Neurosci 2007, 10, pp.647-656 Pandya DN, Van Hoesen GW, Mesulam MM Efferent connections of the cingulated gyrus in the rhesus monkey Exp Brain Res 1981, 42, pp.319-330 Rushworth MF, Behrens TE, Rudebeck PH, Walton ME Contrasting roles for cingulate and orbitofrontal cortex in decisions and social behaviour Trends Cogn Sci 2007, 11, pp.168176 10 Walton ME, Mars RB Probing human and monkey anterior cingulate cortex in variable environments Cogn Affect Behav Neurosci 2007, 7, pp.413-422 ... when one of the behaviors occurred, and sent them to the computer for neuronal recording Social behaviors were also analyzed offline by visual inspection Table 1: Categories and definition of monkeys'... recording monkey approached to the partner monkey - Leaving 1: The recording monkey left from the partner monkey - Grooming 1: The recording monkey groomed by themselves (self-grooming of the... responded to social behaviors (leaving, approaching, contact, communication), while no neurons responded to non -social behaviors (self-grooming, moving around) Figure 1: An example of neuronal