A receptors promotes sleep, whereas their antagonism enhances wakefulness. We explored whether GABAergic mechanisms present in the posterior hypothalamus contribute to the homeostatic and other aspects of sleep-wake regulation. Using micropunches of tissue extracted from either the perifornical (PF) or dorsomedial (DM) regions of the posterior hypothalamus of rats, we determined that mRNA levels for selected subunits of GABAA receptors  were higher at the end of the active period or following sleep deprivation, when the need for sleep is high, than after several hours of sleep, when sleep need is partially fulfilled. Such a pattern was present in the PF region only, and was consistent with changes in β1 subunit and GABA synthesizing enzyme (GAD) protein levels. In contrast, in the DM region, the levels of GABAA receptor subunit mRNAs and proteins  and GAD varied with circadian time, but were not responsive to sleep deprivation. Separate experiments with sleep-wake monitoring and local perfusion of the PF region with the GABAA receptor antagonist bicuculline revealed that the antagonist had a weaker sleep-reducing effect when sleep need was enhanced by sleep deprivation and that the increased amount of REMS characteristic of the late sleep period was dependent on endogenous GABAergic inhibition. These results support the concept that a varying magnitude of GABAergic inhibition exerted within the PF region contributes to the homeostatic regulation of sleep and shapes its temporal pattern, whereas GABAergic mechanisms in the DM region contribute to circadian regulation.Sleep-wake behavior is regulated by a circadian rhythm, homeostatically and by additional mechanisms that determine the timing of slow-wave sleep and rapid eye movement sleep (REMS) episodes. The posterior hypothalamus coordinates the neural and humoral signals with the rest-activity cycle. It contains wake-active neurons, and is a site where stimulation of inhibitory GABA Sleep-wake behavior is regulated by the circadian rhythm and homeostatic drive, with the strength of the latter increasing with the duration of prior time spent awake A receptors (GABAARs) located in the PF region with the agonist muscimol promotes SWS The perifornical (PF) region of the posterior hypothalamus plays an important role in the control of sleep and vigilance and coordinates multiple behaviors that are closely linked to the sleep-wake cycle AR expression in the PF region may increase with the duration of wakefulness and that this may be an important mechanism underlying increased pressure for sleep. Specifically, it appeared plausible that a prolonged wakefulness could lead to an increased expression (or sensitivity) of GABAARs located in the PF region. This, in turn, would enhance the strength of GABAergic inhibition at this site and translate into a stronger sleep-promoting signal, increased subjective sleepiness and deeper sleep. We previously tested selected aspects of this hypothesis by superfusing posterior hypothalamic slices in vitro with either GABAergic agonists (to mimic a period of increased GABA release during sleep), or a GABAAR antagonist (to mimic a state of reduced inhibition) ARs in the PF region.Importantly, in cats made insomniac by anterior hypothalamic lesions, the sleep-promoting effect of GABAergic inhibition exerted within the posterior hypothalamus was augmented in vivo conditions of experimentally varied sleep drive. We quantified selected mRNA and protein levels important for GABAergic transmission in the PF and the adjacent dorsomedial/paraventiricular (DM) region of the posterior hypothalamus. We then functionally tested whether the effects on sleep of pharmacological antagonism of endogenous inhibition mediated within the PF region by GABAARs vary with the pressure for sleep. We found that the strength of GABAergic inhibition in the PF region increases when the drive for sleep is increased, that changes in GABAAR expression likely contribute to this process, and that sleep drive-related changes in the PF inhibition mediated by GABAARs differentially affect SWS, REMS and motor activity during wakefulness. Preliminary results have been published Our present goal was to test our hypothesis under the All animal procedures followed the guidelines of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania (Protocols no. 704337 and 803579). All surgical procedures were performed under anesthesia and with continuous monitoring to ensure stable and pain-free conditions. All efforts were made to minimize any animal discomfort.ad lib access to food and water. All experimental procedures were conducted during the lights-on period, which for rats is the natural sleep/rest period. The 23 animals used in the mRNA studies and the 28 animals used in the protein expression studies were divided into three groups: (1) the “AM” group was sacrificed at 9 am ; (2) the “PM” group was sacrificed between 3 and 4 pm following undisturbed sleep ; and (3) the “PM-SD” group was sacrificed between 3 and 4 pm following 6 h of sleep deprivation (SD) applied from 9 am to 3 pm . To maintain an undisturbed social environment, the rats used in these studies were housed in pairs. During SD, both rats were kept in their home cage, with only one animal targeted for SD until the time when it was euthanized prior to the tissue extraction procedure. SD was achieved by presenting the animals with a novel object  or stroking the targeted animal with a soft brush whenever it assumed a curled posture indicative of intent to enter sleep. Three rats were sacrificed at 3∶00 pm after undisturbed sleep and transcardially perfused; forebrain slices were immunohistochemically stained for glutamic acid decarboxylase . An additional 7 rats were instrumented and, after habituation, their sleep-wake behavior was monitored under different conditions of sleep need, with or without perfusion of the PF region of the posterior hypothalamus with the GABAAR antagonist, bicuculline.The study was conducted with 61 adult male Sprague-Dawley rats (300–370 g) obtained from Charles River Laboratories . The animals were housed on a 12∶12 light/dark schedule with lights on at 7 am and 2, 1 MgCl2, 10 HEPES, 10 glucose, and 20 mannitol; pH 7.4, osmolarity 300±5 mOsm Rats were deeply anaesthetized with isoflurane (3.5%) and decapitated. The forebrain was then rapidly removed and immersed in ice-cold medium containing (in mM): 135 NaCl, 5 KCl, 1 CaClProtein samples were similarly harvested from the PF and DM regions of the posterior hypothalamus from 500 µm-thick slices. They were frozen on dry ice and stored at −80°C. The slices from which the punches were taken were fixed in 10% paraformaldehyde, cryoprotected in 30% sucrose, cut into 25 µm sections, mounted on slides and stained with Neutral red.AR subunits, GAD isoforms 65 and 67, prepro-orexin, and α-tubulin  procedures used for mRNA quantification were described previously PCRs were calibrated using external cDNA standards obtained by amplification of the target cDNAs synthesized from total rat hypothalamic RNA, purification of the product with a gel extraction kit , and then densitometric determination of cDNA concentration in the master standard solution. Calibration curves were then generated for each transcript as previously described A receptor  and β-actin  at 4°C. Primary antibody binding was visualized using donkey enhanced chemiluminescence anti-rabbit horseradish peroxidase-conjugated IgG  and chemiluminescent substrate . Chemiluminescence was detected using autoradiography film . Following documentation of β1 subunit immunostaining, the membranes were washed in TBS and stripped of antibodies by incubation in a stripping buffer (Pierce/Thermo Scientific). The membranes were then washed and incubated overnight with mouse anti-GAD antibody against both 65 kDa and 67 kDa isoforms . They were subsequently incubated with sheep enhanced chemiluminescence anti-mouse horseradish peroxidase-conjugated IgG , and immunostaining was again visualized by chemiluminescence. Films were scanned with 8 bit grayscale resolution, distinct bands were quantified densitometrically  and, for each sample, the amounts of the β1 subunit and GAD65/67 proteins were expressed relative to the optical density of the band for β-actin.For protein quantification, each micropunch sample was sonicated in 12 µl of a solubilizing buffer containing 7 M urea, 2 M thiourea, 0.25 mM Tris base, 4.0% CHAPS and 1% NP-40. The soluble fraction of the homogenate was loaded onto precast polyacrylamide Tris-HCl minigels  in a buffer containing 100 mM Tris-HCl, 200 mM DTT, 4% SDS, 0.2% bromophenol blue and 20% glycerol. Proteins were separated and transferred to nitrocellulose membranes . Membranes were blocked for 60 min in Tris-buffered saline (TBS) containing 5.0% non-fat dry milk and 0.1% Tween-20 (pH 7.4) and then incubated overnight in rabbit antibodies against the β1 subunit of the GABAAll mRNA and protein data sets were tested for normality using the Shapiro-Wilk test. The significance of differences between the conditions was examined using one-way ANOVA with Bonferroni correction. When normality criteria were not fulfilled, nonparametric Kruskal-Wallis ANOVA with Bonferroni correction was applied. Differences were considered significant at p<0.05 . As is customary in sleep and circadian research, mRNA and protein changes were classified as circadian-related when their levels changed with the phase of the circadian cycle but were insensitive to changes in sleep drive, or as sleep-related, if their levels responded to manipulations of the prior amounts of sleep and wakefulness while showing a lesser or no dependence on the circadian time.2, 1.9 MgCl2.Seven rats were anesthetized with ketamine (60 mg/kg) and xylazine (7.5 mg/kg), with anesthesia subsequently maintained with isoflurane (0.5–0.75%). Under aseptic conditions, they were implanted for recording of the cortical EEG, hippocampal activity and neck EMG, as described previously A receptor antagonist bicuculline methiodide . Individual recordings sessions were conducted either between 8∶30 am and 13∶30 pm (AM sessions) or between 11∶30 pm and 4∶30 pm (PM sessions). Prior to selected PM sessions, the animals were subjected to SD from 8∶00 am to noon; SD was continued during the first 30 min after the animal was connected to the recording apparatus. While subjected to SD, the animal remained in its home cage and was gently prompted to maintain wakefulness in the same way as with the animals designated for tissue extractions. Motor activity was quantified in successive 10 min intervals using an infrared beam crossings technique, with the beams spaced at 1 inch in the horizontal plane and each crossing counted as a separate event .All signals were amplified  and digitally acquired using Spike 2 software  at a rate of 100 Hz for the EEG and hippocampal activity, and 1000 Hz for the EMG. All recordings lasted 5 h and included continuous perfusion of the PF region with either acsf or the GABAARs within a limited region surrounding the tip of the perfusion probe.Hypothalamic microperfusion was conducted using a cannula with a 1 mm-long, semi-permeable membrane . A continuous flow of either acsf or BIC at a rate of 0.8 µl/min was maintained throughout each recording session . Each animal was subjected to six time-of-the-day/drug/handling conditions  that were applied in a random order and separated by at least two days. During the AM sessions with hypothalamic perfusion with BIC, the probe was initially perfused with acsf and then, using a liquid switch , BIC flow was switched on at 9∶30 am and switched back to acsf at 12∶30 pm. Similarly, during the PM sessions with BIC, the probe was initially perfused with acsf, and then BIC flow was switched on at 12∶30 pm and switched back to acsf at 3∶30 pm; these times represent the times when the new solution reached the brain after accounting for the delay in the perfusion system. Sufficiently high concentrations of bicuculline perfused into the PF region can strongly suppress sleep and elicit a high level of motor activity At the end of the study, the animals were deeply anesthetized with pentobarbital sodium  and were transcardially perfused with phosphate-buffered saline followed by 4% paraformaldehyde. The forebrains were cryoprotected in 30% sucrose, sectioned in the coronal plane into 35 µm sections, serially mounted, and stained with Neutral red to verify the location of the perfusion site.Sleep-wake states were scored in 10 s epochs as wakefulness, SWS and REMS on the basis of the appearance of the cortical, hippocampal and EMG signals, with the scoring process assisted by simultaneous display of signal powers in selected frequency bands , as described previously AR subunits present in the posterior, lateral hypothalamus ad lib and the SD group.We quantified the effects of circadian time and sleep drive on mRNA levels for most GABAIn contrast to α subunits, the mRNA levels of β1, β3 and ε subunits did not significantly differ between the 9 am and 3–4 pm groups when compared under the conditions of undisturbed sleep, but they were significantly increased in the 3–4 pm group subjected to SD when compared to the same circadian time after undisturbed sleep. Also in contrast to α subunits, the increases occurred in the PF region only, whereas in the DM region there was no evidence of an effect of either time of day or SD on these subunits . The β2 AR subunit mRNA levels in the PF and DM regions depicted in AR subunit mRNAs, GAD65 and prepro-ORX mRNA levels were all lower, or tended to be lower, in both regions at 3–4 pm when compared to 9 am. The difference for GAD65 mRNA was significant for both regions . In contrast, following SD, the β1 subunit protein level significantly increased in the PF, but not the DM, region when compared to the 3–4 pm group with undisturbed sleep  region contain GABAAR in which some subunits increase with increased need for sleep and decrease when sleep need is partially discharged. In contrast, in the more dorsomedially located posterior hypothalamic region (DM), selected GABAAR subunits varied with circadian time, but not with sleep need when it was increased by SD. Furthermore, the mRNA levels for the GABA synthesizing enzymes, GAD65 and 67, varied in both the PF and DM regions with circadian time, but the combined GAD65/67 protein level increased also in response to SD only in the PF region. Consistent with mRNA and protein quantification results, we found that perfusion of the PF region with an antagonist of GABAARs significantly reduced sleep under normal sleep conditions but was less effective when sleep need was enhanced by SD. Lastly, we found that inhibition mediated by GABAARs within the PF region contributes to the progressive increase in the amount of REMS over the duration of a sleep period. Collectively, these results identify dynamic changes in GABAergic mechanisms residing in the PF region of the posterior hypothalamus as potentially important for the homeostatic and time-dependent regulation of sleep and its sub-stages, and GABAergic mechanisms in the DM region of the posterior hypothalamus as important for circadian regulation. In addition, our study specifically suggests that GABAARs containing β1, β3 and ε subunits located in the PF region contribute to sleep regulatory functions.A direct comparison of the pattern of changes in mRNA and protein levels for selected GABAWe designed our study around the comparison between the PF and DM regions of the posterior hypothalamus because the two regions are adjacent to each other and influence a wide range of fundamental regulatory processes that impact the entire organism but have different afferent and efferent connections and different compositions of prevailing cell phenotypes. The PF region receives projections from sleep-active, anterior hypothalamic GABAergic neurons AR, as well as GAD65 and prepro-ORX. Thus, in addition to a clear prevalence of sleep need-sensitive effects in the PF region, certain cells and certain types of GABAARs in both regions participate in circadian regulation. Our data do not provide information about the neurochemical nature of the cells in which changes in GABAARs and GAD occurred, but studies of the distribution of individual GABAAR subunits among hypothalamic cells of different phenotypes offer some guidance. Both ORX and MCH cells express the ε subunit AR subunits and GAD that we found occurred in ORX, MCH and GABAergic neurons located in the PF region, but additional studies at the single cell level are needed to unequivocally associate the PF and DM cells in which GABAAR expression and/or composition distinctly vary with the need for sleep or circadian time.We found an upregulation of mRNA  and protein (β1 subunit and GAD65/67) following SD in the PF region only. Circadian time-related, but sleep need- independent, molecular variations occurred to varying degree in both the PF and DM regions and involved mRNA levels for α1, α2 and α3 subunits of GABAA receptors are pentameric ion channels typically composed of α, β, γ, δ and/or ε subunits, with a total of 19 different subunit types and subtypes available for building receptors with different sensitivities to various modulators  and different dynamic properties ARs with different properties and subunit compositions ARs with different subunit compositions, albeit currently limited, is important for designing improved therapeutic interventions for sleep disorders AR β3 subunit had a normal amount of SWS but a reduced REMS amount during the rest period while their circadian rhythm remained intact for many days even when they were housed under constant dark conditions AR β3 subunit AR α3 subunit-knockout mice A receptors, genetic removal of type B GABA receptors from ORX neurons resulted in fragmentation of sleep-wake episodes but the total amounts of sleep and its circadian pattern were not affected ARs, and the β3 subunit in particular, for the regulation of both SWS and REMS sleep, but they do not provide information about the anatomic and cellular location of the relevant GABAARs. In addition to our data pointing to the PF region of the posterior hypothalamus, an increased immunostaining for the β2/3 subunits of GABAAR was found following SD on neuronal membranes of basal forebrain cholinergic neurons GABAAR containing the β3 subunit, GABAARs located in the PF region and containing the β1 and ε subunits also responded to increased need for sleep. The relevance of our finding that two such subunits were of the β type may be related to the distinct role of these subunits in the assembly of new functional GABAARs, including their phosphorylation and trafficking to the cell surface In addition to the GABAIt has been suggested that, compared to the GAD67, GAD65 levels undergo more rapid changes in response to a varying demand for GABA as a transmitter In vivo molecular changes, such as those detected in the present study, could be driven by circadian inputs orchestrated by the circadian clock, by a sleep-related increase in extracellular GABA levels, or by cellular activity related to the rest-activity cycle. Indeed, our data suggesting a negative feedback between the magnitude of GABAergic inhibition and both the availability of GABAARs and GABA synthesis may represent a special case of the broader concept proposed to explain the mechanisms of plasticity in the central nervous system in which a homeostatic component reactive to varying magnitudes of inhibitory and excitatory inputs plays a central role AR subunit mRNA levels exhibit region- and subunit-specific changes in response to prolonged, systemic elevation of brain GABA levels ARs in different regions of the posterior hypothalamus probably depend on both the nature and timing of the afferent inputs to cells in these two regions and on local interactions. We previously assessed the effects of stimulation or antagonism of GABAARs on GABAAR subunit and GAD mRNAs in the PF and DM regions of the posterior hypothalamus in slices in vitro, which effectively separated the studied regions from the main circadian pacemaker and eliminated any behavioral effects AR subunits and GAD65 in both regions, but the effect was partially or fully antagonized by a selective GABAAR antagonist, gabazine, only in the PF region and for only selected mRNAs . Furthermore, the mRNA level for the β1 subunit of the GABAAR was reduced following incubation of slices with muscimol after synaptic interactions were blocked by tetrodotoxin. These results demonstrated that local interactions occurring within the posterior hypothalamus and involving stimulation of GABAARs can provide negative feedback for the regulation of the strength of GABAergic inhibition in the PF region, whereas in the DM region inhibition mediated by non-GABAAR-dependent mechanisms has a more prominent role.The effects of circadian time and amount of prior wakefulness on GABAARs and reduced GABA synthesis following an increased stimulation of these receptors can provide a negative feedback that would facilitate termination of sleep and restoration of wakefulness. While such a mechanism can operate in many brain areas important for the regulation of sleep, here we provide specific evidence for the wake-promoting region of the posterior hypothalamus being one such a site and for a particular role of GABAARs that contain β1, β3 and ε subunits. ARs, GABA levels and wake-active cells located in the PF region as they may contribute to the regulation of sleep in response to the prior period of wakefulness , as well as in response to circadian, ultradian and other behavioral cues. We propose that, in the PF region, GABA level increases in presynaptic terminals during prolonged wakefulness (through synthesis) and then declines during sleep (through depletion); and that the availability of GABAARs that contain β1, β3 and/or ε subunits on wake-active PF neurons increases when these cells are active and declines as a result of prolonged stimulation of these receptors. Accordingly, when wakefulness is extended by SD, more GABA accumulates in presynaptic terminals and additional GABAARs are synthesized in wake-active neurons (cf., ARs have now been internalized and recycled. As a consequence, sleep can be terminated by relatively weak sensory or endogenous signals and, once awakening occurs, wakefulness is relatively easy to maintain. Our present data show that the interactions incorporated in the scheme in ARs in distinct neuronal populations involved in the regulation of sleep should help further identify the cell types and connections that represent the key substrates of the brain “sleepiness signal.”A downregulation of GABAons cf., . This trons cf., , GABA leAR antagonist at different circadian times and under varying pressure for sleep yielded results consistent with our data showing that a larger pool of GABAARs is available at the end of the active period and even more so after an extended period of wakefulness than when the need for sleep is partially discharged during the second half of the sleep period. Perfusion with a GABAAR antagonist reduced sleep most powerfully under the latter conditions. Considering that we used a relatively low concentration of the antagonist, our findings can be explained on the basis of the interaction between GABA levels and the availability of free and occupied GABAARs on wake active neurons, as schematically shown in AR antagonism in the PF region on sleep is only secondary to sleep need-related processes occurring elsewhere. In support of the latter explanation, c-Fos data suggested that activity of anterior hypothalamic GABAergic neurons that play a key role in the maintenance of sleep is elevated following SD AR-dependent ARs and GAD occur within the PF region shows that negative feedback suitable for a systemic regulation of sleep-wake states can operate in this region. Such a feedback probably converges on multiple wake-active neurons of this region, including ORX and non-ORX excitatory cells whose stimulation leads to multiple behavioral, cardiovascular, respiratory and metabolic signs of arousal Our experiments with microperfusion of the PF region with a GABAAR antagonist had a particularly powerful REMS-reducing effect when it was applied during the second half of the sleep period (AR antagonist was applied to the PF region during the second half of the sleep period despite the natural for this period increase of REMS amount. This suggests that time-dependent changes in inhibition mediated in the PF region by GABAARs importantly contribute to the progressively increasing amount of REMS with the duration of sleep.Our finding that local perfusion of the PF region with a GABAAR availability and levels of activity in wake-active neurons of the PF region interact in such a way that extended wakefulness leads to an increased potential for inhibition of wake-related neurons. The specific GABAARs that are involved in these processes are different from those in the DM hypothalamic region that is important for the regulation of various circadian time-dependent functions but not directly involved in the homeostatic regulation of sleep.Our data demonstrate a potential for GABAergic transmission in the sleep-regulatory PF region of the posterior hypothalamus to vary with both the homeostatic and circadian regulation of sleep. We postulate that dynamically changing levels of GABA, GABA


Alexithymia is a personality trait characterized by difficulties in the cognitive processing of emotions (cognitive dimension) and in the experience of emotions (affective dimension). Previous research focused mainly on visual emotional processing in the cognitive alexithymia dimension. We investigated the impact of both alexithymia dimensions on electrophysiological responses to emotional speech in 60 female subjects.During unattended processing, subjects watched a movie while an emotional prosody oddball paradigm was presented in the background. During attended processing, subjects detected deviants in emotional prosody. The cognitive alexithymia dimension was associated with a left-hemisphere bias during early stages of unattended emotional speech processing, and with generally reduced amplitudes of the late P3 component during attended processing. In contrast, the affective dimension did not modulate unattended emotional prosody perception, but was associated with reduced P3 amplitudes during attended processing particularly to emotional prosody spoken in high intensity.Our results provide evidence for a dissociable impact of the two alexithymia dimensions on electrophysiological responses during the attended and unattended processing of emotional prosody. The observed electrophysiological modulations are indicative of a reduced sensitivity to the emotional qualities of speech, which may be a contributing factor to problems in interpersonal communication associated with alexithymia. Alexithymia is a personality trait characterized by difficulties in the cognitive processing and experience of emotions. With a prevalence rate of up to 10 percent The term alexithymia (‘no words for feelings’) was coined by Sifneos Individuals with alexithymia show a paucity of facial emotional expressions and a somewhat stiff wooden posture The right hemisphere is thought to be more involved in emotion processing than the left hemisphere Electrophysiology with its extremely high temporal resolution in the range of milliseconds is an excellent means to investigate how emotional processing unfolds in time and can give information about whether alexithymia primarily affects overt, appraisal-related aspects of emotional processing or whether it affects already the more automatic, early perceptual-related aspects. The findings of studies employing event-related potentials (ERPs) to address this question will be summarized below. However, it should be kept in mind that previous ERP studies mostly relied on the 20-item Toronto Alexithymia Scale (TAS-20) to assess levels of alexithymia. This scale assesses only the cognitive alexithymia dimension, i.e. difficulty identifying, analyzing, and verbalizing feelings. Therefore, previous findings of ERP studies on alexithymia primarily refer to cognitive deficits in emotional processing, whereas the impact of disturbances in emotional experience  has remained elusive.Franz et al. Pollatos and Gramann Confirming the observation of an impact of alexithymia on both early and late electrophysiological processing of emotions, Walker and colleagues Taken together, ERP studies investigating visual emotional processing suggested that alexithymia influences both early (<300 ms) and late (>300 ms) emotional processing. At late processing stages, thought to reflect cognitive appraisal of emotion, there is converging evidence for reduced emotional processing as a function of alexithymia as reflected in diminished amplitudes of the later occurring P3 component  and the LPP In contrast to the visual domain, auditory emotional processing has rarely been investigated in relation to alexithymia. A recent behavioral study tested the impact of emotional background music on the recognition of emotion words Emotional prosody, the ‘melody of speech’, is an important means to understand the emotional state and intention of others in social communication. A recent meta-analysis showed that emotional prosody perception, though processed by both hemispheres, is relatively lateralized to the right hemisphere The present study was designed to further investigate the impact of alexithymia on the electrophysiological processing of emotional speech, taking its two dimensions into account. In addition to the attended processing of emotional speech , unattended emotional speech processing was tested . An auditory oddball paradigm was employed in both tasks, in which occasional deviant stimuli (20%) were presented in a sequence of frequent standard stimuli (80%). The relation between alexithymia and abilities to identify emotions conveyed by speech was further tested in a behavioral (off-line) task.ERP components of interest during attended emotional prosody processing (deviant detection) are the early components N1 and P2 as well as the late component P3 The ERP component of interest during early unattended processing of emotional prosody (movie watching with prosodic stimuli played in the background) is the Mismatch Negativity (MMN). The MMN, elicited without the participant's attention, occurs between 100 and 200 ms after the onset of a prosodic stimulus and is generated in secondary auditory cortex and inferior frontal cortex Early components (<300 ms) such as the N1 and the P2 have been suggested before to differ as a function of alexithymia by studies on visual emotional processing In light of the existing evidence, we hypothesized a modulation of the early N1 and P2 components by alexithymia during the detection of deviants in emotional prosody, as well as of the MMN during unattended emotional speech processing. We further predicted a reduced sensitivity during overt processing of emotional speech qualities, reflected in reduced amplitudes of the P3 with increasing alexithymia scores. In addition, we hypothesized to find evidence for a left hemisphere preference during the perception of emotional speech with increasing alexithymia scores. Lastly, we wished to determine whether the cognitive and the affective alexithymia dimensions exert a dissociable impact on the attended and unattended processing of emotional prosody and on the ability to identify emotions conveyed by speech.A purely female sample of participants was chosen taking into account previously demonstrated gender differences in emotional prosody perception at the behavioral as well as at the electrophysiological level The 20-item Toronto Alexithymia Scale (TAS-20) was used as a brief assessment tool of alexithymia scores in a total sample of 1039 female students from the University of Groningen. From this total sample, twenty students with high TAS-20 scores  and 20 students with low TAS-20 scores  were selected and invited to the EEG session, along with 20 students with average scores on the TAS-20 . Extreme (low and high) scorers on the TAS-20 scale were chosen in line with previous studies All participants were healthy female native speakers of Dutch (age range 18–25 years), with no neurological or psychiatric disorders in present or past, normal hearing, and normal or corrected-to-normal vision. Participants received € 20 for their participation in the study.The Medical Ethical Committee of the University Medical Center Groningen approved the experimental protocol, and all participants gave written informed consent prior to the study. The study was conducted in accordance with the Declaration of Helsinki.The TAS-20 is the most widely used measure of alexithymia with a demonstrated validity, reliability, and stability The TAS-20 comprises three subscales assessing alexithymia at a cognitive level: (1) difficulty identifying feelings , (2) difficulty describing feelings , and (3) externally oriented thinking . Possible scores range from 20 to 100, higher scores indicate higher degrees of alexithymia.The BVAQ is a 40-item self-report scale, which consists of five subscales with eight items per scale r = .80) The five-subscale structure of the BVAQ corresponds to the original description of alexithymia by Nemiah and Sifneos An auditory oddball paradigm with 80% standards and 20% deviants was created for the present study. Nonsense syllables  spoken in neutral, happy, angry, sad, and disgusted intonation in low  and high  intensity constituted the stimuli of this paradigm. Nonsense syllables were chosen in order to exclude interference by semantic processing, enabling us to specifically measure electrophysiological responses to variations in emotional prosody. The syllables “baba”, “dada”, “gaga” were chosen because they have the same consonant (C) – vowel (V) structure (CVCV), employ the same vowel and contain only voiced consonants, keeping acoustic features of the stimuli constant across conditions.The stimuli were recorded with the help of a semiprofessional actress, who pronounced the syllables in neutral, happy, angry, sad, and disgusted prosody with low and high emotional intensity. The recorded stimuli were cut to a length of approximately 600 ms and amplitude normalized using the Praat speech processing software The stimuli were validated in two pilot studies with 13 independent raters each. The raters were asked to indicate which emotion was conveyed by the respective stimulus  and which emotional intensity the stimuli were spoken in . Only stimuli that were rated by 10 out of 13 raters to convey the correct emotion in the intended intensity were included in the study.The oddball paradigm was presented in E-Prime version 1.2 EEG activity was recorded from 64 tin electrodes mounted in an elastic electro cap organized according to the international 10/20 system. EEG data were recorded with a linked mastoid physical reference and were re-referenced by using an average reference. Electrooculogram (EOG) was recorded for artefact rejection purposes from electrodes placed on the supraorbital and ridges of the left eye. The ground electrode was applied to the sternum. Impedance of all electrodes was kept below 5 kΩ for each participant. EEG was continuously recorded with a sampling rate of 500 Hz, amplified, and off-line digitally low-pass filtered with a cut-off frequency of 30 Hz.Participants were seated in front of a monitor at a distance of approximately 50 cm in a dimly lit, electrically shielded and sound-attenuated cabin. The auditory oddball paradigm was presented via loudspeakers placed at the left and right side of the monitor at approximately 70 dB, while the EEG was recorded.In task 1, participants watched the first 20 minutes of a silent cartoon movie (title: Kiki's delivery service) and were instructed to focus on the story in the movie while ignoring the sounds. In the oddball paradigm used in task 1, neutral prosody served as standards , while happy, angry, sad, and disgusted emotional prosody spoken in low intensity served as deviants . Only stimuli of lower salience  were used in this task to prevent participants from directing their attention to the auditory stimuli, which enabled us to measure electrophysiological responses to subtle prosodic changes during unattended processing. The ERP component of interest in this task was the MMN.In task 2, participants were instructed to press a button as fast as possible whenever they heard an emotion different from the standard emotion (irrespective of intensity). They were asked to look at a fixation point in the center of the screen to prevent eye movements. In this oddball paradigm, sad emotional prosody spoken with low and high intensity represented the standards , whereas happy, angry, and disgusted emotional prosody  in both intensities served as deviants. Sad emotional prosody instead of neutral intonation was used as standard in this task because the aim of this task was to record ERP responses to actual changes in the perception of one emotional intonation to another as it frequently occurs in real life, rather than a change from a neutral to an emotional intonation. The total duration of task 2 was 32 minutes, and participants could take a break after the first 16 minutes, if needed. The ERP components of interest in this task were the early components N1 and P2, and the late component P3.Task 3 was an off-line task, in which participants were given a list to identify both the emotion a nonsense syllable was spoken in as well as the intensity of the emotion. Fifty-four stimuli were presented at an interstimulus rate of five seconds to give participants sufficient time to mark the identified emotion and intensity on the list.EEG data were analyzed with Brain Vision Analyzer (version 1.05) by means of peak analyses. Prior to averaging, trials with eye-movement and blink artefacts were excluded from analysis. Criteria for artefact rejection were a maximal voltage step of 50 µV, a maximal difference between two values in a segment of 100 µV, and a minimal and maximal amplitude of −100 µV and 100 µV, respectively. All averages were aligned to a 100 ms pre-stimulus baseline. In accordance with the MMN literature, MMN parameters were calculated from a difference waveform obtained by subtracting the standard-stimulus ERPs from the deviant-stimulus ERPs.For task 1, a total mean number of 229.5 deviant trials (SD 6.6) in emotional prosody were recorded for each of the 60 participants, with a mean number of 57.4 trials for happy, angry, sad, and disgusted prosodic deviants, respectively. Artefact rejection excluded a mean percentage of 12.2 percent of all trials, leaving a total mean of 201.5 (SD 8.8) deviant trials for analysis, with a mean number of 50.4 trials per emotional deviant condition. For task 2, a total mean number of 172.9 (SD 5.8) deviant trials in emotional prosody was recorded, with a mean number of 57.6 trials per emotional deviant condition. Artefact rejection excluded a mean percentage of 10.6 percent of all trials, leaving a total mean of 154.6 deviant trials (SD 8.5) for analysis, with a mean number of 51.5 trials per condition for each of the 60 participants.Time-windows for peak detection were time-locked to the onset of standards and deviants. The time-windows were chosen in agreement with the existing literature on the respective ERP component and based on visual inspection of the data. The latter revealed that the MMN during unattended processing of emotional prosody consistently exhibited a double-peak across participants. Thus, two MMN peaks were identified in a time-windows of 50–130 ms post-onset for the first peak (in the following referred to as eMMN) and 130–250 ms post-onset for the second peak (in the following referred to as lMMN). Consequently, separate statistical analyses were conducted for amplitudes and latencies of the detected peaks in the eMMN and the lMMN time-windows.During attended processing of emotional prosody (task 2), peaks were identified in the following time-windows: 90–140 ms post-onset for the N1, 150–250 ms post-onset for the P2, and 310–550 ms post-onset for the P3. Because the P3 does not always exhibit a clear peak, results of the peak detection procedure were inspected in each subject and trials that did not show a clear peak in the defined time-window (due to multiple peaks) were excluded. Statistical analyses were conducted for amplitudes and latencies of the detected peaks in the N1, P2, and P3 time-windows. In order to obtain symmetrical coverage of the scalp during statistical analysis, five midline electrodes were chosen covering frontal through parietal areas  along with their corresponding left  and right  counterparts see .As the results of task 1 show, alexithymia scores were found to modulate ERP responses to sad prosody, which served as standards in task 2. To exclude the possibility that differences in ERP responses to sad standards confounded ERP responses to deviants in task 2, peak amplitudes in response to deviants were subtracted by peak amplitudes in response to standards taking intensity difference into account . This procedure ensured that voltage changes elicited by deviants were measured relative to voltage changes elicited by standards. The procedure further led to better comparability of the results of task 1 and 2, as in both tasks standard-related activity was subtracted from activity elicited by deviants and the resulting difference waves were used in subsequent statistical analyses.Statistical analyses were performed in SPSS version 17.0 . Behavioral data were available from 57 (out of 60) subjects. Two subjects did not complete the behavioral task due to time restrictions as a consequence of technical difficulties at the beginning of the session. One subject with an outlier score (67) on the affective dimension of alexithymia was excluded to ensure continuity of scores. Scores of the remaining 57 subjects ranged from 20 to 55 on the affective alexithymia dimension  and from 27 to 93 on the cognitive alexithymia dimension . Pearson's correlations were performed between each alexithymia dimension and error rates as well as RT during prosodic deviant detection (Task 2), and with error rates during emotional prosody identification (Task 3).During unattended processing of emotional prosody (task 1), eMMN and lMMN peak amplitudes and latencies elicited by happy, angry, sad, and disgusted prosodic deviants were analyzed. From the 60 subjects, the data of four subjects had to be discarded due to large amounts of eye blink and motion artefacts leaving an insufficient number of target trials for analysis. Four (emotion: happy vs. angry vs. sad vs. disgusted) by 3  by 5  repeated-measures multivariate analyses of covariance (RM-MANCOVA) were then performed on the data of the remaining 56 participants with scores on the affective and cognitive alexithymia dimensions included as covariates. In case of significant interactions of factors with the covariates, follow up MANCOVAs were conducted for each level of the respective factor in order to identify the sources of the effect. In addition, MANCOVAs including each subscale of the respective alexithymia dimension as covariates were conducted in order to test which subscale significantly contributed to the effect.During attended processing of emotional prosody (task 2), peak amplitudes and latencies of the N1, P2, and P3 elicited by happy, angry, and disgusted prosodic deviants spoken in low (e.g. a bit angry) and high  intensity were analyzed in 57 subjects. The data of three subjects were discarded due to high amounts of eye blink and motion artefacts. Only trials of correctly detected prosodic deviants were included in the analysis. Affective scores of subjects in task 2 ranged from 20 to 55 , cognitive scores ranged from 27 to 93 . Two (intensity: low vs. high) by 3 (emotion: happy vs. angry vs. disgusted) by 3  by 3 (region) RM-MANCOVAs were conducted with affective and cognitive alexithymia scores included as covariates. Follow up MANCOVAs were conducted to identify the sources and the contribution of separate BVAQ subscales to the observed effects.The three levels of the factor region varied for the N1, P2 and P3 in accordance with the known topographic distributions of these components when elicited in the auditory modality: The N1 has a central maximum and was therefore analyzed at frontocentral, central, and centroparietal regions. The P2 is maximal over anterior regions, the analysis therefore comprised anterior electrode sites . The P3 is known to have a centroparietal-parietal maximum, thus central, centroparietal, and parietal regions were included in the P3 analysis.In case of sphericity violations, Greenhouse-Geisser corrected p-values are reported. A Sidak correction of p-values was used in pairwise comparisons between the levels of factors. Results are reported with a focus on main effects and interactions with the affective and cognitive alexithymia dimensions.r = 0.85, p<0.01).Pearson's correlation confirmed a high correlation between the cognitive dimension of alexithymia as assessed by the three cognitive BVAQ subscales and the TAS-20 total score  revealed no significant correlations of the cognitive and affective alexithymia dimensions with accuracy  and reaction time .As shown in In summary, both alexithymia dimensions were associated with significantly worse performance on the identification of disgusted prosody, while performance on emotional intensity identification was unrelated to alexithymia.cognitive dimension × laterality revealed , suggesting that the eMMN peaked significantly earlier in the left hemisphere and tended to peak later in the right hemisphere with increasing scores on the cognitive alexithymia dimension. Follow up tests on each cognitive subscale showed that this interaction was driven by difficulty verbalizing feelings  and difficulty identifying feelings . In addition, difficulty identifying feelings interacted with emotion , indicating that for disgusted prosody only, eMMN peak latencies were delayed with increasing difficulty identifying feelings.RM-MANCOVA showed that peak amplitudes of the eMMN did not differ as a function of alexithymia. EMMN peak latency, however, was associated with the cognitive, but not the affective alexithymia dimension, as a significant interaction Peak latencies and amplitudes of the lMMN did not differ as a function of either alexithymia dimension, suggesting that alexithymia did not affect the global processing of unattended changes in emotional speech.In summary, difficulty verbalizing and identifying feelings as part of the cognitive alexithymia dimension were associated with an earlier left-hemispheric response and a trend toward a delayed right-hemispheric response during the early acoustic encoding (eMMN) of subconsciously perceived variations in emotional speech. Difficulty identifying feelings was further associated with delayed encoding of disgusted prosody. In contrast, the affective alexithymia dimension was not associated with altered unattended processing of emotional speech. Neither alexithymia dimension affected the later stage of global acoustic processing (lMMN).cognitive dimension × region  was found for peak amplitudes of the N1, suggesting larger N1 amplitudes at centroparietal regions with increasing scores on the cognitive alexithymia dimension. Follow up tests on each cognitive subscale revealed that this interaction was driven only by difficulty analyzing feelings . Peak latencies of the N1 showed no difference as a function of the cognitive alexithymia dimension. The affective dimension was not associated with differences in N1 amplitude or latency.For the cognitive dimension, a significant interaction cognitive dimension × region . Follow up tests showed that this interaction was driven only by the subscale difficulty analyzing feelings, which was associated with reduced frontal P2 amplitudes.For the cognitive dimension, RM-MANCOVA on P2 peak amplitudes revealed a significant interaction affective dimension × region . Follow up tests revealed that this interaction was driven by the fantasizing subscale , which was found to be associated with reduced frontal P2 amplitudes   = 9.125, p = .003], suggesting reduced amplitudes of the P3 at parietal regions with increasing scores on the cognitive alexithymia dimension. Follow up tests on each subscale revealed that this interaction was driven by difficulty verbalizing feelings  and difficulty identifying feelings , which were both associated with reduced P3 amplitudes at parietal regions. In addition, the verbalizing subscale showed a significant three-way interaction verbalizing × emotion × region . Follow up tests on each emotion revealed a significant interaction verbalizing × region  for disgusted prosody, indicating that difficulty verbalizing feelings was associated with reduced parietal P3 amplitudes particularly during the detection of disgusted prosodic deviants   = 5.792, p = .019], which was further qualified by the factor region . Follow up tests on each affective subscale revealed significant effects of both the emotionalizing and the fantasizing subscale: For the emotionalizing subscale, there was a significant interaction emotionalizing × intensity , suggesting that the reduction in P3 amplitudes with increasing scores on emotionalizing was even more pronounced for deviants in emotional prosody spoken with high intensity than for those spoken with low intensity. For the fantasizing subscale, there was a three-way interaction fantasizing × intensity × region: F  = 6.414, p = .013]. Follow up tests on the factor region revealed a significant interaction fantasizing × intensity  at central regions, suggesting a reduction in central P3 amplitudes with increasing scores on fantasizing only for high intensity deviants in emotional prosody.For the affective dimension, RM-MANCOVA showed a significant In summary, electrophysiological data for the detection of changes in emotional prosody suggested that alexithymia was significantly associated with alterations of early  as well as late (P3) ERP components. A dissociable impact of the two dimensions of alexithymia was observed: Only the cognitive dimension  was related to larger amplitudes of the early N1 component, while both the cognitive  and the affective dimensions  were associated with reduced frontal P2 amplitudes. Both alexithymia dimensions were associated with reduced amplitudes of the late P3 component, but in a qualitatively different fashion: Individuals with difficulty verbalizing and identifying feelings (cognitive dimension) showed similarly reduced P3 amplitudes at parietal regions for all deviants in emotional prosody irrespective of the specific emotion and intensity, with the exception that individuals with difficulty verbalizing feelings exhibited a particularly diminished P3 response to disgusted prosody. In contrast to the cognitive dimension, the affective alexithymia dimension was sensitive to the difference in intensity with which the prosodic deviants were pronounced: High scores on emotionalizing  were associated with even stronger P3 amplitude reductions for prosodic deviants spoken with high intensity compared to those spoken with low intensity, while high scores on fantasizing  were associated with reduced P3 amplitudes only for prosodic deviants spoken with high emotional intensity.Control analyses of all behavioral and electrophysiological data using the TAS-20 showed highly comparable results with the cognitive alexithymia dimension assessed with the BVAQ.The present study investigated the impact of the cognitive and affective alexithymia dimensions on the electrophysiological processing of attended and unattended emotional prosody. At unattended processing levels, the cognitive dimension was associated with a left-hemisphere bias during the early acoustic encoding of emotional speech, as reflected in overall shorter left-hemispheric eMMN latencies. No effect of the affective dimension was observed during the unattended processing of emotional speech. At attended processing levels, the cognitive alexithymia dimension modulated both early and late ERP components, reflected in larger N1 amplitudes and reduced P2 and P3 amplitudes. In contrast, the affective dimension did not modulate the early N1 component, but was also linked to reduced P2 amplitudes, and further showed a negative association with P3 amplitudes particularly for emotional prosody spoken with high emotional intensity. These results suggest that alexithymia modulates electrophysiological responses to emotional speech at attended as well as unattended processing levels, and provide evidence for a dissociable impact of the cognitive versus the affective alexithymia dimension on the processing of the emotional qualities of speech.Behavioral data of the present study show that the mere detection of deviants in emotional prosody (task 2) was not affected by either alexithymia dimension. This is in line with the two previous studies on the relation between alexithymia and emotional prosody, which did not observe behavioral differences as a function of alexithymia during emotional prosody identification at the sentence level Taken together, our behavioral findings are in line with previous reports of difficulty in the identification of visually displayed emotion in alexithymia Alexithymia was found to affect amplitudes of the eMMN during unattended processing of emotional prosody (task 1). We observed a left-hemisphere bias during early acoustic encoding (as indexed by a faster left-hemispheric eMMN) with increasing scores on the identifying and verbalizing subscale of the cognitive alexithymia dimension for all deviants in emotional prosody , which was additionally paired with a tendency toward a delayed response of the right hemisphere. Difficulty identifying feelings was further associated with overall delayed eMMN latencies to disgusted prosody.These findings are particularly interesting considering that amplitudes of the neuromagnetic equivalent of the MMN in response to changes in emotional prosody have recently been found to be larger in the right hemisphere in healthy individuals During attended processing of emotional speech (task 2), the two alexithymia dimensions modulated early (<300 ms) electrophysiological responses to emotional speech in a qualitatively different fashion. Cognitive alexithymia was associated with larger N1 amplitudes in response to detected deviants, an association that was driven only by the subscale difficulty analyzing feelings. Larger N1 amplitudes were also reported in a previous study on auditory emotion perception in alexithymia in response to aversive white noise Taken together, these results suggest that alexithymia modulates both the unattended as well as the early attended processing of emotional prosody, with a qualitatively different impact of the two alexithymia dimensions: Only the cognitive dimension was associated with a left hemisphere preference during the unattended acoustic encoding of variations in emotional speech, and with a higher amount of attention devoted to the extraction of acoustic cues when detecting emotional prosodic deviants. An effect of the affective dimension was only found starting at approximately 150 ms (P2 time-window) reflected in reduced frontal P2 amplitudes, which were also observed in relation to the cognitive dimension. These results confirm previous reports of early perceptual differences in emotional processing as a function of alexithymia In addition, our observation of a faster left-hemispheric response and a tendency toward a delayed right-hemispheric response during the early acoustic encoding of unattended emotional speech is in line with the hypotheses of a left hemisphere preference during emotional processing Our results further demonstrated that alexithymia is associated with reduced amplitudes of the later (>300 ms) occurring P3, a component reflecting conscious stimulus evaluation. Amplitudes of this component have been shown to be sensitive to the ascribed importance to a stimulus . The P3 is also related to the emotional valence assigned to a stimulus in such a way that higher emotional valence is reflected in larger P3 amplitudes In addition, our results indicate differential effects of the two alexithymia dimensions on P3 amplitudes. The cognitive dimension was related to generally smaller P3 amplitudes in response to changes in emotional prosody, an association driven by the subscales difficulty identifying and verbalizing feelings. Difficulty verbalizing feelings was additionally related to reduced P3 amplitudes particularly to disgusted prosody. In contrast, the affective dimension was found to be sensitive to the intensity with which emotional speech was pronounced, reflected in even stronger reductions of P3 amplitudes for intonations spoken in high emotional intensity. This may suggest that individuals with difficulty identifying and verbalizing feelings generally ascribe less significance to emotional speech qualities, a process that may be particularly prominent in the case of disgusted prosody. Regarding the affective alexithymia dimension, this process might be even more pronounced for emotional prosody spoken with high intensity.Though all prosodic emotions tested in the present study were affected by alexithymia, we observed some indications for a specific role of disgusted prosody. Individuals with difficulty identifying feelings as part of the cognitive alexithymia dimension exhibited overall delayed eMMN peak latencies for disgusted prosody, suggesting a delay in the unattended acoustic encoding of disgust conveyed by speech. Further, individuals with difficulty verbalizing feelings showed particularly reduced P3 amplitudes in response to the detection of disgusted prosodic deviants. Though no special role of disgust at the electrophysiological level was found in relation to the affective alexithymia dimension, both dimensions were associated with impaired identification of disgusted prosody at the behavioral level. These results may indicate a specific role of disgusted prosody during emotional speech processing in alexithymia. However, previous findings on facial emotion recognition do not seem to suggest a specific role of disgusted emotion, but indicate a more general deficit in emotion recognition associated with alexithymia In summary, the present findings hint toward a dissociable impact of the cognitive and affective alexithymia dimensions on the processing of emotional speech qualities. These results provide further support to the notion that the two alexithymia dimensions may be differentially linked to emotional processing It should be kept in mind that the sample of the present study comprised only female participants, and that our results may therefore not be generalizable to male individuals with alexithymia. Also, mood states of participants were not assessed though alexithymia has been associated with reduced positive affect In conclusion, alexithymia seems to modulate electrophysiological responses to emotional speech during attended as well as unattended processing. The two alexithymia dimensions appear to exert a dissociable impact on emotional prosody processing, with a left-hemisphere bias characteristic for the cognitive alexithymia dimension during early stages of unattended processing. The affective alexithymia dimension seems to influence the perception of emotional prosody at later processing stages than the cognitive dimension, and appears to be additionally sensitive to the intensity of emotional speech. These results suggest that alexithymia indeed affects the way emotional speech qualities are processed in the brain, which could be a contributing factor to problems in interpersonal communication associated with this personality construct.


Eulemur rufifrons) to study decision outcomes, coordination of movements, individual foraging benefits and social interactions in response to the presentation of drinking platforms with varying baiting patterns. Behavioral observations were complemented with data from recordings of motion detector cameras installed at the platforms. The animal's behavior in the experimental conditions was compared to natural group movements. We could not determine the type of consensus decision-making because the group visited platforms randomly. The group fissioned during 23.3% of platform visits, and fissioning resulted in more individuals drinking simultaneously. As under natural conditions, adult females initiated most group movements, but overtaking by individuals of different age and sex classes occurred in 67% of movements to platforms, compared to only 18% during other movements. As a result, individual resource intake at the platforms did not depend on departure position, age or sex, but on arrival order. Aggression at the platforms did not affect resource intake, presumably due to low supplanting rates. Our findings highlight the diversity of coordination processes and related consequences for individual foraging benefits in a primate group living under natural conditions.Social animals have to coordinate joint movements to maintain group cohesion, but the latter is often compromised by diverging individual interests. A widespread behavioral mechanism to achieve coordination relies on shared or unshared consensus decision-making. If consensus costs are high, group fission represents an alternative tactic. Exploring determinants and outcomes of spontaneous group decisions and coordination of free-ranging animals is methodologically challenging. We therefore conducted a foraging experiment with a group of wild redfronted lemurs ( Group-living holds a number of benefits, but also costs for individual group members. Many of the benefits, such as shared vigilance or predator confusion, are related to reduced per capita predation risk, whereas increased intra-group feeding competition represents one of the unavoidable main costs of group-living Columba domestica: Myotis bechsteinii) switching communal roosts Papio ursinus) visiting small artificial food patches when followers had weak social ties with the dominant male leading the group Macaca tonkeana) and rhesus macaques (Macaca mulatta) Group fission, i.e. temporary splitting of a group into two or more subgroups, has often been interpreted as the outcome of incompatible individual interests Anser domesticus and heifers, Bos taurus: Suricata suricatta: Most empirical and theoretical studies explore decision-making and group cohesion in the context of group movements because they provide a biologically meaningful model and uniform context to study determinants and consequences of relevant coordination processes  focused on food detection abilities depending on distance, travel speed and resource size While it is already challenging to observe and analyse coordination processes of animal group movements in their natural habitat, it is even more difficult to determine proxies of fitness consequences of the underlying decisions for single group members, such as individual foraging benefits Eulemur rufifrons). Redfronted lemurs provide an interesting model in this context for a number of reasons. First, they live in small egalitarian groups with rather equally distributed resource-holding potential Helogale parvula: Equus burchellii: In the present study, we experimentally investigated decision outcomes, coordination processes, individual foraging benefits and the proximate mediation of conflict in a group of wild redfronted lemurs  to measure the extent to which decisions are shared, and (2) to explore the determinants and efficiency of three potential tactics to decrease the consensus costs of unshared or partially shared decisions, including (i) group fission during group movement, (ii) overtaking during group movement, and (iii) aggressive supplants once the final destination is reached.We first explored the extent to which decisions were shared. To do so, we set up drinking platforms and created conflicts of interest of varying degrees by modifying baiting patterns of the platforms in two different designs, i.e. providing either several group members or only a single individual with drinking opportunities (details below). Our predictions regarding the type of consensus decision-making at departure referred to the decision outcomes, i.e. the observed distribution of the group between the experimental platforms : We hypoWe also explored the circumstances favoring group fissioning, predicting group fission rates – as a means to avoid costly consensus decisions in the first place by foraging in independent subgroups – (i) to decrease when the number of baited platforms was reduced, and (ii) to increase after the group visited a poorly baited platform where only one group member has access to a valuable resource instead of five (details on baiting patterns below). The efficiency of group fission in decreasing consensus costs was measured by comparing the number of individuals accessing foraging rewards simultaneously in the presence versus in the absence of group fission. We expected group fission (iii) to result in more individuals drinking at the same time.Third, we explored individual and ecological factors favoring overtaking during group movements. Redfronted lemurs are characterized by stable leadership of several adult females per group under natural conditions Finally, we explored the circumstances favoring aggressiveness at the platforms – as a way to increase foraging benefits irrespective of coordination during group movements –, expecting its frequency to be negatively correlated with the number of baited platforms, which is here supposed to reflect overall resource abundance and thus the intensity of within-group feeding competition Cryptoprocta ferox), Harrier hawks (Polyboroides radiatus), stray dogs (Canis familiaris) and Malagasy boas (Acranthrophis ssp.) Data were collected at the field station of the German Primate Center (DPZ) at Kirindy Forest, a dry deciduous forest located about 60 km north of Morondava, western Madagascar One group of redfronted lemurs (“group B”) regularly visited the drinking platforms (see below). It consisted of 12 individuals . The study group is part of a habituated population inhabiting a 60-ha study area. The study area features a grid system of foot trails with intersections every 25 m . The memData were collected during the peak dry season (August-September) in 2009, when most trees had shed their leaves and only a few small water holes remained in the nearby Kirindy river bed  in bushes in the camp area in close proximity to each other (approx. 15 cm). The bottles contained different volumes (10–100 ml) of sweet grenadine syrup and water in a 1∶10 ratio and had a light red colour. As soon as the lemurs had realized that the bottles provided drinking opportunities, they usually moved towards them quickly and started drinking as soon as they were attached. Due to this behavior, we considered the value of flavored water as an experimental resource as high, especially because there were other water sources available. During the pilot study we observed in several cases that (i) redfronted lemurs were obviously able to distinguish between different syrup volumes and that they preferably approached bottles containing larger volumes. We never observed (ii) single individuals monopolizing several bottles that were positioned in close proximity, or (iii) individuals displacing conspecifics from other bottles after depleting their own one. We found that single lemurs always emptied volumes of 10 ml. Bottles containing 30 ml were depleted in approx. 70% of cases. In contrast, bottles containing ≥75 ml were only emptied in approx. 50% of cases. Thus, we considered 10 ml as small, 30 ml as medium and 75 ml as large volumes in the experiment .For the actual data collection, we set up 4 drinking platforms placed 75 m apart from each other in the core area of group B's home range . We careAfter a 2-week habituation phase in which all platforms were provided with 5 bottles (each containing 75 ml) every day, the study group visited all 4 platforms regularly, and we started data collection using the camera systems . In the Design 1 started with condition 2 . Here, wCondition 4 marked the beginning of design 2. On the one hand, we reduced the number of baited platforms from 4 to 2 in order to explore the consequences for group fissioning and aggressiveness at the platforms. Additionally, we reduced the resource amount at the 5-bottle-platform from medium (30 ml) to small (10 ml) and at the remaining 1-bottle-platform from large (75 ml) to medium (30 ml). We conducted these changes because we observed in the pilot study that only half of the individuals depleted 75 ml at once, i.e. a platform baited with only 1 bottle containing 75 ml could possibly offer drinking opportunities for at least 2 individuals, whereas bottles containing 30 ml were depleted by 1 individual in 70% of cases. Thus, we aimed at sharpening the conflict among group members when making consensus decisions. As in design 1, we expected shared decision-making to result in preferred visits to the 5-bottle-platform and unshared decision-making to result in visits to the 1-bottle-platform. Condition 5 equaled condition 4 but the baiting patterns of platforms 2 and 3 were shifted to control for habituation effects.Each condition lasted for 1 week. However, data were recorded only during the last 4 days of each condition to give animals enough time to habituate to the new baiting pattern. During the experiment, all platforms were controlled every 30 min. If the focal group depleted the platforms in the morning, bottles were refilled once around 2 pm. If a group other than the focal group visited the platform and drank water, bottles were refilled according to the respective experimental condition. We also refilled bottles when the identity of the group that had depleted them before was unknown. Since this unknown group later turned out to be group B quite often, two or more visits of the group B at the same baited platform during one day occurred regularly .th, 2009, 15∶45∶43 at platform 2 and 15∶46∶47 at platform 1). Therefore, we consider 1 min as a suitable threshold to define a fission event at the platforms. Accordingly, subgroup size was calculated as the number of group members that were recorded at a platform while there were group members at a different platform at the same time or <1 min before or after.Data were collected between August 4 and September 4 , with thBehavioral observations were also conducted during 17 days (85%) of the experiment , usually by two observers (for details of the observation protocol see -test (MWU) to explore whether the number of bottles per platform (1 or 5) affected the number of individuals per visit, i.e. whether more bottles indeed provided more individuals with a foraging opportunity. Whether group fissioning was beneficial in terms of more individuals drinking simultaneously was also evaluated with a MWU test. In order to identify the type of consensus decision-making, we calculated Pearson's chi-squared tests with Yate's continuity correction (corrected for the different proportions of 1- and 5-bottle-platforms in designs 1 and 2) to compare the number of non-fission visits to platforms baited with 5 and 1 bottle(s), respectively. We used Fisher's exact tests to assess whether the study group preferred particular platforms, irrespective of baiting patterns in design 1 and 2. We also used Fisher's exact tests to determine whether overtaking increased with the number of bottles on the platform the movement was directed to . To compare overtaking rates and initiatorship during movements to drinking platforms to observations of the same group, from the same time but from non-experimental contexts, we used chi-squared tests. To test whether the number of baited platforms (2 or 4) impacted fissioning, we used a chi-squared test. To analyze whether the group fissioned more often after visiting a 1- or a 5-bottle-platform , we used Fisher's exact tests. Tests on initiatorship and overtaking were run with PASW Statistics 18 , the other tests were run using R software .We used a Mann-Whitney UIn order to analyse determinants of resource intake and aggression, we ran three linear mixed models (LMM) and one generalized linear mixed model GLMM; . We chosFor models with normal error structure, we used Akaike's Information Criterion All baited platforms were visited frequently throughout the experiment with individual platforms being visited 0–4 times per day . Each grnon-fission visits to platforms baited with 5 bottles compared to platforms baited with only 1 bottle did not differ from random expectations in either design . Therefore, we were not able to assess whether consensus decision-making was shared or unshared based on the decision outcomes observed during the experiment. In fact, the group visited platform 3 more often than the other platforms in each condition, i.e. independent of baiting patterns (non-fission visits to baited platforms). However, differences between the visit numbers at platforms were not significant compared to random expectations .The number of 2 = 3.28, df = 1, p = 0.07). The group did not split into subgroups more often after visiting a 1- compared to a 5-bottle-platform in the experimental designs 0/1 .The sample size dropped for this test because we had to exclude fission visits and visits to non-baited platforms, as well as fission events occurring during the first visits recorded on a day or following immediately after another fission event. Therefore the number of suitable fissions observed during the experimental design 2 precluded any statistical analysis . However, during non-fission visits (nnon-fission = 69) on average less individuals (1.1±1.2) drank simultaneously than during fission visits .We excluded the 4 fission events during which individuals did not stay on different platforms at the same time but arrived at different platforms less than 1 min after one another because they were not able to drink simultaneously. It is not feasible to extend the calculation to individuals drinking <1 min time delayed, either, because individuals often stayed on the platforms for some time after depleting the bottles. Thus, usually no animal drank during the last minutes on a platform.The group split into subgroups 21 times comprising a total of 41 visits at platforms (not 42 because sometimes more than 2 subgroups formed and some visits were part of two different fission events). There were 69 non-fission visits at platforms. If a fission event (n = 21) - when individuals visit different platforms at the same time - is counted as a single visit, fission events make up 23.3% of all visits. We witnessed 6 fission events directly during the 25 movements to platforms that we observed (24%), i.e. we observed movements that resulted in group members being recorded at two different platforms at the same time or <1 min after one another. Two subgroups were recorded at different platforms on 18 occurrences,3 subgroups on two occurrences. Subgroup size ranged from 1 to 5, with a mean of 2.0±1.1 individuals. The proportion of group fissions compared to non-fission visits was larger when 4 platforms were baited (31.4%) instead of only 2 platforms (12.8%) but the difference was not statistically significant  were initiated by adults of both sexes and by one subadult male, with the oldest female of the group being responsible for 72% of all initiations. This high proportion of female initiatorship did not differ from observations of natural group movements of the same group . However, overtaking rates during the experiment (67%) were significantly higher than expected from movements not directed at foraging platforms of the same group during the same time period . In contrast, arriving first was advantageous in terms of foraging benefits . Visit number per platform per day, sex and age were part of the final model but did not contribute significantly . However, a higher aggression rate did not result in increased resource intake. The other factors of the most parsimonious model, visit number, age and sex, did not contribute significantly, either. The final model did not have a better fit than the null model .Agonistic interactions (N = 111) occurred during 30 out of 54 visits during which ≥2 individuals were present at a baited platform (55.6%). In 8 cases (7.2%), a single agonistic action  resulted in the direct displacement of a conspecific from a bottle and offered a drinking opportunity for the aggressor. Individuals that had previously overtaken the leader were never displaced from a bottle. Aggressive interactions were more likely to happen when more individuals were present at a platform and when an individual spent more time at the platform . FurtherAs previous field experiments with single primate groups Most individuals of our study group visited the platforms and depleted the experimental resource regularly, which was a prerequisite for the foraging experiment and reflects the resource value. The proportions of different resource volumes depleted by single individuals during the experiment were similar to those assessed during the pilot study. However, usually only a few group members were visible at the platforms on the video recordings. This could be a result of group fissions during which only one subgroup visited a platform. Unfortunately, we did not capture these fission events with our definition that relied on two subgroups being video-recorded at different platforms at the same time or <1 min after one another. However, the number of individuals recorded increased significantly with the number of bottles at the platforms, which might indicate that the group was usually present around the platform and a larger number of group members only jumped onto the platform, i.e. inside the camera frame, when more than one bottle was present. It also shows that more bottles provided indeed more individuals with a drinking opportunity, i.e. single individuals did not monopolize entire platforms.We tried to identify whether decision-making in groups of redfronted lemurs was shared or unshared using platforms that provided either one or several group members with a foraging opportunity. However, the number of group visits to a platform was not obviously affected by baiting patterns, and we were not able to identify the decision type underlying the observed decision outcomes. There are several reasons that may explain this result.partially shared with only 2–3 individuals contributing to the outcome  that have the same size but offer different foraging benefits could lack ecological relevance because redfronted lemurs usually feed on dispersed fruit and leaves Our experimental setup comprising four platforms was quite complex. Therefore, the lemurs may have failed to react towards variation in baiting patterns because we changed between different conditions too fast.We routinely refilled bottles in the afternoon and often the study group visited platforms even more than 2 times per day due to refilling after depletion by a group that had not been observed. Maybe the total amount of flavored water available was, therefore, more than enough for the whole group.Different platforms could be visited by the same individual subsequently in short time intervals (as little as 64 sec). This might have reduced the incentive for consensus decision-making.Alternatively, it is also possible that consensus decision-making does not present the only means to optimize group foraging benefits. For instance, group members can avoid consensus costs through different strategies such as (1) fissioning, which can presumably maximize the benefits of a majority of individuals  to natural contexts because respective data on inter-individual distances of >20 m under natural conditions are lacking. However, even given the possible shortcomings of our definition of a fission event, it is remarkable that individuals relaxed group cohesion and different subgroups exploited different platforms at the same time in 23.3% of the visits although platforms could be exploited subsequently in short time intervals (64 sec).In accordance with our third prediction, fissioning resulted in a significantly higher number of individuals drinking from bottles at the same time. Hence, fissioning apparently allowed group members to avoid costly consensus decisions and provided instant foraging benefits for a higher number of individuals Equus grevyi, and onagers, Equus hemionus: We predicted predominantly stable leadership by adult females towards the platforms and, accordingly, highest foraging benefits for these individuals. Consistent with our predictions, adult females initiated most movements towards the platforms. Female initiatorship and leadership have also been described in other species  and 9.4% in another study Overtaking rates in the present study were apparently affected by the high value and predictability of the experimental resource compared to natural foraging patches. However, such changes in coordination processes have not been described in comparable studies involving movements to experimental foraging patches by groups of white-faced capuchin monkeys A third tactic to optimize individual foraging benefit could be supplanting of group members from bottles through aggressive behavior at the platforms. As in other studies on redfronted lemurs At first sight, higher aggression levels of adults compared to subadults seem to reflect the proximate importance of physical asymmetry in accessing a food resource Our study permits two more general conclusions regarding the relationships among coordination, foraging behavior and social structure in socially structured groups. First, costs of a temporary lack of coordinated cohesion can be outweighed by high resource quality and predictability, even under supposedly high predation risk. Second, coordination in animal groups characterized by an egalitarian social structure can be highly flexible, allowing the use of alternative behavioral tactics that translate into individual foraging benefits irrespective of sex, age or social status.Table S1Number of visits of the study group on single platforms per study day throughout different conditions/designs.(DOCX)Click here for additional data file.