The cerebellum during provocation and aggressive behaviour: A 7 T fMRI study

Abstract Increasing empirical evidence points towards the involvement of the cerebellum in anger and aggressive behaviour. However, human functional neuroimaging studies so far have emphasised the involvement of subcortical and cortical regions, rather than examining the contributions of the cerebellum. In the present study, 7 T functional magnetic resonance imaging (fMRI) was used to assess cerebellar activation during provocation and aggressive behaviour elicited by the Point Subtraction Aggression Paradigm in 29 healthy adult volunteers. Provocations resulted in left posterior cerebellar activation, while right posterior cerebellar activation was associated with aggressive behaviour. Our findings confirm the involvement of distinct and lateralised non-motor related cerebellar areas during provocation and aggressive behaviour. This study adds to the growing recognition of the posterior cerebellar regions in emotion- and cognition-dedicated processes and to the role of the little brain in human aggression.


INTRODUCTION
Aggression can be defined as behaviour with the intent to cause harm or injury to a person or object in response to provocation or frustration ( Anderson & Bushman, 2002;Berkowitz, 1965).Aggressive behaviour may prove useful in removing threats from the environment, but aggression typically has a negative connotation (e.g., exaggerated and pathological) ( Siever, 2008).The neurobiological cir cuit of aggression is composed of several cortical and subcortical regions, including the hypothalamus, amyg dala, periaqueductal grey (PAG), and medial prefrontal cortex (mPFC), which are associated with the motiva tional, emotional, and cognitive aspects of aggressive behaviour ( Davidson et al., 2000;LeDoux, 2012;Lischinsky & Lin, 2020;Panksepp & Biven, 2012).
Through its reciprocal connections to the hypothala mus ( Çavdar et al., 2018;Dietrichs, 1984;Haines et al., 1984;Kamali et al., 2018), the cerebellum can exert an influence on the hormonal axes and aggressive behaviour forming a cerebello hypothalamic pituitary adrenal (cerebello HPA) axis ( Schutter, 2012( Schutter, , 2020)).Previous endocrinological studies have shown that the steroid hormones testosterone and cortisol are associ ated with aggressive behaviour ( Montoya et al., 2012;Terburg et al., 2009;Van Honk et al., 2010).Testo sterone and cortisol are the main end products of the hypothalamic pituitary gonadal (HPG) and HPA axis, respectively ( Johnson et al., 1992).Acute increases of endogenous testosterone levels can sensitise the hypothalamus and midbrain structures and facilitate fight related motivational tendencies associated with anger and aggression, while cortisol can downregulate the activating effects of testosterone and desensitises the subcortical components of the aggression circuit ( Hermans et al., 2008).Through the mutually inhibitory effects of the HPG and HPA axes, the system may bias itself towards an imbalance between hormone levels, that is, high testosterone and low cortisol or vice versa ( Montoya et al., 2012).An imbalance towards testoster one, that is, a higher ratio between testosterone (T) and cortisol (C), has been associated with higher aggres sive behaviour and this T/C ratio is suggested to pro vide a better marker for aggression than the steroid hormones separately ( Manigault et al., 2019;Platje et al., 2015;Popma et al., 2007;Terburg et al., 2009).In addition to its connections with the hypothalamus, the cerebellum can be considered a target region for ste roid hormonal modulation through the presence of cor ticoid and androgen receptors in the cerebellar cortex ( Sánchez et al., 2000;Webster et al., 2002).
Functional magnetic resonance imaging (fMRI) studies in humans that employ laboratory aggression paradigms have proven valuable to elucidate the neural correlates of human aggression ( Fanning et al., 2017;Nikolic et al., 2022;Wong et al., 2019).Previous fMRI work has demon strated activation of key brain regions implicated in aggression during provocation from a fictional opponent, including the amygdala and prefrontal cortex (e.g., Chen et al., 2021;da Cunha Bang et al., 2017;Kaltsouni et al., 2021;Skibsted et al., 2017).Despite the available empir ical evidence, the cerebellum is not typically considered a region of interest in fMRI studies on aggressive behaviour.The limited amount of laboratory aggression paradigms that have reported cerebellar activation in their whole brain activation were recently summarised in a meta analysis ( Klaus & Schutter, 2021).Results (k = 10) showed evidence for cerebellar activation of the right posterior lobe and bilateral anterior lobes.In addition, recent evidence was found for the involvement of bilat eral Crus I II while viewing threatening faces during an aggression task ( Bertsch et al., 2022).Altogether, fMRI studies that show cerebellar activation during provoca tion or aggressive behaviour remain scarce.This may, in part, be due to not having included the cerebellum as a region of interest, exclusion of the cerebellum in the field of view or to low signal to noise ratio (SNR) in the poste rior fossa ( Diedrichsen et al., 2010;Fair, 2018).With 7 T fMRI, increased SNR and blood oxygen level dependent (BOLD) sensitivity may reveal more subtle BOLD effects ( Cai et al., 2021;van der Zwaag et al., 2009).
The present study therefore investigated cerebellar acti vation associated with provocation and aggressive behaviour using 7 T fMRI in healthy volunteers.In keeping with previous neuroimaging and stimulation studies, we hypothesised activation of the posterior lobules and vermis during provocation (i.e., points were stolen by a fictitious opponent) or aggressive behaviour (i.e., participants stole points from a fictitious opponent).Additionally, relations between cerebellar activation, aggressive behaviour, ste roid hormones, state anger, trait aggression, and trait impulsivity were explored.We anticipated that cerebellar activation during provocation and aggressive behaviour would be positively correlated with trait aggression, impul sivity, and T/C ratios.Additionally, increased aggressive behaviour was expected to correlate with higher trait aggression.Finally, higher T/C ratios were expected to cor relate with increased aggressive behaviour and with higher levels of state anger, trait aggression, and impulsivity.

Participants
Thirty healthy right handed volunteers between 18 35 years old participated in the study that took place at the Spinoza Centre for Neuroimaging in Amsterdam, the Netherlands.Participants were excluded if they had current or previous neurological or psychiatric com plaints, were not MRI compatible (e.g., claustrophobia, electronic implants, pregnancy, metal in their body), or used psychotropic medication or recreational drugs.Written informed consent was obtained.Participants received travel reimbursement and monetary compen sation for participation.The study was approved by the medical ethical committee from the University Medical Center Utrecht (NL77559.041.21) and performed in accordance with the Declaration of Helsinki.Out of the 30 participants, one person was excluded for excessive motion (mean absolute displacement > 1 mm) during scanning, leading to a final sample of 29 participants.

Aggression task
The Point Subtraction Aggression Paradigm (PSAP) is a validated laboratory task, in which participants play a game against a fictional opponent.In the present study, participants were told that the opponent was playing online.The goal for the participants is to earn as many points as possible ( Cherek et al., 1997).Participants can also choose to steal points from their opponent.As par ticipants do not receive the stolen points, this act of retal iation can be considered a form of aggressive behaviour intended to harm an opponent who provokes them.The task was adapted from previous versions ( Geniole et al., 2017;Kose et al., 2015) implemented in E Prime v3.0 (https://www .pstnet .com / eprime .cfm).
During the task, three options were continuously pre sented to the participant: participants could gain points (earn option), steal points from the opponent (steal option), or prevent steals by the opponent (protect option) (Fig. 1A).To earn a point, 40 button presses had to be completed, while 10 button presses had to be completed to steal a point or protect the point total.When a choice for one of the options was made, the participants had to complete the total number of button presses (40 or 10) before switching to another option.Between choices, participants waited for 4 6 seconds (inter trial interval, ITI) (Fig. 1B).Throughout the task, participants could keep track of their point total, current choice made, and number of times a given button had already been pressed (out of 10 or 40, depending on their choice) on the com puter screen.When a point was earned, the point total increased by one and positive visual feedback was pro vided ("+" symbols flashed around the point total for 1000 ms).Throughout the task, participants could have points stolen by the fictional opponent (i.e., a provoca tion).When a point got stolen, one point was subtracted and negative visual feedback was provided (flashing " " symbols shown for 1000 ms).Provocations occurred ran domly every 6 45 seconds, on average 10 times per 9 minute run.At the start of the task, their point total was protected for 45 seconds (provocation free interval, PFI).When participants completed the "steal" or "protect" option, a PFI of 30 seconds occurred.Participants, how ever, were only aware of a protective effect for the "pro tect" option and did not know the length of the PFI beforehand, but thought this was of variable duration.
Before scanning, participants practised the task out side of the scanner for 2 minutes.Here, they were given the opportunity to ask questions and resolve any uncer tainties.In the scanner, a button box was placed under the thumb, index, and middle finger of the right hand, corresponding to the three options during the task.Their monetary reward was 5 eurocents per point earned, which was rounded up to a maximum of 3 euros for each participant.In the scanner, participants performed two runs that each lasted 9 minutes.In our sample of 29 par ticipants, three participants performed just one run due to technical problems.After the PSAP, participants reported what tactic they employed and what they thought of their opponent and their opponent's tactic.Afterwards, they were informed of the aim of the task and that their opponent was a computer.

State Anger
The State Anger (SA) scale of the State Trait Anger Scale (STAS; Spielberger et al., 1983; Dutch version: Van der Ploeg et al., 1982) was administered to measure participants' self reported emotional state and feelings such as anger, irritation, and rage.The SA consists of 10 items with possible answers ranging from "not at all" to "extremely" on a four point scale.The SA was adminis tered before and after the scanning session to acquire a baseline emotional state before the task as well as to quantify the change in SA after being in the scanner and performing two rounds of the PSAP.

Trait aggression
The Buss Perry Aggression questionnaire (BPA; Buss & Perry, 1992;Dutch version: Meesters et al., 1996) was administered after the scanning session to obtain a self reported measure of trait anger and aggression.The BPA consists of four subscales: physical aggression (9 items), verbal aggression (5 items), anger (7 items), and hostility (8 items).Every question is answered on a five point scale ranging from "extremely uncharacteristic of me" to "extremely characteristic of me."

Trait impulsivity
The Barratt Impulsiveness Scale (BIS 11;Patton et al., 1995;Dutch version: Lijffijt & Barratt, 2005) was adminis tered after the scanning session as a self reported mea sure of impulsive behaviour.The BIS 11 consists of three subscales: attentional impulsivity (8 items), motor impul sivity (11 items), and non planning impulsivity (11 items).Every question is answered on a four point scale ranging from "seldom/never" to "almost always."

Steroid hormones
Saliva samples were collected directly before the MRI scanning session by having participants spit in a saliva vial.Participants were instructed to not eat or drink anything besides water for 2 hours before the study.Immediately after collection, the samples were stored in a freezer at 80°C.Testosterone and cortisol levels were determined from the saliva samples by the Central Diagnostic Laboratory (CDL) at the University Medical Center Utrecht.
Salivary cortisol levels were determined by liquid chromatography tandem mass spectroscopy (LC-MS).100 µL saliva was mixed with internal standards Cortisol 9,11,12,12 D4 and Cortisone D8 using MRQ30 vials (Supelco).Samples were evaporated under N 2 at 70 °C.Residues were reconstituted in 30 µL 80% methanol and quantified by LC-MS/MS.Calibrator solutions were pre pared from Sigma Aldrich hydrocortisone and cortisone stock solutions.The UHPLC MS/MS system consisted of an Ultimate 3000 UPLC system coupled with an APCI TSQ Quantiva mass spectrometer (ThermoElectron Corp, West Palm Beach, FL).An Acquity UPLC C18 150 × 2.1 mm, 1.7 µm (Waters) column was used with a gradient elution of water/methanol containing 0.1% for mic acid for separating cortisol and cortisone.The lower limit of detection was 0.5 nmol/L.Day to day impreci sion was < 6% at 1.5 and 23 nmol/L for cortisol, and <10% at 6 and 30 nmol/L for cortisone.Intra assay vari ation was <2% at 1.2 and 9 nmol/L for cortisol, and <3% at 1.1 and 24 nmol/L for cortisone.

fMRI: image preprocessing
All PAR/REC image files were converted to NIfTI with dcm2niix ( Li et al., 2016).Structural T1 weighted data were obtained from the two inversion time images using MATLAB (The Mathworks, Inc.; https://github .com / JosePMarques / MP2RAGE related scripts).Prepro cessing of NIfTI images was performed in FSL (FMRIB's Software Library, Oxford, UK) version 6.0.2 ( Jenkinson et al., 2012): Following distortion correction using FSL's topup ( Andersson et al., 2003;Smith et al., 2004), data were processed with fMRI Expert Analysis Tool v6.00 (FEAT; Woolrich et al., 2001).Functional scans were motion corrected with MCFLIRT ( Jenkinson et al., 2002), smoothed at 3 mm Full Width at Half Maximum (FWHM) with SUSAN ( Smith & Brady, 1997) and a brain mask was created from the mean functional volume with the Brain Extraction Tool (BET; Smith, 2002).Fur ther, functional data were normalised for higher level analyses by a single scaling factor ("grand mean scal ing").From the structural scans, non brain structures were removed with BET.Functional scans were realigned to the structural scan and MNI152 standard space by affine registration using FLIRT ( Jenkinson & Smith, 2001;Jenkinson et al., 2002).ICA AROMA ( Pruim et al., 2015), high pass filter, and nuisance regression were used to remove noise from the data.ICA AROMA was used to generate 100 independent components, which were extracted from the functional data based on spa tial patterns, frequency spectra, and time series.The classification (signal vs. noise) of each component was checked manually and was reclassified if necessary ( Griffanti et al., 2017).ICA AROMA's non aggressive denoising was performed based on these labels.Fur thermore, cerebrospinal fluid (CSF) and white matter (WM) signals were segmented from the structural scans using FAST ( Zhang et al., 2001).Signals from the func tional scans in the CSF and WM were used as nuisance regressors.Finally, a high pass filter at 0.01 Hz was used for data preprocessing.

fMRI: single-subject analyses
A general linear model (GLM) was used to model BOLD activation per participant per task run ( Beckmann et al., 2003;Ogawa et al., 1990).Both 9 minute task runs were modelled separately and combined using fixed effect modelling in FEAT ( Woolrich et al., 2001).In the design matrix for each run, onset times of provocations and each option (i.e., earn, steal, and protect) in the PSAP were included.Per option, two regressors were added: One for the trials directly following a provocation and one for the trials that did not follow a provocation (Fig. 1B).For the trials following a provocation, onset times of the provocations were used instead of the start of earning/ stealing/protecting, to model the reaction and decision making after the provocation.These events lasted until the next option was chosen.Earn, steal, and protect trials without preceding provocation were modelled with 2 second durations after the start of the option (onset time).This duration was taken to be a representative duration of a block of button presses for the shorter options (i.e., steal and protect).Duration for provocations was also modelled as 2 seconds.Furthermore, the onset times for button presses were included as a separate regressor.Due to the quick nature of the button presses, duration was set to zero seconds.All regressors were convolved with a hemodynamic response function and its temporal derivative ( Friston et al., 1998).Temporal autocorrelation was removed by FILM pre whitening and the GLM was fitted voxel wise ( Woolrich et al., 2001).
Similar to previous studies that used the PSAP in an fMRI set up ( da Cunha Bang et al., 2017;Kaltsouni et al., 2021;Skibsted et al., 2017), contrast images of parame ter estimates were generated for BOLD activation after a provocation compared to earning a point (i.e., choosing option "1") and stealing a point (i.e., choosing option "2") compared to earning a point.These two contrasts are hereafter referenced to as Provocation > Earn and Steal > Earn, respectively (Fig. 1B).For the Steal > Earn con trast, earn trials with and without a preceding provoca tion were used in both conditions.However, for the Provocation > Earn contrast, earn trials following a prov ocation were not included to exclude a possible influence of provocation on earn trials.

fMRI: group analyses
Each task run was taken into the group analyses along with the transformations to standard MNI152 space to generate group level contrasts.To constrict the analyses to our research question on the cerebellum as region of interest, we used a binary mask of the cerebellar grey matter from the spatially unbiased atlas template (SUIT; Diedrichsen et al., 2009) normalised to MNI152 space with FLIRT.Secondary whole brain analyses without masking are reported in the Supplementary Material, to put our findings in the context of previous research.
For group analyses, mixed effects modelling was per formed with FLAME 1 in FEAT ( Smith et al., 2004;Woolrich et al., 2004).At the group level, one sample t tests were applied for our contrasts Provocation > Earn and Steal > Earn.Group level contrast images for Provocation > Earn were cluster thresholded at Z = 3.1 (p < 0.001) and cor rected for Family Wise Error (FWE) at p = 0.05 ( Worsley, 2003).Due to behavioural flexibility during the PSAP, not all participants stole from their opponents.For the group analyses of Steal > Earn, we only included participants that stole at least 10 times during the task to provide suf ficient data (n = 19).Due to the smaller sample size, a more liberal cluster based threshold was applied (p < 0.005 instead of p < 0.001 to balance Type I and II errors; Lieberman & Cunningham, 2009).Results were visualised in standard sagittal, coronal, and axial slices, as well as flatmaps rendered in SPM12's SUIT toolbox ( Diedrichsen et al., 2009).

Associations between cerebellar activation and measures of aggression
In the whole sample (Provocation > Earn), associations between cerebellar activation, personality characteristics, and the T/C ratio were explored.For each cluster, the max imal Z value from each participant was extracted from the group level analysis.These values were correlated with the total BPA and BIS 11 scores, change in SA scores, T/C ratio and prescan testosterone and cortisol levels using Pearson correlations for parametric data, and Spearman correlations for non parametric data.An FDR corrected p < 0.05 was considered significant ( Benjamini & Hochberg, 1995), with additional corrections for multiple cerebellar clusters if necessary.

Data reduction and additional behavioural analyses
Behavioural analyses were performed with R version 3.6.0 in RStudio version 1.2.1335 for Windows ( RStudio Team, 2018).Testosterone levels were Z transformed per sex to account for sex differences in testosterone.Additionally, the ratio between testosterone and cortisol levels (T/C) was calculated by dividing Z transformed testosterone levels by cortisol levels.For the PSAP, aggressive behaviour was calculated as the number of steal trials divided by the number of total button presses.
To assess whether there was an increase in SA scores after the PSAP in the MRI scanner compared to pre task levels, a paired Wilcoxon signed rank test was conducted, where p < 0.05 (one sided) was considered a significant increase.We checked whether participants who did not steal often (<10 times) and participants who stole at least 10 times in two runs scored differently on trait aggression or impulsivity (BPA/BIS 11 scores) with a two sample t test.Within the group that used aggressive responses during the PSAP (i.e., participants who stole at least 10 times), aggressive behaviour was correlated with trait aggression and impulsivity (BPA and BIS 11 scores) and percentage changes in state anger using Pearson correla tions for parametric data and Spearman correlations for non parametric data.Exploratively, subscales of the BPA and BIS 11 questionnaires were correlated with aggressive behaviour if there was a significant association with total scores, to take the multidimensionality of these constructs into account ( Patton et al., 1995).For all behavioural analy ses, individual observations were considered outliers if they deviated by at least three standard deviations from the group mean.For these tests, a p value < 0.05 (two sided) was considered significant.For further behavioural analy ses on hormone levels, see Supplementary Section 1.

Study population
Demographic characteristics of the study population are summarised in Table 1.Two participants had undetect able (<0.5 nmol/L) levels of cortisol and their data were not taken into analyses of hormone levels.Furthermore, one participant did not fill in the state anger questionnaire prior to the scanning session and was also excluded.In the remaining group (n = 28), state anger scores were sig nificantly higher after the scan compared to before (Z = 1.93, p = 0.027).

PSAP behaviour
PSAP behaviour is summarised in Table 2.During the task, provocations occurred on average in every five trials (19.6%) and a total of 10 times per run.On average, par ticipants stole 5.8 times [IQR 2.0-7.8]relative to total     button presses.From the 29 participants, 10 participants stole less than 10 times.

Cerebellar activation
When points got stolen from the participants (Provoca tion > Earn), participants showed activation in the left posterior cerebellar lobe (cluster peak in left lobule VI/ Crus I) (Table 3, Fig. 2).Data from the 19 participants who stole at least 10 times were taken into further analyses (i.e., Steal > Earn).For Steal > Earn, activation was pres ent in the right posterior lobe (cluster peak in right Crus II/lobule VIIb) (Table 3, Fig. 2).Whole brain findings are reported in the Supplementary Material.Imaging Neuroscience, Volume 1, 2023

Cerebellar activation-behaviour analyses
Neither the max Z scores of the Provocation > Earn con trast (n = 29) nor the max Z scores from the Steal > Earn contrast (n = 19) were significantly associated with any of the behavioural or hormonal measures (ps = 0.978, see Table S1).

Additional behavioural analyses
Prior to the behavioural analyses, one outlier was removed for more PSAP relative steals and one outlier was removed for a high change in SA scores.There was no association between BPA/BIS 11 scores and the tac tic employed during the task.Participants that employed a tactic of primarily earning and protecting (i.e., total number of steals < 10) did not show a difference in BPA (t 27 = 0.30, p = 0.765) or BIS 11 (t 27 = 0.20, p = 0.843) scores compared to the group that stole at least 10 times (Fig. 3A).Within the group of people that used stealing (>9 times) in their tactic (n = 19), there was no correlation between the number of steals and BPA scores (r = 0.04, p FDR = 0.879; Fig. 3B).Higher BIS 11 scores, however, were correlated with more steals (r = 0.62, p FDR = 0.018; Fig. 3B).Exploratory post hoc analyses showed that this relation appeared to be driven by the non planning impul sivity subscale (r = 0.69, p = 0.002; motor impulsivity: p = 0.176; attentional impulsivity: p = 0.312).Finally, the percentage change in SA scores was marginally cor related with the number of steals during the PSAP, but this was not significant after FDR correction (ρ = 0.38, p FDR = 0.205) (Fig. 3B).

DISCUSSION
The aim of the present study was to investigate cerebellar activation in response to provocation and during aggres sive behaviour.Results showed left posterior cerebellar activation when provocations occurred, while right pos terior cerebellar activation was observed when partici pants engaged in aggressive behaviour.The left hemispheric cluster of cerebellar activation during provocation with a paravermal peak in lobule VI/ Crus I concurs with previous meta analyses that showed activation of left Crus I and II during passive processing of negative emotions, including anger, disgust, and sad ness ( E et al., 2014;Klaus & Schutter, 2021;Pierce et al., 2022).Furthermore, another study in healthy volunteers confirmed the involvement of the left posterior cerebellar areas, mainly Crus I and II, during emotion processing ( King et al., 2019).In emotion processing, arousal and negative valence are suggested to involve left lobule VI (i.e., valence), left Crus II and vermal lobules VI and VIIIa (i.e., arousal), and left lobules V and Crus I (i.e., arousal valence interaction) ( Styliadis et al., 2015).These areas align with our findings in left lobule VI/Crus I II.It is con ceivable that the left posterior cerebellum plays a role in regulating arousal through connections with the reticular system via the cerebellar fastigial nuclei.These connec tions with the fastigial nuclei have been shown to origi nate from both the vermis and hemispheric lobules VI and Crus I II in mice ( Fujita et al., 2020).In processing valence, the cerebellum can communicate with the amygdala ( Schienle & Scharmüller, 2013), hypothalamus ( Moulton et al., 2011), and mPFC ( Etkin et al., 2011; Krienen & Buckner, 2009) through mono and/or polysyn aptic connections.Furthermore, different emotions may be associated with distinct cerebellar patterns.For exam ple, disgust was associated with activation of a left lobule VI cluster slightly anterior to our current cluster ( Baumann & Mattingley, 2012).Provocation can also elicit an aver sive state (e.g., moral disgust), an emotional response to offensive stimuli, which has previously been associated with decreased aggressive responses ( Bondü & Richter, 2016).In addition to arousal and affective responses, provocations are also considered to be threatening.The left posterior Crus I II could contribute to the threat detection circuit via the fastigial nuclei and PAG, through connections that have been evidenced in mice ( Fujita et al., 2020), and thus initiate fight, flight, or freeze responses ( Roelofs, 2017).Provocation may also increase the tendency to physically avoid confrontation by attend ing away from threatening stimuli such as angry faces, stepping away, or pushing a lever away ( Roelofs et al., 2008;Stins et al., 2011;van Honk et al., 1998).The pos terior cerebellum together with the prefrontal cortex (PFC) has been proposed to be a part of a cerebello cortical system involved in approach and avoidance related motivation and emotion ( Schutter, 2020).According to this idea, avoidance related behaviour is lateralised to the left posterior cerebellum and right PFC ( Kelley et al., 2017;Schutter, 2020).In line with this argumentation, left Crus I and II were shown to be structurally connected to the right prefrontal cortex in primates ( Kelly & Strick, 2003;Middleton & Strick, 2001).Furthermore, avoidant behaviour is also a symptom of fear and anxiety disor ders ( Bögels et al., 2010), which also show altered activa tion in the cerebellum ( Ernst et al., 2019;Hoppenbrouwers et al., 2008;Moreno Rius, 2018).In sum, in line with pre vious findings on negative emotion processing and avoidance related behaviour (e.g., Klaus & Schutter, 2021;Schutter, 2020), left posterior cerebellar activation was observed when participants were provoked.
Right cerebellar task activation during stealing from the opponent is in line with previous meta analytic evi dence reporting activation of the right rostral posterior regions, including Crus I II and lobule VIIb, during aggressive behaviour ( Klaus & Schutter, 2021).In further support, a recent volumetry study in healthy volunteers showed that grey matter volumes of the right posterolat eral lobules VIIb and VIIIa were correlated with aggres sive and impulsive personality traits ( Wolfs, Klaus, et al., 2023).Complementary to our findings in the left poste rior cerebellar lobe, we speculate that the right cerebellar hemisphere may be involved in the approach system through its contralateral connections with the left PFC ( Middleton & Strick, 2001).Previous research has shown that relative left to right dominant PFC activity is associ ated with a person's increased tendency for approach related behaviour, which is associated with anger and aggressive behaviour ( Harmon Jones & Sigelman, 2001;Kelley et al., 2017;Schutter & Harmon Jones, 2013).The right posterior cerebellum may thereby be involved in increasing or decreasing the likelihood to approach and behave aggressively ( Schutter, 2020).In addition to the lateral PFC, Crus I II are connected to the default mode network hubs, including the mPFC ( Buckner et al., 2011;Krienen & Buckner, 2009).The mPFC, in turn, has also been found to be activated in laboratory aggression paradigms ( Chen et al., 2021;da Cunha Bang et al., 2017;Kaltsouni et al., 2021;Skibsted et al., 2017) and is suggested to be involved in the subjective experience of conflict between retaliation and non aggressive responses ( Repple et al., 2017).In aggressive veterans, the medial orbitofrontal cortex also showed altered func tional connectivity with the DCN, the collection of areas that relays information from the cerebellar cortex to other parts of the brain ( Wolfs, van Lutterveld, et al., 2023).Furthermore, functional connections exist between the right posterior cerebellum and the fronto parietal net work involved in response inhibition and executive func tioning ( Buckner et al., 2011;Habas et al., 2009;Osada et al., 2019).Interestingly, participants with higher non planning impulsivity scores (e.g., not planning ahead and saying things without thinking) showed increased aggressive behaviour during the PSAP in the current study.This adds to the idea that the right posterolateral lobules may be involved in impulsive behaviour ( Wolfs, Klaus, et al., 2023).In addition to approach related moti vation and impulsivity, aggressive behaviour is arguably associated with a higher reward drive and lower punish ment sensitivity ( Megías Robles et al., 2022).Our right posterior findings, however, do not overlap with a recent meta analysis on reward processing in the cerebellum ( Kruithof et al., 2023) which found involvement of vermal VI/Crus I during reward outcome and the posterior ver mis, bilateral lobules I VI, and lateral left Crus I during reward anticipation.In the present paradigm, it was diffi cult to pinpoint the rewarding aspect of aggressive behaviour, as earning points in itself already serves as a reward.Further studies with aggression paradigms that do not use reward as the main task goal for participants may be considered in future research.In sum, in line with the lateralisation of the approach avoidance system, right posterior cerebellar activation was observed when participants behaved aggressively.The specific underly ing functional system in play, however, remains to be elucidated.
Contrary to our expectations, no evidence was found for activation of the vermis during provocation or aggres sive behaviour.The vermis is thought to be part of the "limbic cerebellum" and has been found to play a role in processes linked to the experience and regulation of emotions ( Adamaszek et al., 2017;E et al., 2014;Frazier et al., 2022;Guell et al., 2018;Leggio & Olivito, 2018).It is involved in arousal and autonomic activation through its connections to subcortical brain structures, such as the amygdala, reticular formation, and hypothalamus, which regulate the sympathetic and parasympathetic branches of the central nervous system arousal and autonomic responses ( Schmahmann, 2000;Styliadis et al., 2015).For example, activity attributed to arousal has been found in vermal lobules VI and VIIIa ( Styliadis et al., 2015).Owing to the nature of provocation in the PSAP, somatic responses to stealing and preparing the body to fight or flight may have been limited.Provoca tions in this study are relatively mild and might elicit mainly a cognitive response, since a defensive response (fight or flight) was neither warranted nor physically pos sible.In future studies, vermal activation may be elicited through the use of stronger provocations such as loud noises, electric shocks, or proximal (e.g., inescapable) threats (e.g., Faul et al., 2020).Additionally, administering a frustration task before the experimental task (e.g., Hortensius et al., 2011) may increase baseline state anger levels and thus provide more insights into the effect of heightened emotional responses on aggressive behaviour.Finally, adding physiological measures of arousal, such as heart rate and pupil dilation, may be interesting to examine if cerebellar activation can be more directly linked to sympathetic arousal in the context of provoca tion and aggressive behaviour.
In accordance with the goal of the PSAP to elicit aggressive behaviour and frustration ( Cherek et al., 1997;Geniole et al., 2017), self reported state anger was higher after the task as compared to baseline.Because aggres sive behaviour during the PSAP was a voluntary act, dif ferent tactics were employed throughout the task regardless of trait aggression or impulsivity scores.Aggressive behaviour costs effort and interferes with the participants' (primary) goal of collecting points and earn ing money.Participants (n = 10) who did not frequently steal (<10 times) may have had a stronger motivation to "win": They reported that earning and protecting was the most efficient way to earn as many points as possible (i.e., the group that did not frequently steal scored on average 23.2 ± 5.7 points, whereas the other group scored 19.5 ± 4.2 points (post hoc difference: t 27 = 1.80, p = 0.094)).Exploratively, between these groups there was no difference in cerebellar activation when partici pants were provoked, suggesting that the initial reaction to provocations was similar regardless of subsequent behavioural responses (see Supplementary Section 5).Statistical power for this post hoc analysis, however, was low and further studies are needed to investigate this dif ference.In addition, the PSAP could be adapted to facili tate immediate stealing whilst being provoked without finishing the current trial.This can provide additional infor mation on aggressive intentions and offer a more natural istic scenario to respond to provocations.
No evidence was found that the T/C ratio was asso ciated with aggressive behaviour during the PSAP.Fur thermore, no correlations between cerebellar activation, behaviour, and steroid hormones were observed.As mentioned earlier, the mild provovations during the task may not have been sufficient to evoke physiological responses to provocation.Our findings also add to the idea that the link between testosterone, cortisol, and aggression is highly complex and that (social) context plays an important role (for reviews, see Geniole et al., 2017Geniole et al., , 2020).The T/C ratio may be more difficult to cap ture in tasks that include provocations, because the association between T/C ratio and aggressive behaviour was shown to be weaker or absent when provoked ( Geniole et al., 2011;Manigault et al., 2019), although opposite relations have also been reported ( Denson et al., 2013).Whilst looking at the hormones separately, lower testosterone levels were associated with higher BIS 11 non planning impulsivity scores (Supplemental Section 1).Speculatively, our findings on the associa tion between higher testosterone levels and lower impulsivity scores could be linked to the involvement of testosterone in goal directed behaviour, risk aversive strategies, and maintaining social status ( Heany et al., 2018;van Honk et al., 2016).In line with this, prior stud ies provide inconclusive evidence on the association between aggressive behaviour in the PSAP and testos terone levels, but also emphasise the importance of looking at moderating factors (e.g., sex, anxiety levels, sleep deprivation) in more detail ( Geniole et al., 2017).To better establish the role of testosterone and cortisol within the cerebellar framework of anger and aggressive behaviour, larger sample sizes and more sensitive meth ods to assess steroid hormone levels are necessary.Finally, administration studies (e.g., Goetz et al., 2014; Imaging Neuroscience, Volume 1, 2023 Hermans et al., 2008) may be another way to examine the proposed links between steroid hormones, the cer ebellum, provocation, and aggressive behaviour.
In conclusion, provocation and aggressive behaviour were linked to two spatially distinct regions of activation in the human cerebellum.Our findings provide evidence for the involvement of distinct non motor related cerebel lar areas during both provocation and aggressive behaviour and add to the growing recognition of the pos terior cerebellar regions in emotion and cognition dedicated processes.

Fig. 1 .
Fig. 1.Point Subtraction Aggression Paradigm set up. (A) Presentation of the three options for each trial: earn, steal, and protect.(B) Possible schematic timeline of the task, including visualisation of the onsets and durations of the regressors used in the Provocation > Earn and Steal > Earn contrasts.The ITI was of variable 4 6 second duration.Abbreviations: ITI = Inter Trial Interval; no prov.= no preceding provocation; PFI = Provocation Free Interval; prot.= protect; prov.= provocation; s = seconds.

Fig. 3 .
Fig. 3.The association between PSAP stealing behaviour and questionnaire scores.(A) No difference in BPA and BIS 11 scores between participants that stole less than 10 times and at least 10 times.(B) PSAP steals (relative to total button presses) correlations with BPA and BIS 11 total scores and change in SA scores within the group that stole at least 10 times.Abbreviations: BIS 11 = Barratt Impulsiveness Scale; BPA = Buss Perry Aggression questionnaire, PSAP = Point Subtraction Aggression Paradigm, SA = State Anger.

Table 1 .
Characteristics of the study population.
Data are presented as mean ± standard deviation or median [interquartile range (IQR)] for continuous variables and as number (percentage of total) for categorical variables.Displayed steroid hormone levels are not standardised.†data available in n = 27;‡ data available in n = 28.Abbreviations: BIS 11 = Barratt Impulsiveness Scale; BPA = Buss Perry Aggression questionnaire; SA = State Anger, T/C = Testosterone/Cortisol.

Table 2 .
Behaviour during the Point Subtraction Aggression Paradigm.

Table 3 .
Task activation in the Point Subtraction Aggression Paradigm in the cerebellum.