Cannabis is the illicit drug most widely used worldwide, and has been associated with significant acute and long-term cognitive and motor adverse effects¹. The neurophysiological basis of impairments may reside in focal and/or more extensive alterations of brain networks. Recent studies have pointed out the functional and anatomical alterations of specific brain regions due to cannabis consumption^{2 3 4}. In particular, the anterior insula (AI), part of the Salience Network (SN), has a role in salient elements detection, in attentional patterns and decision-making. Moreover, it has a key role as a hub, mediating the dynamic interactions between Central Executive Network (CEN) and Default Mode Network (DMN)⁵. Thus, its anatomical alteration might have extensive effects on brain functional connectivity. The purpose of the present study was twofold: first to reveal the changes of functional networks due to chronic cannabis use and highlight the specific involvement of AI, then to evidence the effects of acute cannabis inhalation.

Twenty-three young male regular cannabis users and 21 matched cannabis-free subjects participated in the study. All subjects underwent a baseline RS-fMRI acquisition; the regular cannabis users had two additional acquisitions after smoking a joint of cannabis or a placebo. A standardized experimental setting included a controlled cross-over design and a fixed-pace inhalation procedure⁶. Data were acquired on a Siemens 3T Trio MR scanner. RS-fMRI was performed during 10 minutes of continuous acquisition (single-shot EPI gradient-echo sequence, 300 volumes, TR=2000ms, TE=30ms, FA=90°, inplane resolution=3x3mm², 32 slices of 3mm). High-resolution T1-weighted 3D gradient-echo sequence (MPRAGE), 160 slices (1x1x1mm³ voxel size), was acquired as structural basis. Imaging data were pre-processed according to standard procedure using Statistical Parametric Mapping (SPM12, Welcome Department of Cognitive Neurology, London, UK). Pre-processed data were analysed using the GIFT group ICA toolbox version 3.0a. We estimated 69 components using the Infomax algorithm. Group-level ICs were then back-reconstructed for each subject using GICA. To identify valid RSNs, group-level ICs were spatially sorted in GIFT toolbox according to their maximum overlap with templates of RSNs of interest (Brainmap database). Components representing standard brain networks were chosen by verifying maximum spatial correlation with SPM probability maps for the gray matter, minimum for white matter and cerebrospinal fluid, and consistent spectral power⁷. Seed-based network analyses were performed on AI toward whole brain using the CONN-fMRI fc toolbox v15.a. AI mask was defines as from the SN component in cannabis users. In both analyses long term effects of consumption were assessed by comparing regular users at the basic state (C), with the control group (ctrl). Acute effects of consumption were established by comparing the different sessions acquired on regular users: basic state (C), after placebo consumption (P), and after cannabis consumption (THC). Statistical analyses were performed in SPM12 according to Random Field Theory, maps were thresholded for peak height at p<0.005 (k>30) or p<0.05 (k>30) and corrected at cluster level for between or within group comparisons, respectively.ICA showed that chronic cannabis consumption significantly decreases the AI activation in the SN. Indeed, the comparison of SN between different sessions of smokers subjects, allowed us to highlight that AI was less activated among regular cannabis users at the basic state than in control subjects (figure1). When comparing acute cannabis consumption and baseline or placebo conditions, we showed a new decrease of activation in AI, in rostral anterior cingulate cortex (ACC) and supplementary motor area (figure2). Connectivity seed-based analysis pointed out a decrease of connectivity between AI and ACC, thalamus and striatum, and an increase between AI and superior parietal lobule (SPL) in chronic cannabis users compared to control subjects (figure3). After acute cannabis consumption, we observed a slight increase (recovery) of connectivity between AI and ACC or the striatum and a decrease between AI and SPL. The connectivity remaining altered compared to control subjects.Long term cannabis use leads to an attenuation of the engagement of brain regions of the Salience Network that mediate response inhibition far beyond the brain regions showing anatomical modifications. The further decrease of activity after acute THC consumption can reflect the decrease of awareness of subjects of their own errors and performances, as an alteration of the balance between the different networks. Modifications of the connectivity between AI and ACC and AI and SPL revealed by seed-based analysis could be explained by a slight compensatory effect due to acute cannabis consumption in regular cannabis users.