Anesthesia is usually required in preclinical functional magnetic resonance imaging (fMRI) studies as it prevents motion and stress experienced by the animals. Anesthesia, however, directly affects brain function and complicates data interpretation.¹ To avoid these confounding effects, several groups have implemented awake protocol in which subject is habituated to MRI environment. Despite habituation, animals are prone to movement and likely to experience stress which may also disturb brain function and functional connectivity (FC). Therefore, we investigated whether light sedation protocol would be a potential alternative to anesthetized and awake rat fMRI connectivity studies.The animal procedures were approved by the National Animal Experiment Board. Male Wistar rats were used. For 8 rats, small cannulas were inserted into femoral artery, and tracheostomy was performed under 2% isoflurane. Mechanical ventilation was adjusted to maintain normal blood gas values. These rats were subsequently imaged under 1.3% isoflurane. Rest of the rats were habituated to the fMRI in mock scanner at 4 consecutive days prior to imaging under 0.1% (n=6) or 0.5% (n=7) isoflurane. The custom-made restrainment kit was fully compatible with standard Bruker rat holder and quadrature receiver coil. The length of habituation session gradually increased from 15 min to 45 min. Breathing, heart rate, movement, weight, and corticosterone levels were monitored. The fMRI data (300-750 volumes) were acquired with 7T Bruker Pharmascan with single-shot spin-echo echo planar imaging (TR 2 s, TE 45 ms, FOV 2.5x2.5 cm2, 64x64 matrix, and 9-11x1.5 mm slices). The MRI data were converted to NIfTI (http://aedes.uef.fi), slice-timing corrected, motion-corrected, spatially smoothed, co-registered (SPM8), and band-pass filtered (0.01-0.15Hz). The correlation values between 12 regions of interest (from 300 motion-free volumes) were calculated to obtain measures for FC. All values in figures are mean±SEM.Representative seed-based correlation maps are shown in Figure 1. The group-level maps obtained under 0.1% and 0.5% isoflurane are very similar. The maps obtained under 1.3% isoflurane include mostly strong global correlations. However, the hypothalamic connectivity is missing under 1.3%, whereas visible in the 0.1% and 0.5% groups. Similar observations were made in correlation matrices (Figure 2) and in mean FC values (Figure 3). The FC under 1.3% is high throughout the cortex and striatal parts, whereas absent in hypothalamus. The more detailed analysis of hypothalamic FC (Figure 3B) confirms that connectivity to cortical and striatal regions is non-existent under 1.3% isoflurane. No significant differences were observed in any FC measures between the 0.1% and 0.5% groups. The physiologic measures related to habituation are shown in Figure 4. Although the values indicate adaptation, several measures (e.g. weight, corticosterone, and movement) suggest that rats in the 0.1% group were more stressed than rats in the 0.5% group.Our results demonstrate that FC, affected by anesthetic isoflurane dose, is organized in different manner than in conscious brain. Isoflurane (1.5-1.8%) is known to induce bursting neuronal activity, which is reflected directly in hemodynamics ^2,3 and explains the abnormally high correlation values in the 1.3% group. Cortico-striatal synchronization is the dominant feature, heavily masking the intrinsic connectivity. By contrast, the connectivity between cortico-striatal and hypothalamic regions appears to be disrupted by the anesthetic dose, which may be linked to the anesthetic mechanisms. With subanesthetic doses, the bursting activity was not apparent, enabling more detailed assessment of connectivity. The results also suggest, that the FC is not significantly different between the investigated subanesthetic doses. In other words, 0.5% isoflurane has minimal confounding effects on FC. The corticosterone level and physiologic measures clearly indicate that the rats in 0.5% group were well adapted to restrainment and imaging.Anesthetic dose of isoflurane (1.3%) was confirmed to significantly modify the organization of FC by inducing extensive cortico-striatal activity and disconnecting these regions from hypothalamic areas. No significant differences in FC were observed between the subanesthetic doses (0.1% and 0.5%). However, rats in the 0.1% group showed increased corticosterone levels and altered physiological parameters as markers of stress, whereas rats in the 0.5% group did not. Therefore, the light isoflurane sedation protocol may provide the best combination for connectivity studies: minimal stress and motion with normal brain function.