Resting-state functional magnetic resonance imaging (rsfMRI) is a translational imaging method with great potential in several neurobiologic applications. Most preclinical rsfMRI studies are performed in anesthetized animals, but the confounding effects of anesthesia on the measured functional connectivity (FC) are poorly understood. Therefore, we measured FC under six commonly used anesthesia protocols and compared the findings with data obtained from awake rats. The results demonstrated that each anesthesia protocol uniquely modulated FC. Connectivity patterns obtained under propofol and urethane anesthesia were most similar to that observed in awake rats. FC patterns in the α-chloralose and isoflurane-medetomidine combination groups had moderate to good correspondence with that in the awake group. The FC patterns in the isoflurane and medetomidine groups differed most from that in the awake rats. These results can be directly exploited in rsfMRI study designs to improve the data quality, comparability, and interpretation.

Functional magnetic resonance imaging (fMRI) is a powerful noninvasive tool for studying spontaneous resting state functional connectivity (RSFC) in laboratory animals. Brain function can be significantly affected by generally used anesthetics, however, rendering the need for awake imaging. Only a few different awake animal habituation protocols have been presented, and there is a critical need for practical and improved low-stress techniques. Here we demonstrate a novel restraint approach for awake rat RSFC studies. Our custom-made 3D printed restraint kit is compatible with a standard Bruker Biospin MRI rat bed, rat brain receiver coil, and volume transmitter coil. We also implemented a progressive habituation protocol aiming to minimize the stress experienced by the rats, and compared RSFC between awake, lightly sedated, and isoflurane-anesthetized rats. Our results demonstrated that the 3D printed restraint kit was suitable for RSFC studies of awake rats. During the short 4-day habituation period, the plasma corticosterone concentration, movement, and heart rate, which were measured as stress indicators, decreased significantly, indicating adaptation to the restraint protocol. Additionally, 10 days after the awake MRI session, rats exhibited no signs of depression or anxiety based on open-field and sucrose preference behavioral tests. The RSFC data revealed significant changes in the thalamo-cortical and cortico-cortical networks between the awake, lightly sedated, and anesthetized groups, emphasizing the need for awake imaging. The present work demonstrates the feasibility of our custom-made 3D printed restraint kit. Using this kit, we found that isoflurane markedly affected brain connectivity compared with that in awake rats, and that the effect was less pronounced, but still significant, when light isoflurane sedation was used instead.

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During deep anesthesia, the electroencephalographic (EEG) signal of the brain alternates between bursts of activity and periods of relative silence (suppressions). The origin of burst-suppression and its distribution across the brain remain matters of debate. In this work, we used functional magnetic resonance imaging (fMRI) to map the brain areas involved in anesthesia-induced burst-suppression across four mammalian species: humans, long-tailed macaques, common marmosets, and rats. At first, we determined the fMRI signatures of burst-suppression in human EEG-fMRI data. Applying this method to animal fMRI datasets, we found distinct burst-suppression signatures in all species. The burst-suppression maps revealed a marked inter-species difference: in rats, the entire neocortex engaged in burst-suppression, while in primates most sensory areas were excluded—predominantly the primary visual cortex. We anticipate that the identified species-specific fMRI signatures and whole-brain maps will guide future targeted studies investigating the cellular and molecular mechanisms of burst-suppression in unconscious states.