An Open-Source Restraint System for Magnetic Resonance Imaging in Awake Rats

Magnetic resonance imaging (MRI) is a critical tool for translational neuroscience, but preclinical studies frequently rely on anesthesia, which alters neural activity and limits comparison with human studies. Awake rodent functional MRI (fMRI) enables investigation of brain function under physiologically relevant conditions; however, its implementation is constrained by the need for anesthesia during restraint setup. We developed and evaluated a restraint system and habituation protocol for awake rat fMRI. Ten rats were studied: an awake group and an anesthetized group (three males and two females per group). The protocol included head post implantation and an 11 d habituation period. T2-weighted anatomical and functional scans were acquired. Head motion and functional connectivity were analyzed using the RABIES pipeline and compared between groups. The modular 3D-printed restraint system developed can be assembled in under 5 min; eliminates the need for anesthesia, ear bars, and bite bars; and supports several behavioral paradigms. High-quality anatomical and functional images were obtained for awake rats. Anesthetized rats exhibited significantly lower translation, rotation, and framewise displacement. Functional connectivity differed between awake and anesthetized rats, with some region pairs showing higher (e.g., left-right primary somatosensory cortex and hypothalamus-insula) and lower (e.g., cingulate-prelimbic cortex and retrosplenial-motor cortex) correlations in awake rats. However, these differences did not survive network-based statistics correction. This work presents a scalable, reproducible, and animal-friendly platform for awake rat fMRI that enables high-quality, behaviorally enriched imaging without anesthesia, while highlighting the effects of anesthesia on functional connectivity.