Significance: Functional brain imaging in awake animal models is a popular and powerful technique that allows the investigation of neurovascular coupling (NVC) under physiological conditions. However, ubiquitous facial and body motions (fidgeting) are prime drivers of spontaneous fluctuations in neural and hemodynamic signals. During periods without movement, animals can rapidly transition into sleep, and the hemodynamic signals tied to arousal state changes can be several times larger than sensory-evoked responses. Given the outsized influence of facial and body motions and arousal signals in neural and hemodynamic signals, it is imperative to detect and monitor these events in experiments with un-anesthetized animals.

Aim: To cover the importance of monitoring behavioral state in imaging experiments using un-anesthetized rodents, and describe how to incorporate detailed behavioral and physiological measurements in imaging experiments.

Approach: We review the effects of movements and sleep-related signals (heart rate, respiration rate, electromyography, intracranial pressure, whisking, and other body movements) on brain hemodynamics and electrophysiological signals, with a focus on head-fixed experimental setup. We summarize the measurement methods currently used in animal models for detection of those behaviors and arousal changes. We then provide a guide on how to incorporate this measurements with functional brain imaging and electrophysiology measurements.

Results: We provide a how-to guide on monitoring and interpreting a variety of physiological signals and their applications to NVC experiments in awake behaving mice.

Conclusion: This guide facilitates the application of neuroimaging in awake animal models and provides neuroscientists with a standard approach for monitoring behavior and other associated physiological parameters in head-fixed animals.