Background

Heart and respiratory rates reflect dynamic responses of the autonomic nervous system to external and internal stimuli [1,2]. Cardiogenic pulsatility, frequently assessed via non-invasive electrocardiogram (ECG) measurements, plays a crucial role in driving various homeostatic processes in the brain, including the regulation of blood flow and cerebrospinal fluid (CSF) dynamics [3]. While ECG is a state-of-the-art method in clinics and preclinical settings, in small animal research models, it is predominantly performed under anesthesia to avoid the confounding effects of stress and motion [4]. Furthermore, simultaneous ECG and brain imaging in awake conditions can provide valuable correlative measures that allow a closer understanding of the relationship between brain function, cognitive processes, motor behavior, CSF transport, and the complex cardiovascular dynamics associated with autonomous nervous system activity [5].

With the advent of glymphatic system research [6], simultaneous in vivo ECG and microscopy recordings in awake animal models are crucial to underpin the influence of complex heart–brain interaction and to bring higher translational value to basic glymphatic research.

Existing methods for obtaining ECGs in fully awake mice typically involve restraint or telemetry devices, which often represent a suboptimal solution or introduce confounding effects into the acquired signals by inducing stress or altering recovery after implantation of the device [2,4,7,8]. To address these limitations, we designed a technique that enables rapid, minimally invasive ECG recordings in awake head-fixed mice. This approach enables long-term, simultaneous ECG and respiratory recordings alongside in vivo microscopy, which is highly desired in neuroscientific applications. Therefore, the proposed and validated setup allows a comprehensive platform for studying cardiovascular dynamics alongside cerebrovascular changes during full wakefulness. This integrated methodology surpasses historical constraints, offering a powerful tool for understanding the interplay between cardiovascular and neural activities during various physiological and pathological conditions.