Recent advances in ultrasound Doppler imaging have facilitated the technique of functional ultrasound (fUS) which enables visualization of brain-activity due to neurovascular coupling. As of yet, this technique has been applied to rodents as well as to human subjects during awake craniotomy surgery and human newborns. Here we demonstrate the first successful fUS studies on awake pigeons subjected to auditory and visual stimulation. To allow successful fUS on pigeons we improved the temporal resolution of fUS up to 20,000 frames per second with real-time visualization and continuous recording. We show that this gain in temporal resolution significantly increases the sensitivity for detecting small fluctuations in cerebral blood flow and volume which may reflect increased local neural activity. Through this increased sensitivity we were able to capture the elaborate 3D neural activity pattern evoked by a complex stimulation pattern, such as a moving light source. By pushing the limits of fUS further, we have reaffirmed the enormous potential of this technique as a new standard in functional brain imaging with the capacity to unravel unknown, stimulus related hemodynamics with excellent spatiotemporal resolution with a wide field of view.

Recently, a new functional neuroimaging method called fUS was proposed, which is based on high-frame-rate Power Doppler imaging. So far, fUS has only been performed on rodents, but major issues in neuroscience such as cerebral asymmetries and language learning are mainly studied in birds. Here, we show the first successful fUS measurements on a non-mammalian species without cortical brain architecture, such as pigeons. These measurements are based on a framerate enhanced fUS acquisition algorithm, which was necessary to suppress the signal variations originating from the slower heartrate of pigeons.