Development of functional hindbrain oculomotor circuitry independent of both vascularization and neuronal activity in larval zebrafish

In nearly all adult vertebrates, the vasculature is of key importance for supplying nutrients and oxygen to the brain. Throughout the nervous system, neurons and blood vessels align closely to form the neurovascular unit [1]. Little is known, however, about the causal role of the vasculature during the embryonic establishment of neuronal pathways and functional activity. In addition, little information exists about the interplay between neuronal excitability and embryonic development of the neurovascular unit. Several studies have shown that endothelial cells can stimulate the formation of neurons [2-4] and that neurons can instruct blood vessel development [5,6]. Moreover, neuronal activity can regulate vascular tonus in mouse brain slices, suggesting that the supply of nutrients and oxygen to the brain is tightly controlled by local energy demands in the brain [7]. We investigated the contribution of blood vessel formation and neuronal excitability to the development of functional neural circuitry in larval zebrafish by analyzing oculomotor performance in response to visual and vestibular stimuli. To address the dependence of neuronal function on the presence of blood vessels, we compared wild type embryos to reck and cloche mutants that lacked intracerebral blood vessels. To test how neuronal excitability impacts neuronal development and intracerebral vascularization, we blocked neural activity using Tetraodotoxin (TTX) and Tricaine. In reck mutants, we found both slow phase horizontal tracking and fast phase resets with only a slightly reduced amplitude and bandwidth. Spontaneous saccades, eye position holding and vestibular gravitoinertial induced eye rotation were also present. All of these behaviors except for visual tracking were observed in cloche mutants that lacked any head vasculature. Thus, numerous oculomotor neuronal circuits spanning the forebrain, midbrain and hindbrain compartments, ending in motor innervations of the eye muscles, were correctly formed and generated appropriate oculomotor behaviors without blood vessels. However, our observations indicate that beginning at approximately six days, circulation was required for sustained behavioral performance. We further found that blocking neuronal excitability with either TTX or Tricaine up to 4-5 days post fertilization did not noticeably interfere with intracerebral blood vessel formation in wild type larvae. After removal of drug treatments, the oculomotor behaviors returned within hours. Thus, development of neuronal circuits that drive oculomotor performance does not require neuronal spiking or activity. Together these findings demonstrate that neither vascularization nor neuronal excitability are essential for the formation of numerous oculomotor nuclei with intricately designed connectivity and signal processing. We conclude that a genetic blueprint specifies early larval structural and physiological features, and this developmental strategy may be viewed as a unique adaptation required for early survival. Abstract