Label‐Free Multiphoton Imaging Reveals Volumetric Shifts Across Development in Sensory‐Related Brain Regions of a Miniature Transparent Vertebrate

Animals integrate information from different sensory modalities as they mature and perform increasingly complex behaviors. This may parallel differential investment in specific brain regions depending on the changing demands of sensory inputs. To investigate developmental changes in the volume of canonical sensory regions, we used third harmonic generation imaging for morphometric analysis of forebrain and midbrain regions from larval through juvenile and adult stages in Danionella dracula, a transparent, miniature teleost fish whose brain is optically accessible throughout its lifespan. Relative to whole-brain volume, increased volume or investment in the telencephalon, a higher order sensory integration center, shows the most dramatic increases between 30-60 days postfertilization (dpf) and again at 90 dpf as animals reach adulthood. The torus longitudinalis (TL), a midbrain visuomotor integration center, also significantly increases between 60 and 90 dpf. In contrast, investment in the midbrain optic tectum (TeO), a retinal-recipient target, progressively decreases from 30 to 90 dpf, whereas investment is relatively consistent across all stages for the midbrain torus semicircularis (TS), a secondary auditory and mechanosensory lateral line center, and the olfactory bulb (OB), a direct target of the olfactory epithelium. In sum, increased investment in higher-order integration centers (telencephalon, TL) occurs as juveniles reach adulthood (60-90 dpf) and exhibit more complex cognitive tasks, whereas investment in modality-dominant regions occurs earlier (TeO) or is relatively consistent across development (TS, OB). Complete optical access throughout Danionella's lifespan provides a unique opportunity to investigate how changing brain structure over development correlates with changes in connectivity, microcircuitry, or behavior.