Characterisation of transgenic lines labelling reticulospinal neurons in larval zebrafish

From lamprey to monkeys, the organization of the descending control of locomotion is conserved across vertebrates. Reticulospinal neurons (RSNs) form a bottleneck for descending commands, receiving innervation from diencephalic and mesencephalic locomotor centres and providing locomotor drive to spinal motor circuits. Given their optical accessibility in early development, larval zebrafish offer a unique opportunity to study reticulospinal circuitry. In fish, RSNs are a small, highly stereotyped, uniquely identifiable group of large neurons spanning from the midbrain to the medulla. Classically labelled by tracer dye injections into the spinal cord, recent advances in genetic tools have facilitated the targeted expression of transgenes in diverse brainstem neurons of larval zebrafish. Here, we provide a comparative characterization of four existing and three newly established transgenic lines in larval zebrafish. We determine which identified neurons are consistently labelled and offer projection-specific genetic access to subpopulations of RSNs. We showcase transgenic lines that label most or all RSNs ( nefma, adcyap1b ^ccu96Et ) or subsets of RSNs, including ipsilateral ( vsx2, calca ^ccu75Et ), contralateral ( pcp4a ^ccu97Tg ) or all ( tiam2a ^y264Et ) components of the Mauthner array, or midbrain-only RSNs ( s1171tEt ). In addition to RSNs, selected transgenic lines ( nefma, s1171tEt, calca ^ccu75Et ) labelled other potential neurons of interest in the brainstem. For those, we performed in situ hybridisation to show expression patterns of several excitatory and inhibitory neurotransmitters at larval stages as well as glutamatergic expression patterns in juvenile fish. We provide an overview of transgene expression in the brainstem of larval zebrafish that serves to lay a foundation for future studies in the supraspinal control of locomotion.