Characterisation of transgenic lines labelling reticulospinal neurons in larval zebrafish

Abstract

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 few, highly stereotyped, uniquely identifiable, 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, adcyap1bccu96Et) or subsets of RSNs, including ipsilateral (vsx2, calcaccu75Et), contralateral (pcp4accu97Tg) or all (tiam2ay264Et) components of the Mauthner array, or midbrain-only RSNs (s1171tEt). In addition to RSNs, selected transgenic lines (nefma, s1171tEt, calcaccu75Et) 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. Significance StatementGenetic access to subpopulations of brainstem neurons greatly facilitates the dissection of supraspinal circuitry and function. Here, we present several new transgenic lines and rigorously describe existing ones, all in terms of their degree of overlap with the reticulospinal system, variability in transgenic labelling and neurotransmitter identity. Having transgenic access to different subpopulations of reticulospinal neurons enables targeted functional calcium imaging, anatomical tracing and optogenetic manipulations to decipher the role of individual reticulospinal neurons in movement production, ultimately clarifying existing understanding and facilitating future studies in the supraspinal control of locomotion. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/629714v2_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@d2161eorg.highwire.dtl.DTLVardef@aa4b97org.highwire.dtl.DTLVardef@48c9b9org.highwire.dtl.DTLVardef@b394ee_HPS_FORMAT_FIGEXP M_FIG C_FIG