Afferent connectivity of the Zebrafish Habenulae

The habenular nuclei are bilateral nuclei located in the dorsal diencephalon. The habenulae and their associated circuitry, the dorsal diencephalic conduction system (DDC) are conserved across all vertebrates. As part of the DDC, the habenulae act as a major relay station between forebrain regions and monoaminergic centres in the midbrain. Zebrafish habenulae display prominent neuroanatomical asymmetries and asymmetries in circuit microarchitecture have been described in both afferent and efferent projections. In this study, I characterise two of the main forebrain afferent nuclei of the habenula: the ventral entopeduncular nucleus (vENT) and nucleus rostrolateralis (RL). Through fate mapping, transgenic line analysis and gene expression studies, I show that the vENT is diencephalic in origin and extends into the telencephalon. vENT is the main afferent telencephalic nucleus of the habenula in zebrafish and is homologous to the entopeduncular nucleus in mammals. I show the afferent nucleus conveying visual information to the habenula is RL. RL is prethalamic and retina and tectal recipient. RL and parapineal afferents asymmetrically innervate the left dorsal habenula. Using parapineal ablations and mutants in which habenula asymmetry is disrupted, I show that RL asymmetric innervation is not parapineal dependent. RL afferents arborise close to differentiating “left sided” habenula neuronal sub-types. Previous studies in zebrafish claim the habenula receives pallial innervation. Using a pan-pallial enhancer -trap line, Tg(gata2:EGFP)bi105, I show that this is not the case. I mapped the insertion to egr3, a gene linked to schizophrenia and important for hippocampal learning and memory and characterise the expression pattern of this line at larval stages. To look at the morphologies and projection patterns of individual habenula afferent and pallial neurons I have adapted a method for switching EGFP to Gal4 in transgenic lines using CRISPR/Cas9. The neuroanatomical characterisation of these habenulo-afferent areas lays important groundwork for further functional characterisation of this important circuit.