Abstract
The cerebellum is involved in some forms of motor coordination and learning, and in cognitive and emotional functions. To elucidate the functions of the cerebellum, it is important to unravel the detailed connections of the cerebellar neurons. Although the cerebellar neural circuit structure is generally conserved among vertebrates, it is not clear whether the cerebellum receives and processes the same or similar information in different vertebrate species. Here, we performed monosynaptic retrograde tracing with recombinant rabies viruses (RV) to identify the afferent connections of the zebrafish cerebellar neurons. We used a G-deleted RV that expressed GFP. The virus was also pseudotyped with EnvA, an envelope protein of avian sarcoma and leucosis virus (ALSV-A). For the specific infection of cerebellar neurons, we expressed the RV glycoprotein (G) gene and the envelope protein TVA, which is the receptor for EnvA, in Purkinje cells (PCs) or granule cells (GCs), using the promoter for aldolase Ca (aldoca) or cerebellin 12 (cbln12), respectively. When the virus infected PCs in the aldoca line, GFP was detected in the PCs’ presynaptic neurons, including GCs and neurons in the inferior olivary nuclei (IOs), which send climbing fibers (CFs). These observations validated the RV tracing method in zebrafish. When the virus infected GCs in the cbln12 line, GFP was again detected in their presynaptic neurons, including neurons in the pretectal nuclei, the nucleus lateralis valvulae (NLV), the central gray (CG), the medial octavolateralis nucleus (MON), and the descending octaval nucleus (DON). GFP was not observed in these neurons when the virus infected PCs in the aldoca line. These precerebellar neurons generally agree with those reported for other teleost species and are at least partly conserved with those in mammals. Our results demonstrate that the RV system can be used for connectome analyses in zebrafish, and provide fundamental information about the cerebellar neural circuits, which will be valuable for elucidating the functions of cerebellar neural circuits in zebrafish.
Highlights
We previously revealed that cerebellin 12, a zebrafish ortholog of Cerebellin1 in mammals, is expressed in granule cell (GC) in the cerebellum (Takeuchi et al, 2017)
Sagittal sections of the adult Tg brain showed that Venus was detected in cell bodies in the torus longitudinalis (TL) in the tectum, in the granular layer (GL) and lobus caudalis cerebelli (LCa) in the cerebellum where the GC somata were located, and in nerve fibers in the stratum marginale (SM) of the tectum, in the molecular layer (ML) of the corpus cerebelli (CCe), and in the crista cerebellaris (CC) in the dorsal hindbrain where the GC axons were present (Figure 2F)
In this study: (1) we established a transgenic system in which the cbln12 promoter drives transgene expression in GCs in the cerebellum; (2) we developed an Rabies Virus (RV) tracing method that labeled only the presynaptic neurons in zebrafish; and (3) using our RV tracing method, we identified the bona fide precerebellar nuclei giving rise to mossy fiber (MF) in zebrafish
Summary
The cerebellum is involved in smooth and skillful movements and in cognitive and emotional functions, such as fear conditioning and reward expectations (Ito et al, 1982; Raymond et al, 1996; Yoshida et al, 2004; Ito, 2006; Glickstein, 2007; Voogd, 2014; Strata, 2015; Adamaszek et al, 2017; Matsuda et al, 2017; Wagner et al, 2017; Wylie et al, 2018; Schmahmann, 2019). Comparative studies of the cerebellum of different vertebrate species, those having a simple cerebellar structure, may provide clues for understanding the general functions of the cerebellum. The zebrafish brain is a versatile model for studying the functions and development of the vertebrate brain, including the cerebellar neural circuits, since it is smaller than the mammalian brain, and the zebrafish body is transparent during the early larval stages (and at late larval stages in a pigment-less mutant background). Ca2+ imaging and optogenetic manipulation can be used to study zebrafish neural circuits (Matsui et al, 2014a; Kawashima et al, 2016; Song et al, 2016; Cong et al, 2017; Knogler et al, 2017; Matsuda et al, 2017)
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