Tracing of Afferent Connections in the Zebrafish Cerebellum Using Recombinant Rabies Virus
Tracing of Afferent Connections in the Zebrafish Cerebellum Using Recombinant Rabies Virus
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DOI：
10.3389/fncir.2019.00030
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发表时间：
2019-04-24
影响因子：
3.5
通讯作者：
Hibi, Masahiko
中科院分区：
文献类型：
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作者：
Dohaku, Ryuji;Yamaguchi, Masahiro;Hibi, Masahiko
小脑参与某些形式的运动协调和学习，以及认知和情感功能。为了阐明小脑的功能，阐明小脑神经元的详细连接非常重要。尽管小脑神经回路结构在脊椎动物中普遍保守，但目前尚不清楚不同脊椎动物物种中小脑是否接收和处理相同或相似的信息。在这里，我们用重组狂犬病病毒（RV）进行单突触逆行追踪，以识别斑马鱼小脑神经元的传入连接。我们使用表达 GFP 的 G 缺失 RV。该病毒还用EnvA（禽肉瘤和白血病病毒（ALSV-A）的包膜蛋白）进行了假型化。对于小脑神经元的特异性感染，我们分别使用醛缩酶 Ca (aldoca) 或小脑蛋白 12 (cbln12) 的启动子在浦肯野细胞 (PC) 或颗粒细胞 (GC) 中表达 RV 糖蛋白 (G) 基因和包膜蛋白 TVA（EnvA 的受体）。当病毒感染阿尔多卡系中的 PC 时，在 PC 的突触前神经元中检测到 GFP，包括 GC 和下橄榄核 (IO) 中的神经元，这些神经元发送攀爬纤维 (CF)。这些观察结果验证了斑马鱼的 RV 追踪方法。当病毒感染cbln12系中的GC时，在其突触前神经元中再次检测到GFP，包括顶盖前核、外侧瓣膜核（NLV）、中央灰质（CG）、内侧八外侧核（MON）和降八瓣核（DON）中的神经元。当病毒感染羊驼系中的 PC 时，在这些神经元中没有观察到 GFP。这些小脑前神经元通常与其他硬骨鱼物种的报道一致，并且至少部分与哺乳动物中的神经元相同。我们的结果表明，RV系统可用于斑马鱼的连接组分析，并提供有关小脑神经回路的基本信息，这对于阐明斑马鱼小脑神经回路的功能具有重要价值。
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.