Article Figures & Data
Figures
- Figure 1.
Neurons retrogradely labeled via tracer injection into the VC. a1, b1, Schematic drawings of the injection of dextran-conjugated Alexa Fluor 488 into the FL (a1) and UN (b1) of rats. a2, b2, Fluorescence photomicrographs of the injection sites. Each photo is separated by 300 μm in the rostrocaudal direction. a3, b3, Confocal images of frontal sections that include the PHN. The dashed line approximately indicates the boundary of the PHN. a4, b4, Enlarged images of the box in 3. a5, b5, Fluorescence photomicrographs of frontal sections that include the inferior olive. a6, b6, Confocal images of frontal sections that include the INC. The dashed line approximately indicates the boundary of the INC. a7, b7, Enlarged images of the box in 6. Scale bars: a2, b2, 500 μm; a3–7, b3–7, 100 μm. 4 V, fourth ventricle; FL, flocculus; UN, uvula and nodulus; r-MAO, rostral part of the medial accessory olive; beta, subnucleus beta of the inferior olive. The Roman numerals indicate the cerebellar lobules.
- Figure 2.
Neurons retrogradely labeled following tracer injection into lobules I–II in the vermis and crus I–II. a1, b1, Schematic drawings of the injection of dextran-conjugated Alexa Fluor 488 into lobules I–II in the vermis (a1) and crus I–II (b1) of rats. a2, b2, Fluorescence photomicrographs of the injection sites. Each photo is separated by 300 μm in the rostrocaudal direction. a3, b3, Fluorescence photomicrographs of frontal sections that include the PHN. The dashed line approximately indicates the boundary of the PHN. a4, b4, Enlarged images of the box in 3. a5, b5, Fluorescence photomicrographs of frontal sections that include the inferior olive. a6, b6, Bright-field images of frontal sections that include the INC. The dashed line approximately indicates the boundary of the INC. a7, b7, Enlarged fluorescence images of the box in 6. Scale bars: a2, b2, 500 μm; a3–7, b3–7, 100 μm. 4 V, fourth ventricle. c-MAO, caudal part of the medial accessory olive; v-PO, ventral lamella of the principal olive; d-PO, dorsal lamella of the principal olive. The Roman numerals indicate the cerebellar lobules.
- Figure 3.
Indirect connections from the INC to the VC via the PHN and MVN. a, Experimental design of retrograde monosynaptic tracing from the PHN and MVN projecting to the FL or UN of a rat. b1, c1, Fluorescence photomicrographs of GFP at the sites of injection of two helper AAV2retro viruses (AAV2retro-G and AAV2retro-GFP-TVA) on the right side of the FL (b1) and the midline region of the UN (c1). b2,3, c2, Example of starter cells (arrows) in the right PHN and MVN expressing both GFP and RFP. EnvA-RVdG-RFP was injected into the right side of the PHN and MVN. b4, c3, Examples of RFP-expressing neurons in the INC that received retrograde transsynaptic infection with rabies virus from starter cells. d1,2, Numbers of starter cells in the PHN and MVN (1) and RFP-expressing neurons in the INC (2), which provide inputs to the starter cells, for each rat analyzed. “FL” and “UN” represent groups where helper AAVs were injected into the FL and UN, respectively. The numbers following “FL” and “UN” are used to distinguish individual rats within each group. d3, Relationships between the number of starter cells and RFP-expressing neurons in the INC. Scale bars: b1, c1, 500 μm; b2–4, c2,3, 100 μm.
- Figure 4.
Control experiments for rabies transsynaptic tracing. a, Frontal section of the cerebellum at the site of injection of AAV2retro-GFP-TVA in the UN (AAV2retro-G was not injected). b, Enlarged image of the box in a. c, Frontal section showing the PHN and MVN after AAV2retro-GFP-TVA injection into the UN and after EnvA-RVdG-RFP injection into the PHN and MVN. The dashed line approximately indicates the boundary of the PHN and MVN. d, e, RFP (d) and GFP (e) images from c. The arrows indicate neurons that express both GFP and RFP. Scale bars: a, b, 500 μm; e, 100 μm.
- Figure 5.
Distribution of RFP-expressing neurons in the rostral–caudal regions of the INC. a1–3, b1–3, Fluorescence photomicrographs of frontal sections that include the INC after rabies virus transsynaptic tracing (related to Fig. 3). RFP-expressing neurons in the rostral (1), intermediate (2), and caudal (3) regions of the INC following injection of the two helper AAV2 retroviral vectors into the FL (a) and UN (b). (a4, b4) Comparison of the percentages of RFP-expressing (RFP+) neurons in each region with respect to the total number of RFP-expressing neurons in the INC (a4, n = 3; b4, n = 4). Individual plots indicate data obtained from individual rats. The error bars represent the SDs. Scale bars: a3, b3, 100 μm. Asterisks indicate a significant difference between groups (*p < 0.05; **p < 0.01).
- Figure 6.
The indirect INC-FL pathway is mediated via the bilateral PHNs and MVNs. a1, Fluorescence photomicrograph of GFP at the site of injection of the two helper AAV2retro viruses (AAV2retro-G and AAV2retro-GFP-TVA) on the right of the FL. a2,3, Example starter cells (arrows) expressing both GFP and RFP in the left PHN and MVN. EnvA-RVdG-RFP was injected into the left PHN and MVN. Figure 3 shows that the rabies virus was injected into the right PHN and MVN. a4,5, Fluorescence photomicrographs of frontal sections that include the INC. RFP-expressing neurons, which received retrograde transsynaptic infection with rabies virus from starter cells, were observed in the right (4) and left INC (5). b, Schematic diagram of the pathway from the INC to the FL via the bilateral PHNs and MVNs revealed in this study. The diagram shows the neural pathway to the right FL only. Scale bars: a1, 200 μm; a2–5, 100 μm.
- Figure 7.
Inferior olive neurons retrogradely labeled following helper AAV2 retrovirus injection into the FL. a, Fluorescence photomicrographs at the sites of injection of the two helper AAV2 retroviral vectors (AAV2retro-GFP-TVA and AAV2retro-G) in the right FL (related to Fig. 3). Each photo is separated by 300 μm in the rostrocaudal direction. b–d, Fluorescence photomicrographs of frontal sections that include the CDC and VLO of the inferior olive and the r-MAO. The arrows indicate the midline. Scale bars: a, 200 μm; b–d, 100 μm. FL, flocculus; CDC, caudal dorsal cap; VLO, ventrolateral outgrowth; r-MAO, rostral part of the medial accessory olive.
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