Table 1.
Labeled Purkinje cell axonal boutons after FL lentivirus injections.
Fig 1.
Anterogradely labeled FL P-cell axons in the medial vestibular nucleus (MVN) and prepositus hypoglossi (PrH) in case (#) 94R.
A, GFP expression in FL at 3 weeks after lentiviral injection. Sections were stained with anti-GFP (green) and neuronal marker anti-NeuN (red) antibodies. B, GFP was preferentially expressed in FL P-cells. C, GFP-labeled axons of FL P-cells in the ventromedial MVN. FL, flocculus; icp, inferior cerebellar peduncle; MVMC, magnocellular MVN; MVPC, parvocellular MVN; P-cell, Purkinje cell; PFL, paraflocculus; PrH, prepositus hypoglossi nucleus; 4V, fourth ventricle. Scale bars, 200 μm (A and C) and 50 μm (B).
Fig 2.
Distribution of FL P-cell axons and their boutons in MVN/PrH after FL lentivirus injections.
A–D, Tracing of anterogradely-labeled FL axons in four mice. Despite the difference of density of GFP expression among four cases shown in the photographs in the top row, labeled FL P-cell axons were distributed throughout MVN and PrH in all of the four cases. Distributions of the labeled axons in MVN and PrH for each injection are summarized in 4 images that are shown from the rostral to caudal MVN/PrH. A, case (#) 12L; B, #14L; C, #74R; D, #94R. Labeled FL P-cell axons and their boutons are respectively shown by black lines and red circles. E–G, Summary of distribution of FL P-cell axonal boutons at rostral (E), middle (F), and caudal (G) MVN. The averaged density of labeled axonal boutons was calculated from cases 12L, 14L, 74R, and 94R. Although the FL P-cell axonal boutons were observed throughout MVN, they were densely present in the medial part of parvocellular MVN/PrH. MVMC, magnocellular MVN; MVPC, parvocellular MVN; PrH, prepositus hypoglossi nucleus; 4V, fourth ventricle. Scale bars, 200 μm (A–D) and 100 μm (E–G).
Fig 3.
Two distinct types of FL P-cell axonal boutons observed in MVN/PrH (case (#) 94R).
A–C, Merged images of GFP-fluorescent FL P-cell axonal boutons (green) and anti-NeuN-labelled somata (red) of MVN neurons. In A, anterogradely labeled FL P-cell axonal boutons were observed in the magnocellular MVN, in which the somata and proximal dendrites of MVN neurons were surrounded by a small group of axons, showing basket like structures (arrowheads in B and C). D–F, Similar to A–C, but for those observed in the parvocellular MVN/PrH, in which axonal boutons showing en passant structures were dominantly observed (arrows in E and F). MVMC, mgnocellular NVN; MVPC, parvocellular MVN; PrH, prepositus hypoglossi nucleus; 4V, fourth ventricle. Scale bars, 200 μm (A and D) and 20 μm (B, C, E and F).
Fig 4.
Neurons that receive FL P-cell projections on their distal dendrites in the parvocellular MVN/PrH are predominantly glutamatergic.
A and B, Representative images of MVN/PrH neurons of Thy1-GFP M-line transgenic mouse that were stained with anti-GFP (green) and anti-PKCγ (red) antibodies. C, D, and E, Images of coronal sections of MVN/PrH stained with either one of three antibodies against neurotransmitters (anti-Glu, anti-Gly, and anti-GABA). The majority of GFP-labeled neurons were stained positive for anti-Glu antibody (C), and some of the remaining neurons were positive for anti-Gly antibody (D), but not positive for anti-GABA antibody (E). Scale bars, 200 μm (A), 100 μm (B), and 50 μm (C–E). GABA, γ-aminobutyric acid; Glu, glutamate; Gly, glycine.
Fig 5.
Axodendritic synapses between FL P-cells and parvocellular MVN/PrH neurons.
A1–A4, B1–B4, Photographs of two examples of EM serial sections (interval, 0.14 μm) for FL P-cell axonal boutons forming symmetrical synapses on parvocellular MVN/PrH neurons. Edges of synapses were shown by arrowheads. C, Distribution of the mean diameters for post-synaptic dendrites. Note that FL P-cell axonal boutons formed synapses on relatively small diameter dendrites (mean, 0.73 μm; n = 40 dendrites from three mice). den, dendrite of MVN/PrH neuron; Pb, P-cell axonal bouton. Scale bars, 0.2 μm.
Fig 6.
The location of FL P-cell axonal boutons and excitatory synapses on the dendrites of parvocellular MVN/PrH neurons.
A1–A4, B1–B4, EM photographs of two examples of serial sections (interval, 0.21 μm) for the FL P-cell axonal boutons forming symmetrical synapses on MVN/PrH neurons. Note that the FL axonal boutons (arrow heads) were located close to the excitatory synapses on the same dendrites (arrows). * indicates excitatory synapses. den, dendrite of MVN/PrH neuron; Pb, P-cell axonal bouton. Scale bars, 0.2 μm.
Fig 7.
Distribution of vestibular nerve axonal boutons and FL P-cell axonal boutons in the parvocellular MVN/PrH.
A, Low magnification image of MVN/PrH showing two distinct types of synaptic inputs. BDA was injected into the horizontal semicircular canal after the lentivirus injection into FL. FL P-cell axons and vestibular nerve axons were visualized by anti-GFP antibody (green) and Alexa 594 streptavidine (red), respectively. Note that the distribution of FL P-cell axons overlapped partially with that of the vestibular nerve axons in MVN/PrH. B, High magnification confocal image of the region marked by square in A. Some of the FL P-cell axonal boutons were closely located to those of the vestibular nerve axonal boutons (arrowheads). C1–C3, D1–D3, Two examples of serial EM photographs (interval, 0.14 μm in C1–C3, 0.21 μm in D1–D3) of MVN/PrH neuronal dendrites. GFP (FL P-cell axons) was detected by DAB reaction products, and BDA (vestibular nerve axons) was detected by silver-enhanced gold particles. Note that the FL P-cell axons formed symmetrical synapses (arrowheads), whereas the vestibular nerve axons formed asymmetrical synapses (arrows) on the same MVN neuronal dendrites. den, dendrite of MVN/PrH neuron; MVN, medial vestibular nucleus; Pb, P-cell axonal bouton; PrH, prepositus hypoglossi nucleus; Vb, vestibular nerve axonal bouton; 4V, fourth ventricle. Scale bars, 200 μm (A), 20 μm (B), and 0.2 μm (C1–C3, D1–D3).
Fig 8.
A putative scheme for the two different types of FL-targeted HVOR/HOKR-relaying neurons in mouse MVN.
In magnocellular MVN, FL P-cell axons make synaptic contacts on the proximal dendrites and somata of neurons [23,34], Figs 2 and 3 in the present study), which are presumably inhibitory glycinergic neurons projecting to the ipsilateral abducens nucleus [34]. By contrast, in parvocelliar MVN/PrH, FL P-cells make synaptic contacts on the distal dendrites of neurons (Figs 2, 3 and 5), which are presumably excitatory glutamatergic (Fig 4 and S2 Fig) neurons projecting to the ipsilateral oculmotor nucleus [53]. Also see S1 Table. ABN, abducens nucleus; Glu, glutamatergic (excitatory) neuron; Gly, glycinergic (inhibitory) neuron; OMN, oculomotor nucleus.