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. 2018 Feb 7:12:6.
doi: 10.3389/fncir.2018.00006. eCollection 2018.

Anatomical Evidence for a Direct Projection from Purkinje Cells in the Mouse Cerebellar Vermis to Medial Parabrachial Nucleus

Mitsuhiro Hashimoto  1   2   3   4 , Akihiro Yamanaka  3 , Shigeki Kato  5 , Manabu Tanifuji  4   6   7 , Kazuto Kobayashi  5 , Hiroyuki Yaginuma  1
Affiliations

Anatomical Evidence for a Direct Projection from Purkinje Cells in the Mouse Cerebellar Vermis to Medial Parabrachial Nucleus

Mitsuhiro Hashimoto et al. Front Neural Circuits. .

Abstract

Cerebellar malformations cause changes to the sleep-wake cycle, resulting in sleep disturbance. However, it is unclear how the cerebellum contributes to the sleep-wake cycle. To examine the neural connections between the cerebellum and the nuclei involved in the sleep-wake cycle, we investigated the axonal projections of Purkinje cells in the mouse posterior vermis by using an adeno-associated virus (AAV) vector (serotype rh10) as an anterograde tracer. When an AAV vector expressing humanized renilla green fluorescent protein was injected into the cerebellar lobule IX, hrGFP and synaptophysin double-positive axonal terminals were observed in the region of medial parabrachial nucleus (MPB). The MPB is involved in the phase transition from rapid eye movement (REM) sleep to Non-REM sleep and vice versa, and the cardiovascular and respiratory responses. The hrGFP-positive axons from lobule IX went through the ventral spinocerebellar tract and finally reached the MPB. By contrast, when the AAV vector was injected into cerebellar lobule VI, no hrGFP-positive axons were observed in the MPB. To examine neurons projecting to the MPB, we unilaterally injected Fast Blue and AAV vector (retrograde serotype, rAAV2-retro) as retrograde tracers into the MPB. The cerebellar Purkinje cells in lobules VIII-X on the ipsilateral side of the Fast Blue-injected MPB were retrogradely labeled by Fast Blue and AAV vector (retrograde serotype), but no retrograde-labeled Purkinje cells were observed in lobules VI-VII and the cerebellar hemispheres. These results indicated that Purkinje cells in lobules VIII-X directly project their axons to the ipsilateral MPB but not lobules VI-VII. The direct connection between lobules VIII-X and the MPB suggests that the cerebellum participates in the neural network controlling the sleep-wake cycle, and cardiovascular and respiratory responses, by modulating the physiological function of the MPB.

Keywords: aden-associated virus; anterograde tracing; cerebellar vermis; cerebellum circuits; medial parabrachial nucleus; retrograde tracing.

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Figures

Figure 1
Figure 1
Regions projected from cerebellar lobule IX. Axons from cerebellar lobule IX were anterogradely labeled by AAV-CMV-hrGFP. (A–I) Series of transversal sections from posterior to anterior cerebellum. Fluorescence of hrGFP on each section was imaged. (A'–I', A”, B”) The magnified images of quadrilateral regions in each section. Neurons in lobule IX are selectively and densely labeled by hrGFP (A', B'). Inferior olive (IO) neurons are not retrogradely labeled by AAV-CMV-hrGFP (A”, B”). hrGFP-positive neurons in cerebellar lobule IX project their axons to medial (fastigial) cerebellar nucleus (Med; C'–F'), posterior interposed cerebellar nucleus (IntP; D', E'), spinal vestibular nucleus (SpVe; C'–E'), superior vestibular nucleus (SuVe; F', G'), lateral vestibular nucleus (LVe; F'), lateral parabrachial nucleus (LPB; G'–I'), and medial parabrachial nucleus (MPB; G'–I'). (J–J”) Magnified images of MPB. (J') The magnified image of the region of MPB and scp in (J). (J”) The magnified image of a quadrilateral region in (J'). Arrowheads indicate hrGFP-positive axonal varicosities in MPB. 4v, fourth ventricle; IX, cerebellar lobule IX; X, cerebellar lobule X; cbc, cerebellar commissure; Cu, cuneate nucleus; ECu, external cuneate nucleus; FL, flocculus; Gi, gigantocellular reticular nucleus; MVePC, parvocellular part of medial vestibular nucleus; IntA, anterior interposed cerebellar nucleus; icp, inferior cerebellar peduncle; Lat, lateral (dentate) cerebellar nucleus; LRt, lateral reticular nucleus; mcp, medial cerebellar peduncle; MdV, ventral part of medullary reticular nucleus; PCGS, paracochlear glial substance; PF, paraflocculus; Pr, prepositus nucleus; Pr5, principal sensory trigeminal nucleus; scp, superior cerebellar peduncle; Sol, nucleus of the solitary tract; Sp5C, caudal part of spinal trigeminal nucleus; SP5I, interpolar part of spinal trigeminal nucleus; XN, X nucleus. Scale bar, 1 mm in (A–J), (A'–I', A”, B”); 500 μm in (J'); 50 μm in (J”).
Figure 2
Figure 2
Definition of a region of interest. Schematic drawings indicate serial transverse sections at intervals of 200 μm. The diamond pattern area is defined as a region of interest (ROI). ROI is located in the ventral side of scp and identical to anterior region of the MPB. The posterior region of MPB that is adjacent to the vestibular nuclei was eliminated from the ROI (lower panel). 4v, fourth ventricle; 7n, root of facial nucleus; d, dorsal; DTg, dorsal tegmental nucleus; g7, genu of facial nucleus; l, lateral; Mo5, motor trigeminal nucleus; MVeMC, magnocellular part of medial vestibular nucleus; py, pyramidal tract; s5, sensory root of trigeminal nucleus; spt5, spinal trigeminal tract; Su5, supratrigeminal nucleus; v, ventral. Scale bar, 500 μm.
Figure 3
Figure 3
MPB projected from lobule IX but not from lobule VI. (A–C) Anterograde labeling from cerebellar lobule IX by AAV-CMV-hrGFP. (D–E) Anterograde labeling from cerebellar lobule VI by AAV-CMV-hrGFP (B,E). The transversal sections of each labeled brain were immunostained with anti-TH antibody, which is a marker for locus coeruleus (LC) (C,F). Merged images are indicated in (A) and (D). (A,B) In the case of cerebellar lobule IX labeling, hrGFP-positive axons are abundant in LPB, MPB, the medial-most region of superior cerebellar peduncle (mscp), and ventral spinocerebellar tract (vsc). There are no hrGFP-positive axons in TH-positive LC, scp excluding mscp, and uncinate fasciculus of cerebellum (unc). (E) In the case of cerebellar lobule VI labeling, hrGFP-positive axonal terminals are absent in LPB, MPB, and vsc. Scale bar, 500 μm.
Figure 4
Figure 4
Immunohistochemistry with anti-synaptophysin (SYP) and anti-vesicular glutamate transporter 2 (VGLUT2) antibodies. The transversal sections of brain that are anterogradely labeled by the AAV-CMV-hrGFP injection into lobule IX are immunostained with anti-SYP (A–I), anti-VGLUT2 (J–L), and anti-CGRP (M–R) antibodies. Z-stacked confocal images of hrGFP-fluorescence (A,D,G,J,P), z-stacked confocal images of immunostaining (B,E,H,K,Q), and merged images of the hrGFP-fluorescence and the immunostaining (C,F,I,L,R) are indicated. (A–C) vsc immunostained with anti-SYP antibody. The hrGFP-positive axonal fibers in vsc are negative for SYP. (D–F) LPB immunostained with anti-SYP antibody. The hrGFP-positive axonal fibers in LPB are negative for SYP. (G–I) MPB immunostained with anti-SYP antibody. The hrGFP-positive axonal terminals in MPB are positive for SYP (arrows). (J–L) MPB immunostained with anti-VGLUT2 antibody. Axonal terminals labeled by hrGFP are observed around VGLUT2-positive MPB neurons (arrows). (M–O) Immunostaining with anti-CGRP antibody. The hrGFP-positive axonal fibers (M) and the CGRP-positive cells [horizontal arrows in (N,O)] are observed in ventrolateral region of MPB. (P–R) Z-stacked confocal images of the rectangular regions in (M–O). The images rotated counterclockwise by 90 degrees are shown. Axonal terminals labeled by hrGFP are observed around CGRP-positive MPB neurons (horizontal arrows). Scale bar, 50 μm.
Figure 5
Figure 5
Injection sites of Fast Blue into MPB and MVe. (A–D) The Fast Blue-injection site into MPB. (E–H) The Fast Blue-injection site into MVe. Schematic drawings (A–D, E–H) indicate serial sections at intervals of 200 μm. (A) is identical to Figure 6A. (G) and (H) are identical to Figures 8A,B respectively. The Fast Blue injection sites are shown in blue regions. The intensity of Fast Blue-labeling of (B,G) is weaker than it of (A,H). The Fast Blue-injection site to MPB does not overlap with the Fast Blue-injection site to MVe. 7n, root of facial nucleus; DTg, dorsal tegmental nucleus; g7, genu of facial nucleus; Mo5, motor trigeminal nucleus; py, pyramidal tract; s5, sensory root of trigeminal nucleus; spt5, spinal trigeminal tract; Su5, supratrigeminal nucleus. Scale bar, 500 μm.
Figure 6
Figure 6
Projection neurons to MPB. Neurons projecting their axons to MPB were retrogradely labeled by Fast Blue. (A) Local injection of Fast Blue to MPB. The injection point is indicated by an arrow. (B) Magnified image of region including LPB, scp, and vsc. Fas Blue-labeled axons pass through LBP and along vsc. There are no Fast Blue-labeled axons in scp and unc. (C–E) Series of transversal sections from posterior to anterior cerebellum. (C'–E', D”, E”) The magnified images of quadrilateral regions in each section. Arrowheads indicate Purkinje cells labeled retrogradely from MPB by Fast Blue. Fast Blue-labeled Purkinje cells are observed on the ipsilateral side of lobules VIII–IX (C', D', D”), lobules II–IV (E”), ventral PF (E, E'), and FL (E, E'). No Purkinje cells in lobules VI and VII are labeled by Fast Blue (C', D”). Dashed lines in (C'), (E”), and (F) indicate the midline. Dotted line in (E') indicates posterolateral fissure (plf). (F) Magnified image of bilateral cerebellar nuclei. Ipsilateral IntP is labeled by Fast Blue. By contrast, neurons in contralateral Med are more labeled than in ipsilateral Med. II–IX, numbers of cerebellar lobules; ipsi, ipsilateral; MdD, dorsal part of medullary reticular nucleus; Pa5, paratrigeminal nucleus; pcn, precentral fissure; PCR, parvocellular reticular nucleus; pcuf, preculminate fissure; ppf, prepyramidal fissure; RVL, rostral ventrolateral reticular nucleus; sf, secondary fissure. Scale bar, 500 μm.
Figure 7
Figure 7
Projections to MPB from lobule VIII. Axons from cerebellar lobule VIII are anterogradely labeled by AAV-CMV-hrGFP. (A) Local injection of AAV-CMV-hrGFP to lobule VIII. Lobule VIII (VIII) is selectively and densely labeled by hrGFP. Inferior olive (IO) neurons are negative for hrGFP. (B) hrGFP-positive axons are abundant in LPB, MPB, and vsc, but not in scp and unc. VII, cerebellar lobule VII; IX, cerebellar lobule IX. Scale bar, 500 μm.
Figure 8
Figure 8
Projection neurons to medial vestibular nuclei. (A–D) Series of transversal sections from the anterior to posterior cerebellum. Fluorescence of Fast Blue on each section was imaged. The images are reversed to enhance visibility of Fast Blue labeling. Black dots are Fast Blue labeled neurons. (A, B) Fast Blue injection site. Fast Blue is locally injected into MVePC. The neurons projecting to MVePC are retrogradely labeled by Fast Blue. The arrowheads indicate the Fast Blue-positive Purkinje cells in lobules I–X, copula pyramidis (Cop), crus1 of ansiform lobule (Crus1), FL, and PF. The Fast Blue-positive Purkinje cells are observed on the bilateral side to the Fast Blue injected MVe. II–IX, numbers of cerebellar lobules; cont, contralateral; g7, genu of facial nucleus; ipsi, ipsilateral; LVe, lateral vestibular nucleus; MdD, dorsal part of medullary reticular nucleus; Pa5, paratrigeminal nucleus; pcn, precentral fissure; pcuf, preculminate fissure; ppf, prepyramidal fissure; sf, secondary fissure; SuVe, superior vestibular nucleus. Scale bar, 500 μm.
Figure 9
Figure 9
Projection neurons to MPB. Neurons projecting their axons to MPB were retrogradely labeled by AAV2retro-CAG-EGFP. (A) Local injection of AAV2retro-CAG-EGFP to MPB. The injection site is labeled by EGFP. (B) Schematic drawing of the AAV2retro-CAG-EGFP injection site. Green regions indicate AAV2retro-CAG-EGFP injected region. (C) Purkinje cells retrogradely labeled by AAV2retro-CAG-EGFP. The posterodorsal view of cerebellum is shown. Arrowheads indicate the EGFP-positive dendrites of Purkinje cells. The EGFP-positive dendrites are observed on the ipsilateral side (ipsi) but not on the contralateral side (cont) to the MPB injected with AAV2retro-CAG-EGFP. Unfilled arrowheads indicate the EGFP-negative lateral regions of lobules VIII and IX. (D–G) Series of transversal sections from posterior to anterior cerebellum immunostained with an anti-GFP antibody. The immunoreactivity to GFP is visualized by brown. Arrowheads indicate EGFP-positive bands in lobule IV/V. Arrows indicate the EGFP-negative hemispheres and lobules VI and VII. Unfilled arrowheads indicate the EGFP-negative lateral regions of lobules VIII–X. plf, posterolateral fissure; ppf, prepyramidal fissure; psf, posterior superior fissure; RVL, rostroventrolateral medulla; sf, secondary fissure. Scale bar, 1 mm.
Figure 10
Figure 10
Projection neurons to cerebellar lobule IX. Neurons projecting to cerebellar lobule IX were retrogradely labeled by Fast Blue. (A) Local injection of Fast Blue to cerebellar lobule IX. The injection site is indicated by an arrow. (B–J) Retrogradely labeled neurons from cerebellar lobule IX in dorsolateral Pn (B, arrowhead), beta subnucleus of IO (C, arrowhead), paraventricular hypothalamic nucleus (PVN; D, arrowheads), periaqueductal gray (PAG; E, arrowheads), parvocellular part of red nucleus (RPC; F, arrowheads), PCGS (G, arrowhead), LC (H, arrowheads), MPB (I, arrowheads), and XN (J, arrowheads). (F) White line indicates the shape of medial lemniscus (ml). (G) White lines indicate the shape of unc and vsc. (K) Fast Blue-negative LPB. LPB neurons are not retrogradely labeled from lobule IX by Fast Blue. (L) Fast Blue-negative cerebellar nuclei. The cerebellar nuclei (IntA, IntP, Lat, and Med) were not labeled by Fast Blue. The left side in (B,C,E–L) is the medial side of brain section. 3v, third ventricle; 4v, fourth ventricle; Aq, aqueduct; DMSp5, dorsomedial spinal trigeminal nucleus; spt5, spinal trigeminal tract. Scale bar, 500 μm in (A), 200 μm in (B–L).
Figure 11
Figure 11
Beaded fibers from MPB. Axons from MPB are anterogradely labeled by AAV-CMV-hrGFP. (A) Local injection of AAV-CMV-hrGFP to MPB. An arrow indicates the injection site. MPB is densely labeled by hrGFP. (B) The transversal section of cerebellum. The cerebellar lobules VII–X are indicated. (C) hrGFP-positive beaded fiber in lobule IX. The magnified image of the quadrilateral region in (B) is indicated. Arrowheads indicate the hrGFP-positive beaded fiber, which is along the Purkinje cell layer (PL). (D) hrGFP-positive beaded fiber in lobule VIII. The cerebellar section different from (B) is indicated. The arrowheads indicate the hrGFP-positive beaded fibers extending from PL to ML. 4V, fourth ventricle; GL, granular layer; ML, molecular layer. Scale bar, 200 μm.
Figure 12
Figure 12
Putative circuit associated with lobule IX of cerebellar vermis and MPB. Precerebellar nuclei of lobule IX, cerebellar nuclei projected by lobule IX, and brain regions projected by lobule IX are indicated according our results. Purkinje cells in lobule IX project to the ipsilateral IntP and Med. IntP projects to the ipsilateral MPB but Med mainly projects to the contralateral MPB. Lobule IX also directly projects to the ipsilateral MPB and vestibular nuclei. MPB interconnect with many brain regions, including ipsilateral lobule IX of the cerebellar vermis. Efferent projections of MPB (asterisk) refer to previous reports (Saper and Loewy, ; Fulwiler and Saper, ; Bernard et al., ; Feil and Herbert, ; Chamberlin et al., ; Len and Chan, 1999). Periaqueductal gray (PAG) and Paraventricular hypothalamic nucleus (PVN) get inputs from MPB and project to lobule IX. Therefore, a loop circuit is formed among PAG/PVN, lobule IX, and MPB. AI, Agranular insular cortex; BNST, Bed nucleus of the stria terminalis lateral division; CeA, Central amygdaloid nuclei; CL, Claustrum; IL, Infralimbic cortex; IO, inferior olive; LC, locus ceruleus; LH, Lateral hypothalamic area; MdD, dorsal part of medullary reticular nucleus; PCGS, paracochlear glial substance; PLN, Paralemniscal nucleus; Pn, Pontin nucleus; POA, Preoptic area; RPC, Parvocellular part of red nucleus; RVL, rostroventrolateral medulla; SN, Substantia nigra; VTA, Ventral tegmental area; XN, X nucleus; ZI, Zona incerta.
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