Neuronal migration depends on blood flow in the adult mammalian brain
- Department of Developmental and Regenerative Neurobiology, Nagoya City University Graduate School of Medical Sciences, Japan
- Division of Neural Development and Regeneration, National Institute for Physiological Sciences, Japan
- Division of Homeostatic Development, Department of Developmental Physiology, National Institute for Physiological Sciences, Japan
- Laboratory of Comparative Neurobiology, Cavanilles Institute, University of Valencia, Spain
- Department of Cell Biology, Functional Biology and Physical Anthropology, University of Valencia, Spain
- Laboratory of Stem Cell and Neuro-Vascular Biology, Genetics and Developmental Biology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, United States
- Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical Science, Japan
Figures
Figure 1 with 9 supplements
New neurons migrate along blood vessels with abundant flow in the adult brain.
(A) Experimental scheme. (B) Three-dimensional reconstructed images of a new neuron (green) and blood vessels (red) in the rostral migratory stream (RMS) (B) and glomerular layer (GL) (C) of 6- to 12-week-old adult mice. (D) Distance between new neurons and nearest vessels in the olfactory bulb (OB) and RMS (one-way repeated measures ANOVA followed by Bonferroni’s test; three and four mice for the analysis in the OB and RMS, respectively). (E) Transmission electron microscopy image of a new neuron (green) in close contact with a blood vessel (red) in the GL of a 6- to 12-week-old adult mouse. Astrocytes (clear arrowheads). (F) Time-lapse images of a migrating neuron (indicated by asterisks) in the GL of a 6- to 12-week-old Dcx-EGFP mouse. Red blood cell (RBC) flow is recorded as a two-photon line-scan image as shown in the right panel. Stationary cells are indicated by sequential numbers. (G) Average distance between migrating cells and nearest blood vessels (41 cells from 38 mice). (H) Density of perivascular migrating cells (Wilcoxon signed-rank test; 10 mice). (I) Average migration speed (Welch’s t-test; low, 19 cells, high, 25 cells from 39 mice). (J) Percentage of migratory period (Mann–Whitney U-test; low, 19 cells, high, 24 cells from 38 mice). (K) Maximum migration speed (unpaired t-test; low, 22 cells, high, 24 cells from 39 mice). (L) Fluorescent images in the NG2-DsRed mouse GL. Arterioles, capillaries, and venules were characterized by band-like smooth muscle cells (solid arrowhead), pericytes (arrows), and fenestrated smooth muscle cells (clear arrowhead), respectively. CD31 (blue), DsRed (red). (M) Fluorescent image of a Dcx+/BrdU+ new neuron (solid arrowhead) attached to a capillary. Dcx (green), BrdU (blue), CD31 (blue, tube-like structures), DsRed (red). (N) Density of BrdU+/Dcx+ cells in the perivascular region of arteriole- and venule-side capillaries (paired t-test; three mice). (O) Two-photon images of GABAergic neurons (white) and a blood vessel (red) in the VGAT-Venus mouse GL. Circles show positions of added (yellow) and lost (pink) Venus+ cells. Added and lost neurons are indicated by yellow and pink arrows, respectively. RBC flow on Day 21 is shown in the right panel. (P) Density of newly added neurons in the perivascular region (paired t-test; seven mice). Data are presented as the means ± standard error of the mean (SEM). *p < 0.05, **p < 0.01. Scale bars: B, 30 μm; C, 40 μm; E, 1 μm; F, 10 μm; M, 20 μm; N, 20 μm; P, 10 μm. See also Figure 1—figure supplements 1–3.
Figure 1—figure supplement 1
Directional migration of new neurons relative to local blood flow.
New neuron–vessel interactions were categorized into four groups according to the angle between the migration direction and vessel axis (small: 0°–45°, 135°–180°; large: 45°–135°), and whether the new neurons were migrating toward or away from the direction of higher red blood cell (RBC) flow. Percentages represent the proportions of total interactions (n = 39 from 26 mice).
Figure 1—figure supplement 2
New neurons migrate along endomucin-negative vessels.
(A, B) Representative images of vasculature in the glomerular layer (GL) of the olfactory bulb (OB). Red blood cell (RBC) flow was recorded in a live animal (A), followed by immunostaining of endomucin/CD31 in a fixed brain section (B). Identical vessels are indicated by different numbers (endomucin-positive; 2, 6, 7, endomucin-negative; 1, 3, 4, 5). (C) Average RBC flow in endomucin-positive and endomucin-negative vessels (Mann–Whitney U-test; endomucin-negative, 24 vessels, endomucin-positive, 46 vessels). (D) Fluorescent image of new neurons distributed in the vasculature in the GL. BrdU+/Dcx+ cells are shown in the perivascular region of endomucin-negative vessels (white arrowheads), endomucin-positive vessels (clear arrowhead), and distant from vessels (arrow). CD31 (magenta), endomucin (green, tube-like structures), Dcx (green), and BrdU (red). (E-H) Density of BrdU+/Dcx+ cells in the vicinity of endomucin-positive and endomucin-negative vessels in the GL (E), granule cell layer (F), and rostral migratory stream (OB core) (G) (paired t-test; four mice). (H) Density of BrdU+ mature neurons at 28 days post injection (dpi) in the vicinity of endomucin-positive and endomucin-negative vessels in the GL (paired t-test; four mice). Data are presented as the means ± SEM. *p < 0.05, ***p < 0.005. Scale bars: 20 μm (A, B); 20 μm (D).
Figure 1—figure supplement 3
New neurons exhibit a preference for arteriole-side vessels.
(A) Fluorescent image of immunostained tissue sections from the glomerular layer of the olfactory bulb. Dcx (green), BrdU (deep blue), CD31 (red), and SLC16A1 (green, tube-like structures). (B) Density of perivascular new neurons in the vicinity of SLC16A1-positive and SLC16A1-negative vessels (paired t-test; four mice). (C) Schematic illustration of the distribution of new neurons and vessel identification. (D) Fluorescent images of the ventral striatum from a 4-month-old common marmoset. Immature neurons are indicated by solid arrowheads. Dcx (green), SLC16A1 (deep blue), CD31 (red). (E, F) Density of BrdU+/Dcx+ cells in the vicinity of SLC16A1-positive and SLC16A1-negative vessels in the ventral striatum (E) (paired t-test; five animals) and in the neocortex (F). Data are presented as mean ± SEM. *p < 0.05. Scale bars: 20 μm (A); 20 μm (D).
Figure 1—video 1
A three-dimensional image from the rostral migratory stream of a Dcx-EGFP (green) mouse infused with RITC-Dex-GMA (red).
Figure 1—video 2
High-magnification view extracted from Figure 1—video 1.
Arrows indicate EGFP+ cells interacting with blood vessels.
Figure 1—video 3
A three-dimensional image from the rostral migratory stream of a Dcx-EGFP/Flt1-DsRed mouse.
EGFP (green), DsRed (red).
Figure 1—video 4
A three-dimensional, high-magnification image of chain-forming new neurons in the rostral migratory stream.
Dcx-EGFP (green), Flt1-DsRed (red). Arrows indicate EGFP+ cells interacting with blood vessels.
Figure 1—video 5
A three-dimensional image of immature neurons leaving the ventral ventricular–subventricular zone in a 1-month-old common marmoset infused with RITC-Dex-GMA (red).
Dcx (green). Arrows indicate Dcx+ cells interacting with blood vessels.
Figure 1—video 6
A three-dimensional image of an immature neuron from the ventral striatum in a 1-month-old common marmoset infused with RITC-Dex-GMA (red).
Dcx (green). Arrows indicate Dcx+ cells interacting with blood vessels.
Figure 2 with 1 supplement
Blood flow inhibition attenuates neuronal migration.
(A, D) Experimental schemes. (B) Fluorescent images of Venus+ new neurons (green) in the rostral migratory stream and olfactory bulb (OB). (C) Proportion of Venus+ cells in the OB in the Sham and bilateral carotid artery stenosis (BCAS) groups (Mann–Whitney U-test; Sham, six mice, BCAS, five mice). (E) Two-photon images of neuronal migration (arrows) before and after photothrombotic clot formation in a Dcx-EGFP mouse. A new neuron (green), a blood vessel (red). (F) Line-scan images from a blood vessel shown in (E). Comparison of migration speed before and after laser irradiation in the control (G) (paired t-test; six cells from six mice) and photothrombosis groups (H) (paired t-test; four cells from four mice). Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, n.s., not significant. Scale bars: B, 100 μm; E, 10 μm.
Figure 2—figure supplement 1
Bilateral carotid artery stenosis (BCAS) affects cell proliferation and survival in the ventricular–subventricular zone (V-SVZ) and rostral migratory stream (RMS).
(A, B) Fluorescent images of immunostained RMS tissue sections showing Ki67 (green) (A) and cleaved caspase-3 (green) (B). (C, D) Density of Ki67+ cells (C) and cleaved caspase-3+ cells (D) in the RMS and V-SVZ (unpaired t-test; Sham, seven mice, BCAS, seven mice). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01. Scale bars: 10 μm (A, B).
Figure 3 with 2 supplements
Ghrelin is delivered from the bloodstream to the rostral migratory stream (RMS) and olfactory bulb (OB) in the adult brain.
(A) Representative images of the OB of a fluorescent ghrelin-infused mouse (6- to 12-week-old). CD31 (red), fluorescent ghrelin (green). (A′, A′′) High-magnification images of boxed areas in (A). Arrowheads indicate fluorescent signals in parenchymal areas adjacent to the vascular endothelium. (B) Fluorescent images of neuronal migration along blood vessels in the external plexiform layer (EPL) and the RMS. CD31 (red), Dcx (magenta), fluorescent ghrelin (green). (C) Fluorescent images of blood vessels in the glomerular layer (GL). CD31 (white), endomucin (red), fluorescent ghrelin (green). (D) Normalized fluorescence signal intensity in vascular endothelial cells (paired t-test; three mice). (E) Schematic diagram for analyzing signal gradients in extra-vascular areas. (F) Percentage of vessel segments with >1.5-fold increases in Area I relative to Area II (paired t-test; three mice). Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005. Scale bars: A, 50 μm; B, 20 μm (EPL), 10 μm (RMS); C, 20 μm. See also Figure 3—figure supplements 1 and 2.
Figure 3—figure supplement 1
New neurons express Ghsr1a mRNA in the adult brain.
(A) Fluorescent images from the ventricular–subventricular zone (V-SVZ), rostral migratory stream (RMS), and olfactory bulb (OB) showing Dcx (green) and Ghsr1a mRNA (red). Arrowheads indicate Dcx+ cells with Ghsr1a mRNA puncta. (B) Quantification of Ghsr1a mRNA puncta per Dcx+ cell in the OB of the ad libitum (AL) and calorie restriction (CR) mice (AL, 68 cells from 3 mice, CR, 68 cells from 3 mice). Data are presented as mean ± SEM. n.s., not significant.Scale bars: 10 μm.
Figure 3—figure supplement 2
Blood-derived ghrelin enters the rostral migratory stream (RMS) and olfactory bulb (OB).
(A) Representative images of the OB from mice with saline injection (A) and with fluorescent ghrelin injection (B). High-magnification images are shown in (A′) and (B′). It was found that the experimental process did not affect the brightness of sections. CD31 (red), fluorescence (647 nm) (green). (C) Representative images of the OB section showing ghrelin fluorescence (647 nm, green), CD31 (red), and Dcx (magenta), covering the RMS, granule cell layer (GCL), external plexiform layer (EPL), and glomerular layer (GL). Scale bars: 100 μm (A–C).
Figure 4 with 2 supplements
Ghrelin promotes neuronal migration by activation of actin cup formation.
(A) Fluorescent images of Matrigel culture. Dcx (white). (B) Percentage of Dcx+ cells >200 μm distant from the edge of pellets (unpaired t-test; three independent cultures prepared on different days). (C) Time-lapse images of cultured new neurons expressing DsRed (red). The number above each panel indicates minutes after initiation of migration. (D-J) Migration speed (D), percentage of migratory phase (E), migration cycle (F), length/speed of leading process extension (G, H), and stride/speed of somal translocation (I, J) in neuronal migration (one-way ANOVA followed by Turkey–Kramer test; D–F; control/Ghrelin (−), 15 cells, control/Ghrelin (+), 13 cells, KD/Ghrelin (−), 13 cells, KD/Ghrelin (+), 18 cells, I, J; control/Ghrelin (−), 17 events, control/Ghrelin (+), 18 events, KD/Ghrelin (−), 14 events, KD/Ghrelin (+), 23 events). (K) Time-lapse images of actin cup formation (arrowheads) in the cell soma of new neurons. EGFP-UtrCH (green). Condensed dots of F-actin were scattered throughout the elongated cell soma in a control cell with ghrelin application. (L, M) Average duration of actin cups (L) and migration distance during actin cup formation (M) in new neurons (Kruskal–Wallis test followed by the Steel–Dwass test; control/Ghrelin (−), 79 cells, control/Ghrelin (+), 31 cells, KD/Ghrelin (−), 39 cells, KD/Ghrelin (+), 44 cells). Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005. Scale bars: A, 100 μm; C, 5 μm; K, 5 μm.
Figure 4—video 1
Actin cup imaging in control cells.
EGFP-UtrCH (green).
Figure 4—video 2
Actin cup imaging in ghsr1a-knockdown cells.
EGFP-UtrCH (green).
Figure 5 with 4 supplements
Ghrelin signaling promotes neuronal migration in the adult brain.
(A, D) Experimental schemes. Lentivirus injection into the olfactory bulb (OB) core (A) and the ventricular–subventricular zone (V-SVZ) (D) was performed in 6- to 12-week-old adult mice. (B) Fluorescent images of new neurons in the OB in (A). (C) Proportion of labeled cells in the GL at 5 days post injection (dpi) in (A) (paired t-test; three mice). (E, G) Fluorescent images of new neurons in the GL (E) and rostral migratory stream (RMS) (G) for the experiments shown in (D). Control cells (white arrowheads), Ghsr1a-KD cells (clear arrowheads). (F, H) Proportion of labeled cells in the GL (F) and the RMS (H) at 10 dpi. in (D) (paired t-test; four mice). (I) Experimental scheme. (J, K) Proportion of labeled cells in the GL at 8 dpi in the ad libitum (J) and calorie restriction (K) groups (Control, unpaired t-test; AL, four mice, CR, three mice) (KD, unpaired t-test; AL, four mice, CR, three mice). Control (green), Ghsr1a-KD (red). GL (glomerular layer), EPL (external plexiform layer), MCL (mitral cell layer), IPL (internal plexiform layer), GCL (granule cell layer), RMS (rostral migratory stream), AL (ad libitum), CR (calorie restriction). Data are presented as the means ± SEM. *p < 0.05, n.s., not significant.Scale bars: B, 100 μm; E, 40 μm; G, 40 μm.
Figure 5—figure supplement 1
Ghrelin signaling is required for neuronal migration in the adult olfactory bulb (OB).
(A) Experimental scheme. (B) Proportion of labeled cells in the glomerular layer (GL) at 5 days post injection (dpi) (Welch’s t-test; Control, four mice; Ghsr1a-KD, five mice). Data are presented as mean ± SEM. *p < 0.05.
Figure 5—figure supplement 2
Ghsr1a-KD does not affect cell proliferation of new neurons in the rostral migratory stream (RMS).
(A) Fluorescent image of immunostained RMS tissue sections showing Ki67 (red) and labeled cells (green). Ki67+ labeled cells are indicated by clear arrowheads. (B) Quantification of Ki67+ cells as a percentage of the total labeled cells in the RMS at 5 days post injection (dpi) (Control, five mice; Ghsr1a-KD, six mice). Data are presented as mean ± SEM. n.s., not significant. Scale bars: 5 μm (A).
Figure 5—figure supplement 3
Ghsr1a-KD does not affect the influence of bilateral carotid artery stenosis (BCAS) on neuronal migration.
(A) Experimental timeline. (B) Proportion of Ghsr1a-KD cells in the olfactory bulb (OB) at 5 days post injection (dpi) (unpaired t-test; Control, five mice; Ghsr1a-KD, five mice). (C) Normalized density of labeled cells in perivascular regions of the rostral migratory stream (RMS) (paired t-test; Control + Sham, five mice; Control + BCAS, seven mice; Ghsr1a-KD+Sham, five mice; Ghsr1a-KD + BCAS, five mice). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01.
Figure 5—figure supplement 4
Calorie restriction (CR) promotes neuronal maturation in the olfactory bulb (OB).
(A) Experimental scheme. (B) Fluorescent images of new neurons in the OB. BrdU (green), NeuN (red), Dcx (magenta). A NeuN-/Dcx+ cell (white arrow), NeuN+/Dcx+ cells (white arrowheads), NeuN+/Dcx− cells (yellow arrowheads). (C) Proportion of NeuN+/Dcx− cells among total BrdU+ cells in the OB (KD, unpaired t-test; ad libitum [AL], three mice; CR, four mice). IPL (internal plexiform layer), GCL (granule cell layer). Data are presented as mean ± SEM. *p < 0.05. Scale bars: 50 μm (B).
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Neuronal migration depends on blood flow in the adult mammalian brain
eLife 13:RP99502.
https://doi.org/10.7554/eLife.99502.3
