BDNF stimulates migration of cerebellar granule cells
Paul R. Borghesani1,3,*,
Jean Michel Peyrin2,*,
Robyn Klein4,*,
Joshua Rubin1,
Alexandre R. Carter1,3,
Phillip M. Schwartz1,
Andrew Luster4,
Gabriel Corfas2,
and
Rosalind A. Segal1,3,5,
1 Department of Pediatric Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA
2 Division of Neuroscience, The Children’s Hospital, Boston, MA 02115, USA
3 Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA
4 Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Charlestown, MA 02129, USA
* The first three authors contributed equally
The last two authors contributed equally
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Fig. 1. Granule cell migration out of the EGL is delayed in Bdnf–/– mice. (A) Dividing granule cells in P11 wild-type and Bdnf–/– littermates were labeled by systemic injection of 50 mg/kg BrdU. Cerebellar tissue was then collected and processed for BrdU immunohistochemistry at the indicated times post-injection. Adjacent sections were stained with Cresyl Violet. EGL, external germinal layer; ML, molecular layer; PC, Purkinje cell layer; IGL, internal granule cell layer. Scale bar, 50 µm. (B) At 42 hours post-BrdU labeling, there is a significant reduction in the number of granule cells in the molecular layer and IGL in Bdnf–/– mice. *P<0.005 by two-tailed t-test. (C) At 96 hours post-BrdU labeling, fewer granule cells have migrated into the IGL while more BrdU-positive cells remain in the EGL and molecular layer of Bdnf–/– mice. The total number of labeled granule cells did not differ between genotypes. *P<0.005 by two-tailed t-test.
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Fig. 2. Granule cells differentiate appropriately in Bdnf–/– mice. Mid-sagittal cerebellar sections from P12 wild-type and Bdnf–/– mice were processed for in situ hybridization using an antisense probe for the early granule cell marker, ATOH1(Math1), or the marker of later differentiation, NEUROD1. Parallel sections were processed for immunohistochemistry using antibodies to ZIC or SNAP25 (double labeled with DAPI, blue). Granule cells in the EGL and IGL of Bdnf–/– mice show appropriate patterns of expression of all markers.
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Fig. 3. Purified Bdnf–/– granule cells have a decreased migratory index. Freshly isolated wild-type and Bdnf–/– granule cells were plated on previously established wild-type and Bdnf–/– glial cultures, respectively. After 1 day in vitro, attachment and migration were analyzed by time-lapse video microscopy. One frame every 3 minutes was recorded for 90 minutes. (A) The same proportion of wild-type (230 of 1508) and Bdnf–/– (205 of 1280) granule cells attached to glial processes. (B) The proportion of attached Bdnf–/– (36 of 205) granule cells that migrated along glial fibers (the migratory index) was significantly lower than that for wild-type (96 of 230) granule cells. *P<0.0001 by  2 analysis. (C) Once moving, the migratory rate was the same in wild-type and Bdnf–/– cells. White bars, wild-type animals; black bars, Bdnf–/– littermates.
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Fig. 4. BDNF induces granule cells to initiate migration. (A) The same proportion of wild-type (124 of 1163 control; 144 of 1720 with added BDNF) and Bdnf–/– (103 of 1195 control; 69 of 823 with added BDNF) attached in the absence or presence of exogenous BDNF. (B) Exogenous BDNF increases the migratory index in cultures of both wild-type (45 of 124 control; 75 of 144 with added BDNF) and Bdnf–/– (25 of 103 control; 36 of 69 with added BDNF) cells, restoring migration of mutant cells to wild-type levels. **P<0.01 and ***P<0.0002 by 2 analysis. (C) BDNF does not affect the speed of granule cell migration. White bars, wild-type animals; black bars, Bdnf–/– littermates.
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Fig. 5. BDNF is acutely motogenic for granule cells. Freshly isolated Bdnf–/– granule cells were plated on previously established Bdnf–/– glial cultures. After 1 day in vitro without added BDNF, attachment and migration were analyzed by time-lapse video microscopy for 90 minutes (pre addition). BDNF (10 ng/ml) or vehicle control was then added to the medium and migration of the same cells was followed for a second 90 minute period (post addition). (A) An individual Bdnf–/– granule cell was followed over time. The granule cell (arrow) remains stationary until BDNF is added then rapidly began to migrate along the glial cell. Arrow indicates the migrating granule cell, arrowhead indicates a fixed position within the images. (B) Cumulative data show that BDNF, but not vehicle control, increased the migratory index of granule cells and thus is acutely motogenic. We monitored the acute response of 212 cells to BDNF and 100 cells to vehicle control. **P<0.0006 by 2 analysis.
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Fig. 6. Purified Bdnf–/– granule cells have defects in motility that are rescued by BDNF. Purified wild-type and Bdnf–/– granule cells were grown for 24 hours in Boyden chambers without (control) or with (+BDNF) 30 ng/ml BDNF in both chambers. Granule cells that migrated through the porous membrane into the lower chamber were directly quantified by light microscopy. Purified Bdnf–/– granule cells exhibited reduced migration in the control medium that could be rescued to wild-type levels by the addition of BDNF. *P<0.05 (Student’s t-test) for wild-type versus Bdnf–/– cells in control conditions. White bars, wild-type animals; black bars, Bdnf–/– animals. Pooled results from three separate experiments are shown.
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Fig. 7. Graded expression of BDNF may stimulate directed migration of granule cells. (A) Wild-type and Bdnf–/– cerebellar sections from P7 mice were immunostained with an antibody to BDNF. BDNF protein is present in a non-uniform distribution in the cerebellar cortex, with higher levels in the molecular layer and the IGL than in the EGL. (B) Purified wild-type granule cells were grown for 24 hours in Boyden chambers without BDNF (-), with 30 ng/ml BDNF added to both compartments (both) or with 30 ng/ml BDNF added only to the lower compartment (lower). As shown, a gradient of BDNF increased directed movement of wild-type granule cells through the porous membrane as compared to the movement with BDNF in both compartments, or to no BDNF. *P<0.05 (Student’s t-test) for control versus +BDNF, and for BDNF in both compartments versus BDNF in lower compartment only. Pooled results from three separate experiments are shown.
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© The Company of Biologists Ltd 2002
