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Motility and Cytoskeletal Organization of Migrating Cerebellar Granule Neurons

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Motility and Cytoskeletal Organization of Migrating Cerebellar Granule Neurons The Journal of Neuroscience, February 1995, f5(2): 981-989 Motility and Cytoskeletal Organization of Migrating Cerebellar Granule Neurons Rodolfo J. Rivas and Mary E. Hatten The Rockefeller University, New York, New York 10021-6399 To characterize CNS neuronal precursor migration along as- The cerebellar granule neuron has provided an experimental troglial fibers, we examined the motility of the migratory lead- model for CNS migration, in part becausethe specializedmove- ing process and cytoskeletal-based mechanisms of loco- ment of granule neurons along glial fibers can be reproduced in motion of early postnatal mouse cerebellar granule neurons an in vitro system(Edmondson and Hatten, 1987;Hatten, 1990). in vitro. To visualize the surface motility of the leading pro- During migration, the granule neuron is highly polarized in the cess, granule neurons were labeled with the fluorescent li- direction of cell movement. The rostra1portion of the elongated pophilic dye, PKH-26, and imaged by time lapse fluores- cell body tapers in the direction of migration into a thickened cence microscopy. The motile behavior and cytoskeletal migratory leading processthat extends along and wraps around organization of the migrating neuron had several distinctive the glial fiber (Rakic, 1971; Edmondsonand Hatten, 1987). The features. As the migrating neuron moved along the glial fiber, saltatory movement of the neuronal cell soma, moving and the leading process rapidly extended, projecting up to 40 pausing in regular intervals (Edmondson and Hatten, 1987) pm, and retracted, withdrawing towards the cell soma. Broad resemblesthe cyclic advance of migrating fibroblasts (Aber- lamellipodia were common along the entire length of the crombie et al., 1970; Abercrombie, 1980). This view is sup- leading process, giving it a ruffled appearance. Within the ported by the findings of Dunn and Heath (Dunn and Heath, cell soma, a cage-like distribution of microtubules encircled 1976) who showed that while fibroblast locomotion is random the nucleus and actin filaments formed a subcortical rim on flat substrates, movement is oriented when the cells move underneath the plasma membrane. Disruption of actin fila- on a thin-cylindrical substrate. In this model, the growing tip ments with cytochalasin B inhibited migration, suggesting of the leading processwould advance along the thin glial fiber involvement of actin subunit assembly in neuronal migration. by rapid extension and retraction of the leading process,anal- Both microtubules and actin filaments were heavily concen- ogous to the rapid advance and retreat of the fibroblast leading trated in the leading process; the leading process did not lamella, with a net movement in the direction of migration. show the development of a distinct actin-rich domain at its Two lines of evidence suggesta role for the cytoskeleton in tip. neuronal motility along astroglial fibers. First, by correlated [Key words: neuronalmigration, cytoskeleton, cellpolarity, video and electron microscopy, a system of longitudinally ori- cerebellum] ented microtubules extends from a juxtanuclear basalbody into the leading process(Gregory et al., 1988). The microtubule sys- In the developing mammalian brain, neuronal migration estab- tem has been proposed to underlie an oriented flow of mem- lishes the laminar pattern of cortical regions, ushering young branous elements and vesicles from the soma into the leading neurons from ventricular zones where they are generatedto the process,generating a net forward flow of the neuronal cytoplasm layers where they establish synaptic relationships (Ramon y (Hatten, 1993). Second, cytoskeletal elements are thought to Cajal, 1911; Sidman and Rakic, 1973). Although considerable underlie the two classesof neuron-glia appositions seenduring progresshas been made towards identifying the primary path- migration-a specializedinterstitial junction along the interface ways for neuronal migration (Rakic, 1990; Gray and Sanes, of the cell soma and glial fiber, and punctae adherentia along 1991; O’Rourke et al., 1992; Hatten, 1993) and the adhesion the length of the leading process.Along the interstitial junction, systemsthat function in directed migration along the radial glial the intracellular space contains thin fibers that are contiguous fiber system (Edmondson et al., 1988; Sanes,1989; Fishell and with, or are transmembranousextensions of, submembranous Hatten, 1991; Fishman and Hatten, 1993) the cytoskeletal elements that appear to attach to microtubules (Gregory et al., structures underlying the specialized motility of migrating neu- 1988). Moreover, antibody perturbation studies provide evi- rons during CNS development remain uncharacterized. dence that antibodies against the neuron-glia ligand astrotactin significantly reduce the rate of neuronal migration, induce dis- organization of cytoskeletal components, and causewithdrawal of the leading process(Fishell and Hatten, 1991). In particular, Received Mar. 3, 1994; revised June 30, 1994; accepted July 28, 1994. treatment of granule neurons with anti-astrotactin antibodies We gratefully acknowledge the advice of Drs. Gord Fishell and Renata Fishman results in tangles of cytoskeletal elementsin the area rostra1to and thank Dr. Fishell and Dr. Sharon Powell for readina the manuscript. Peter Peirce provided expert assistance with photomicrographs. This work was sup- the nucleus(Fishell and Hatten, 1991). This result has suggested ported by NIH Postdoctoral Fellowship NS08834 (R.J.R.) and NIH Grant NS that the neuronal cytoskeleton interacts with neuron-glia re- 15429 (M.E.H.). ceptor systemsto produce movement of the cell soma. Correspondence should be addressed to Mary E. Hatten, The Rockefeller Uni- versity, 1230 York Avenue, New York, New York 10021-6399. To provide a detailed view of the motile behavior of the Copyright 0 1995 Society for Neuroscience 0270-6474/95/150981-09$05.00/O leading process,we labeled granule neurons, purified from early 982 Rivas and Hatten * Organization of Migrating Cerebellar Granule Neurons postnatal mouse cerebellum, with the fluorescent lipophilic dye, 568 nm illumination from a krypton-argon multiline laser. A Nomarski PIU-I-26 (Horan and Slezak, 1989; Gao et al., 1992). The labeled image was obtained by transmitting the laser light through an optic fiber cable. Alternatively, PKH-labeled cells were imaged using a Hamamatsu neurons were plated on unlabeled glial fibers and migration was 2400-50/80 I-CCD camera head and controller. Epifluorescent excita- visualized by time lapse fluorescence microscopy. This allowed tion/emission was at 510/550 nm. Three neutral density filters were us to view the motility of the leading process separately from added to reduce the excitation light level by 99%. For time lapse re- the surface activity of the underlying glial fiber. During migra- cordingsusing the I-CCD, oneimage (100 msec exposure) was acquired every 3-5 min. tion the leading process rapidly extended, projecting up to 40 Disruption of cytoskeletal structures of migrating neurons with cyto- pm, and retracted, withdrawing towards the rostra1 portion of chalasin B or nocodazole. To infuse agents that disrupt cytoskeletal the cell soma. Lamellipodial and filopodial extension occurred elements, the cover of the microculture system was modified. Rather along the entire length of the leading process. Treatment of than sealing the microwell with a second #l coverslip, the petri dish migrating neurons with cytochalasin B, a drug that disrupts actin was filled with glial-conditioned medium (14 ml). The culture was sealed with a petri dish cover fitted with a “window” (obtained by drilling a filaments (Forscher and Smith, 1988; Letoumeau et al., 1987), hole (14 mm) in the top and sealing with a # 1 coverslip). Additions of rapidly arrested neuronal migration, suggesting that actin sub- concentrated stocks of nocodazole or cytochalasin were made through unit assembly functions in neuronal locomotion along the glial a second hole (6 mm) in the plastic petri dish cover which was sealed substrate. In contrast to the growth cone, where actin filaments with Parafilm before and after drug additions. For drug addition ex- periments, Nomarski images were acquired with a Zeiss Axiovert mi- and microtubules are separated into distinct peripheral and cen- croscope fitted with a long working distance condenser. tral domains, the leading process did not show the development Immunocytochemical procedures. Tubulin and actin were localized of a distinct actin-rich domain at its tip. by a procedure modified from Wang et al. (1982). In brief, cells were washed once with calcium-free Hanks’ balanced salt solution and once Materials and Methods with cytoskeleton buffer (CB; Hanks’ BSS plus 2 mM MgCl,, 2 mM Purijication of cerebellar granule neurons and astroglial cells. Cell sus- EGTA, 5 mM PIPES, pH 6.9). The cells were then treated for 1 min pensionsenriched in cerebellargranule neurons (95%) and astroglia (5%) with CB plus 0.25% (w/v) glutaraldehyde, 0.5% (w/v) Triton X-100, were purified by Percollgradient sedimentation from C57B1/6J mice rinsed in CB, and then postfixed (1% glutaraldehyde in CB, 10 min). on postnataldays 5 or 6 (Hatten, 1985).Cells were plated in serum- After further permeabilizationin 0.5% Triton X-100 in CB (10 min), supplementedmedium (BME plus 10%horse serum, 9 mg/mlglucose, aldehyde autofluorescence was quenched with 1 mg/ml NaBH, in cy- 0.3 mg/ml glutamine,50 U/ml penicillin,and 50 j&ml streptomycin) toskeleton buffer (three
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