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Cytology and Neuron-Glial Apposition of Migrating Cerebellar Granule Cells in Vitro

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Cytology and Neuron-Glial Apposition of Migrating Cerebellar Granule Cells in Vitro The Journal of Neuroscience, May 1988, 8(5): 1728-l 738 Cytology and Neuron-Glial Apposition of Migrating Cerebellar Granule Cells in vitro William A. Gregory, James C. Edmondson, Mary E. Hatten, and Carol A. Mason Department of Pharmacology, New York University Medical Center, New York, New York 10016 In developing mammalian brain, many neurons migrate to their cerebellum provides a paradigm for glial-guided neuronal mi- final position by moving in direct apposition to radially ori- gration, that of the granule neurons along Bergmann glia. Cer- ented glial cells. Glial-guided migration can be visualized in ebellar granule cells undergo their final cell division in the ex- microcultures of mouse cerebellar cells by the combined use ternal granule layer, after which they become bipolar and of cellular antigen markers and high resolution time-lapse elaborate processes that become the parallel fibers of the mo- video microscopy (Hatten et al., 1984; Edmondson and Hat- lecular layer (Ramon y Cajal, 19 11). The granule cell soma then ten, 1987). Such studies have demonstrated the behavior of translocates perpendicular to the parallel fibers, migrating in- migrating cells and revealed a motile leading process on the ward through the developing molecular layer in association with migrating neuron that resembles an axonal growth cone and Bergmann glia (Rakic, 197 1). This migration is disrupted in the grows along extended glial fibers. To study the fine structural neurological mutant weaver mouse (Rakic and Sidman, 1973; details of the migrating neuron and its neuron-glial apposi- Sotelo and Changeaux, 1974), where neuron-glial interactions tion, we identified and monitored neurons in microcultures appear to be defective (Hatten et al., 1986). with video microscopy and examined the cytology and cel- The cellular and molecular mechanisms of neuron-glial in- lular contacts of the same cells with transmission electron teraction and migration can be studied with microculture sys- microscopy. tems (Trenkner and Sidman, 1977; Hatten and Liem, 1981; The cytology of the soma and leading process of migrating Hatten et al., 1984, 1986; Trenkner et al., 1984; Hatten, 1985; cells closely matches that described for granule cells in intact Mason et al., 1988). Recently, a detailed view of migration of brain (Rakic, 1971). Newly observed structures include the cerebellar granule neurons along elongated astroglia has been presence of longitudinally oriented microtubules extending observed in culture in real time by the use of high-resolution from a basal body in the soma into the leading process, and video-enhanced differential interference microscopy (Edmond- microfilament-rich filopodia arising from the soma and lead- son and Hatten, 1987). ing process. To learn how cerebellar granule neurons move and what cel- The most striking feature of actively migrating neurons is lular structures are used during motility, we observed migrating a specialized junction between the neuronal cell soma and granule neurons in vitro with video microscopy, then examined apposing glial fibers. At this junction, here termed “inter- the cytology and appositions of the same cells with electron stitial density,” the extracellular space is dilated to 20 nm microscopy. As a comparison, we also studied cells that did not and filamentous material in the intracellular cleft either spans have a recent history of migration. A preliminary report of this the cleft or runs parallel to the cell membranes. Some in- work has been made (Gregory et al., 1986). terstitial fibrils are contiguous with, or are transmembranous extensions of, submembranous cytoskeletal elements that Materials and Methods attach to microtubules. Interstitial junctions were not found Cell cultures and video. Microculture dishes were prepared by punching between neurons that did not translocate in the observation an I-mm-diameter hole in the center of culture dishes (Falcon 1006), and affixing an 1 I-mm-diameter No. 1 coverslip to the bottom of the period before fixation. Instead, stationary cells formed des- dish with a 3: 1 mixture of paraffin:Vaseline (Hatten and Liem, 198 1). mosomes (puncfa and macula adhaerentia) at appositions Monolayer microcultures of postnatal mouse cerebellum were prepared with glial processes. as described elsewhere (Hatten, 1985; Edmondson and Hatten, i987). Briefly.__- Durified cultures of astrodia derived from P3-4 C57BW6J mice were grown for 24 hr on a polyly&e-treated (100 &ml) glasscoverslip. The migration of neurons along glial fibers is fundamental to Purified granule cells were then added, and after 1 additional day, cul- mammalian brain development (Rakic, 197 1, 1972, 1985; Ra- tures were observed. Cultures were maintained in a humidified incu- kit et al., 1974). Among cortical regions in the young brain, the bator (Napco) at 35.X in 5% CO,/95% air. For video-microscopic analysis, a glass coverslip was placed directly over the surface of the microculture and was sealed in place with silicon vacuum grease (Dow Coming). The microscope stage,objective, and condenser were enclosed Received Apr. 27, 1987; revised July 29, 1987; accepted Aug. 25, 1987. in a heating chamber that was thermostatically controlled at 37°C. Cul- This work was supported by Grants NS- 15429 and NS-2 1451. We thank our tures were examined using a Zeiss IM inverted microscope equipped colleagues, Drs. Ekkhart Trenkner, Ron K. H. Liem, Michael V. L. Bennett, and with differential interference optics (Edmondson and Hatten, 1987). The David C. Spray for many helpful suggestions, and Mr. Jose Sanchez for technical assistance. Mr. Peter Peirce provided photographic assistance and Ms. Julia Cohen microscope was equipped with an analog contrast-enhancing video cam- provided assistance with word processing. era (Hamamatsu C1965-01). The video signal was passed through a Correspondence should be addressed to Mary E. Hatten or Carol A. Mason at time-date generator (MS1 Video Systems), and was recorded on a time- their present address: Department of Pathology, College of Physicians and Sur- lapse optical disc recorder (Panasonic TQ 2025fl, which sampled 1 geons, Columbia University, 630 West 168th St., New York, NY 10032. frame/4 set, and was viewed on a high-resolution video monitor (Sony Copyright 0 1988 Society for Neuroscience 0270-6474/88/051728-l 1$02.00/O PVM- 122). Tha Journal of Neu-. May 1988,8(5) l?thB Figure 1. Electron micrographs of a migrating neuron that was fixed after observation with video microscopy. A, B, An elongated neuron (N) is observed with video. In B, 30 min after A, the neuronal soma is positioned to the right, indicating that migration toward a cluster of cells (arrows) has recently occurred. C, A low-magnification electron micrograph of the migrating neuron (NJ shows correspondence with the video representation in B. Arrows indicate cells in the cluster shown in video (arrows, A, B). The leading process (lp) is shown at higher magnification in D. D, The leading process (Zp) is a direct extension of the soma. The nucleus is present at far lef with adjoining Golgi (G). The leading process contains primarily longitudinally oriented microtubules (m), rosettes of ribosomes and mitochondria. The soma of this cell is shown in Figure 2. 1730 Gregory et al. - Granule Neurons Migrating on Astrcglia Figure 2. Electron micrographs of a neuron migrating along a glial process. The culture was fixed after observation of these cells with video microscopy. These are different sections through the same neuron shown in Figure 1. A, The rostra1 end of the soma contains Golgi (G), basal body (bb), coated vesicles (cv), and ribosomes. Microtubules (m) course around the nucleus and extend into the leading process (Zp). B, A different section through the same migrating neuron (N) shows the characteristic caudal placement of the nucleus during migration, with a nuclear indentation on the forward surface (arrow). A region of pronounced interstitial density (id) is formed between the neuron and adjoining glial process (GI) in the indicated rectangular region shown at higher magnification in C. C, Fibrillar interstitial material is evident between membranes of a migrating neuron (NJ and glial process (Gr). Fibrils span membranes (smaZler solid arrows). Interstitial material is also located parallel to membranes (between The Journal of Neuroscience, May 1988, 8(5) 1731 Cultures were scanned for cells having the elongated (bipolar) shape nation, and from several cultures that were fixed without video obser- typical of migrating neurons (Hatten et al., 1984; Trenkner et al., 1984; vation. Edmondson and Hatten, 1987). A video record was made for 30-60 The details of structure and the quality of fixation showed no con- min of selected cells, using a 63 x planapochromat oil-immersion ob- sistent variation between the cells that were subjected to video micros- jective projected through a 20 x eyepiece onto the video camera. Ac- copy and those that were not. Preservation was good even for cultures tively migrating neurons were defined as cells whose somata translocated subjected to video observation for l-2 hr prior to the selection of a cell at least one somatal length (approximately 10 pm) during the obser- for video-electron-microscopic analysis. No changes in cellular behav- vation period. Cultures containing migrating neurons were then pro- ior were observed with video after several hours of observation (un- cessed for electron microscopy. After video observation, the region was published observations and Edmondson and Hatten, 1987). Thus, the scribed with a false objective, and a 10 x objective was used to record migration of neurons in this tissue culture system appears to be robust a low-magnification video record of the field showing the observed cell(s) with respect to possible short-term effects of light-microscopic video and surrounding landmarks. In some cases, cells that failed to migrate examination on the structure, health of cells, or fixation. in the observation period were also fixed in this manner.
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