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Rapid Sprouting of Filopodia in Nerve Terminals of Chromaffin Cells, PC1 2 Cells, and Dorsal Root Neurons Induced by Electrical Stimulation

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Rapid Sprouting of Filopodia in Nerve Terminals of Chromaffin Cells, PC1 2 Cells, and Dorsal Root Neurons Induced by Electrical Stimulation The Journal of Neuroscience, October 1994, 14(10): 59175928 Rapid Sprouting of Filopodia in Nerve Terminals of Chromaffin Cells, PC1 2 Cells, and Dorsal Root Neurons Induced by Electrical Stimulation Shanthi Manivannan and Susumu Terakawa Department of Cell Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki 444, Japan Rapid morphological changes induced by direct electrical Electrical activity plays a prominent role in communications stimulation of nerve terminals were studied by using video- within the nervous system. It has been suggestedthat electrical enhanced differential interference contrast microscopy at a activity might influence the morphological characteristics and very high magnification (12,000 x ). We used mainly cultured functional connectivity in the nervous system during develop- bovine chromaffin cells, which developed neurite-like pro- ment and in neuroplastic events. In fact, electrical activity in- cesses, and PC 12 cells, which showed neuronal differenti- fluences various neuronal properties like synthesis of neuro- ation upon NGF treatment. In a few cases, primary neurons transmitters (Walicke et al., 1977; Ip and Zigmond, 1984) of the rat dorsal root ganglion were also examined. Brief expressionof neurotransmitter receptors (Lomo and Rosenthal, pulse stimulation of the terminals and varicosities induced 1972; Zigmond and Bowers, 198I), the rate and direction of exocytosis accompanied by rapid formation of filopodia. neurite outgrowth (Borgens et al., 1981; Pate1and Poo, 1982; These filopodia, 0.1-0.2 Am in diameter and up to 10 pm in Cohan and Kater, 1986) formation and pattern of synaptic length, formed within a few hundreds of milliseconds and connections(Changeux and Danchin, 1976; Archer et al., 1982) then retracted within tens of seconds. They could also be and sprouting at the neuromuscularjunction (Brown and Iron- induced by K depolarization. This rapid filopodial sprouting ton, 1977; Brown et al., 1981). Receptor-mediated changesin strongly depended on the presence of extracellular Ca2+ and the morphology and the activity of the neuronal growth cone could be abolished in a medium containing a Ca chelator have been shown to be induced by serotonin (Haydon et al., (EGTA) or La3+. Anti-cytoskeletal agents colchicine and cy- 1984) and nerve growth factor (NGF) (Connolly et al., 1987). tochalasin B failed to block this response completely but Glutamate, an excitatory neurotransmitter, induces filopodia lidocaine fully suppressed it. Quantitative analysis of exo- formation in the growth cone (Cornell-Bell et al., 1990). Ap- cytosis and filopodial sprouting showed that they were in- plication of an electrical field or a K-rich solution increasesthe dependent events, not directly linked to each other, having number, facilitates elongation, and induces disposition of filo- different thresholds usually higher for filopodial formation. podia (Davenport and Kater, 1992; Rehder and Kater, 1992). In PC12 cells, the extent of filopodial sprouting varied with Thus, motility and structural changesof the growth cone have the state of differentiation of the cells, suggesting a func- been extensively studied. However, very little is known about tional role of rapid sprouting during a particular phase of the immediate or rapid morphological changesof the growth their differentiation. Filopodia could be induced with greater cone resulting from electrical activities as brief as the action ease by repetitive stimulation. The same responses may potential. Since brief action potentials are usual under physio- occur at growth cones approaching the target cells or even logical conditions in vivo, effects of such electrical activities, if at mature synapses particularly after repetitive electrical ac- any, on growth cones would be important for their steering tivity, possibly playing a role in use-dependent synapse for- toward targets or for their transformation into synaptic termi- mation or plasticity. nals. [Key words: chromaffin ce//, sprouting, growth cone, filo- In this study, we employed bovine chromaffin cells and a podium, exocytosis, lidocaine] pheochromocytoma cell line PC12 cultured in a low density as a model neuron, and observed the terminal of neurites under a video-enhanced differential interference contrast microscopeat a very high magnification (12,000 x). An electrical pulse of 1 Received June 7, 1993; revised Mar. 15, 1994; accepted Mar. 24, 1994. msec duration was used to stimulate the terminal to mimic or We thank Dr. K. Kumakura for providing dissociated chromaffin cells, Dr. K. actually induce such an action potential as those normally gen- Yamaguchi for providing dorsal root ganglion neurons, Ms. R. Sakurai and Ms. A. Miwa for maintaining the chromaffin cells in culture, Mr. M. Ohara for elec- erated under physiological conditions. We found a rapid sprout- tronical assistance, and Messrs. A. Ito, F. Mizutani, and M. Mori for programming ing of filopodia in nerve terminals immediately after this brief the digital photographic printer. S.M. was supported by a fellowship from Uehara electrical stimulation. In order to gain some insights into the Memorial Life Science Foundation. This work was partly supported by a grant- in-aid (0 1480 118) from The Ministry of Education, Science and Culture, Japan activities of filopodia, their rapid sprouting was studied with to ST. special emphaseson its ionic and chemical sensitivities and on Correspondence should be addressed to Susumu Terakawa, Photon Medical Research Center, Hamamatsu University School of Medicine, 3600 Handa, Ha- its relationship with exocytosis occurring at the sameterminal. mamatsu, 43 l-3 1, Japan. Here, we describethe basic properties of this unusual response, Copyright 0 1994 Society for Neuroscience 0270-6474/94/145917-12$05.00/O and present evidence for its biological significance. 5918 Manivannan and Terakawa * Rapid Filopodial Sprouting in Neurite Terminals Figure 1. Exocytosis of a granule and sprouting of filopodia in a terminal of a chromaffin cell neurite. A, Low-magnification Nomarski image of differentiated chromaffin cells in culture. Several cells (C) with nuclei and a neurite (N) with a swollen terminal (7) are shown. B, Series of high- magnification images of the terminal in A taken 1 set after electrical stimulation (1.40 PA, 1 msec). A granule (arrow, left) abruptly changed in brightness and in shape, giving an impression of popping (arrow,right; taken at a 66 msec interval). More than 50 of such responses continued to appear in 5 sec. C, Images of the same terminal immediately before (left) and 8 set after (right) stimulation with a current pulse of a slightly larger amplitude (1.54 PA). Several filopodia sprouted rapidly (arrows).Asterisk, micropipette. The images were recorded as described in Materials and Methods. Scale bars in C: A, 5 pm; B, 1 pm; C, 2 pm. Preliminary results were reported in the 3rd IBRO World Materials and Methods Congressof Neuroscience, Montreal, 1991, and in the 17th Ceil culture.Chromaffin cells were isolated from bovine adrenal me- Seiriken Conference,Okazaki, 1992 (seeManivannan and Ter- dullae by the collagenase digestion method (Waymire et al., 1983). The akawa, 1993). cells were differentially plated and cultured on collagen-coated cover- The Journal of Neuroscience, October 1994, 14(10) 5919 Figure 2. A sequence of filopodial sprouting and retraction in a terminal of a chromaffin cell neurite. Images were taken before (A) and at various times after (indicated in seconds, B-F) after electrical stimulation. Several filopodia started to sprout in a fraction of second (arrows in B) and fluctuated off focus (arrows in C and D). Conspicuous raster lines (arrows in E) are due to the rapid movement of filopodia during the interlace scan. Asterisk, micropipette. Scale bar, 2 pm. slips in Dulbecco’s modified Eagle’s medium (DMEM) supplemented was maintained at 32-36°C. The optical image was detected with a 0.5 with 101 fetal calf serum (FCS). PC12 cells (subclone PC12h having inch CCD video camera (TL23P, NEC, Tokyo), and the contrast of the an NGF-sensitive tyrosine hydroxylase activity; Hatanaka, 198 1) were image was enhanced with a high-speed digital image processor (PIP- also cultured on collagen-coated coverslips in DMEM supplemented 4000, ADS, Osaka). The processed image was observed on a slightly with 5% FCS. The latter cells were further treated with nerve growth overscanned video monitor (14 inches, B/W, Hitachi, Tokyo), and si- factor (NGF; 50 @ml; Sigma, St. Louis, MO) from the second day of multaneously videotaped with an S-VHS format recorder (AG-7750, culture onward and were studied after various periods of NGF treat- Panasonic, Osaka). The ratio of the effective scanning width of the ment. Primary neurons were dissociated from the dorsal root ganglion monitor to that of the CCD detector reached 48, and thus increased the of adult rats by the collagenase digestion method. They were cultured total magnification to 12,000 x . Pictures were reproduced by digitizing on a polylysine-coated coverslip in a serum-free Nl medium for 3 d necessary video frames and printing with a digital photographic printer (for details, see Yamaguchi, 1990). (Pictrography 2000, Fujix, Tokyo). Video microscopy. The cells were examined under an inverted No- Stimulation. Neurite terminals and varicosities were stimulated di- marski microscope equipped with a 100 x DIC objective lens and a rectly with an electrical pulse from a glass micropipette placed extra- 2.5 x insertion lens (Axiovert 35, Zeiss). The coverslip plated with the cellularly. The micropipette was 0.5-1.5 pm in diameter, filled with the cultured cells was fixed with petroleum jelly (Vaseline) to a square hole normal solution (see below) and connected to a piece of Ag-AgCl wire. made in the center of a plastic slide. The slide was placed on the mi- It was held by a water-driven micromanipulator (WR-3, Narishige, croscope stage warmed with an electric heater. Using a peristaltic pump Tokyo). The tip of the micropipette was pressed gently to the surface (SJ- 1220, Atto, Tokyo), the cells were perfused continuously with warmed of the terminal or varicosity.
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