Visualization of calcium channels involved in transmitter release from neuronal growth cones

Neuroscience Letters 251 (1998) 93–96

Visualization of calcium channels involved in transmitter release from neuronal growth cones Hiromitsu Soeda a , b ,*, Hitoshi Tatsumi b,1,2, Yukishige Kozawa a, Hiroyuki Mishima a, Yoshifumi Katayama b a

Department of Anatomy, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakaecho-Nishi, Matsudo, Chiba 270, Japan b Department of Autonomic Physiology, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101, Japan Received 14 April 1998; received in revised form 13 June 1998; accepted 13 June 1998

Abstract Immunocytological localization of q-agatoxin IVA (q-aga IVA)-sensitive Ca2 + channels involved in glutamate release from growth cones of cultured rat dorsal root ganglion (DRG) neurons was studied with field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM). The q-aga IVA-sensitive Ca2 + channels were visualized by labeling with immuno-gold particles (30 nm). FE-SEM and TEM images showed that immuno-gold particles were present in the area of growth cones as well as somata, and generally absent on neurite stem and fibroblasts. TEM images of vertical ultra-thin sections showed that the immuno-gold particles were present on the surface of the plasma membrane. Since the gold particles indicate the immunological presence of q-aga IVA-sensitive Ca2 + channels, the Ca2 + channels involved in transmitter release are present on growth cones before making synapse formation.  1998 Elsevier Science Ireland Ltd. All rights reserved

Keywords: Ca2 + channel; Growth cone; Neurotransmitter release; Field emission scanning electron microscopy; Immunocytochemistry; q-Agatoxin IVA

Our previous study [15] demonstrated that growth cones of cultured dorsal root ganglion (DRG) neurons release glutamate even before forming synapses, and that release is almost completely inhibited by a P- and/or Q-type Ca2+ channel blocker, q-agatoxin IVA (q-aga IVA) [8,10–12]. This indicates that q-aga IVA-sensitive Ca2+ channels involved in glutamate release are present in the growth cones. A recent advance of electron microscopic techniques [6,12] has provided a new approach for visualizing virusproteins on the plasma membrane surface. In the present study, localization of the q-aga IVA-sensitive Ca2+ channels on the growth cones of cultured DRG neurons was immu* Corresponding author. Tel.: +81 3 52808078; fax: +81 3 52808077. 1 PRESTO Japan Research Development Corporation. ‘The Intelligence and its Origin’. 2 Present address: Department of Physiology, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan.

nocytologically demonstrated with this new approach, using field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM). This is the first immunological visualization of Ca2+ channels on the growth cones using the FE-SEM. Wistar rats (0–3 days after birth, Saitama Experimental Animals Supply, Japan) were anaesthetized with Nembutal. Dorsal root ganglia were isolated and divided into two pieces with a razor blade under a dissecting microscope. The dissected tissue was treated with papain (20.3 units/ ml; Worthington, USA) in low-Ca2+ and low-Mg2+ Krebs solution (see below) for 20 min at 37°C, washed with modified Krebs solution (see below) and triturated using pipettes. DRG neurons were plated in these ways: (1) on glass coverslips for FE-SEM observation, (2) on membrane-coated (Pioloform, Agar, UK) single hole carbon grids for whole-mount preparations [17] observed with TEM, and (3) on plastic membrane (Saran Wrap, Japan) for TEM observation of ultra thin sections. All of these

0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00525- 4

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Fig. 1. FE-SEM images of a growth cone of a cultured DRG neuron immunocytologically labeled with 30 nm gold particles. (a) A secondary electron image (SEI) shows a fine structure such as a neurite stem (white arrow head), a lamillipodium (black arrow head) and filopodia (open arrow heads). (b) A backscattered electron image (BEI) shows immuno-gold particles indicated as white dots (arrow heads). (c) A superimposed image of the SEI (a) and the BEI (b) shows that the gold particles (arrow heads) are widely observed on the growth cone.

three, the glass coverslips, the carbon glids and the plastic membrane were coated with poly-l-lysine and laminin before plating. These procedures were carried out in Dulbecco’s MEM (10% fetal calf serum). The DRG neurons were kept in culture at 37°C for 1 day before experiments. The ionic composition of the modified Krebs solution was (mM): NaCl, 117; KCl, 4.7; CaCl2, 2.5; MgCl2, 1.0; glucose,11; 3-[N-morpholino]propanesulfonic acid (MOPS), 25; and pH 7.2 adjusted with NaOH. The low-Ca2+ and low-Mg2 + Krebs solution was made by adding ethylenediaminetetraacetic acid (EDTA, 2.5 mM) to the modified Krebs solution. Cultured DRG neurons were treated with q-aga IVA (300 nM, Peptide Inst., Japan) in modified Krebs solution for 15 min at 37°C, and washed with modified Krebs solution for 3 min at 37°C. The DRG neurons were fixed with 1% paraformaldehyde in phosphate-buffered saline (PBS) solution

for 45 min, incubated with rabbit anti-q-aga IVA IgG (Alomone, Israel) as a first antibody at a 1:10 dilution recommended for immunocytochemistry in PBS solution for 1 h. The DRG neurons treated with the first antibody were immunologically visualized with goat anti-rabbit IgG coupled to 30 nm colloidal gold (British Bio Cell, UK) as a second antibody for 1 h, and fixed again with 1% paraformaldehyde in PBS solution for 45 min at room temperature. The preparations were rinsed with PBS solution for 1 min at room temperature between each treatment. FE-SEM observations. Immuno-gold-labeled DRG neurons were fixed with 1% osmium tetroxide in PBS solution for 1 h at 4°C, rinsed with distilled water for 1 min at 4°C, and dehydrated in graded ethanol series (70–100%). Finally, after two washes with absolute ethanol, the DRG neurons were immersed in isoamyl acetate and dried using a critical point drying method. The samples were coated with

Fig. 2. FE-SEM images of a DRG neuron and a fibroblast. (a) A superimposed image of SEI and BEI of the DRG cell body, showing immuno-gold particles (arrow heads). (b) A superimposed image of SEI and BEI of the fibroblast cell body shows immuno-gold particles (arrow heads) are barely visible.

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Fig. 3. TEM images of a whole-mount preparation and a vertical ultra-thin section (100 nm) of DRG neurons. (a) Immuno-gold particles are observed as black dots in the growth cone in the whole-mount preparation (an arrow head indicates a typical clusters of gold particles). (b) Immuno-gold particles (arrow heads) are present on the surface of the plasma membrane using a vertical ultra thin section (100 nm) of the DRG cell. Images in (a) and (b) were obtained from different DRG neurons.

carbon (JEE-400; JEOL, Japan) just before FE-SEM observation (S-800; Hitachi, Japan). Fine structures were observed with a secondary electron image (SEI), and the presence of immuno-gold particles was clearly detected with a backscattered electron image (BEI). An SEI (Fig. 1a) indicated the fine structures of the growth cones; neurite stem, lamillipodia and filopodia were all evident. On the other hand, a BEI (Fig. 1b) showed the presence of immuno-gold particles as white dots. Superimposed images of the SEI and the BEI (Fig. 1c) showed that the immunogold particles were present on the growth cones. Fig. 2 shows FE-SEM images of a DRG neuron and a fibroblast on the same culture coverslip; immuno-gold particles were present on DRG cell body (Fig. 2a) but barely detectable on a fibroblast cell body (Fig. 2b). This indicates the presence of q-aga IVA-sensitive Ca2+ channels on the DRG cell body. Indeed, our previous study [15] showed that elevation of intracellular calcium concentration ([Ca2 + ]i) was recorded in the soma, when stimulated electrically. However, glutamate release was not detected there. Therefore, it is considered that q-aga IVA-sensitive Ca2 + channels on soma might not be involved in transmitter release; that is the DRG soma is not always endowed with all the machinery necessary for transmitter release, such as vesicles containing glutamate and vesicle-binding proteins. TEM observations. The immuno-gold-labeled DRG neurons for whole-mount preparations and for preparations of ultra-thin sections were fixed with 1% osmium tetroxide, rinsed, and dehydrated in the same way as those for FESEM observations (see above). Whole-mount preparations were immersed in isoamyl acetate and dried using a critical point drying method. Preparations of ultra-thin sections were embedded in super resin (EPON 812; Taab Laboratories Equipment, USA). Whole-mount preparations and

vertical ultra-thin sections (100 nm) of DRG neurons were observed with TEM (200CX and 1200EX JOEL, Japan, respectively). TEM images of whole-mount preparations showed clusters of immuno-gold particles in the area of the growth cone (Fig. 3a). These immuno-gold particles were observed only on the plasma membrane surface, as shown in a TEM image of a vertical ultra-thin section (Fig. 3b). Both TEM and FE-SEM observations of immuno-gold particles indicated the presence of q-aga IVA-sensitive Ca2 + channels on the surface of growth cone membrane. Thus, the main finding of the present study is that q-aga IVA-sensitive Ca2 + channels involved in transmitter release locate on growth cone membrane surface. It is known that q-aga IVA-sensitive Ca2 + channels localize in mature presynaptic terminals [4,14,16]. Our previous study [15] demonstrated that growth cones of cultured rat DRG neurons could release glutamate even before making synapses and that the release was almost completely inhibited by q-aga IVA (300 nM). The present immunocytological study shows that growth cones are endowed with q-aga IVA-sensitive Ca2 + channels. q-Aga IVA-sensitive Ca2 + channels involved in transmitter release may be involved in growth cone functions which play an important role in path finding of developing neurites [1,5]. Various factors are reported to affect behavior of the growth cone [2,3,7,9,13]. In this context, neurotransmitter release from growth cones may be a trigger to arrange several factors in the vicinity of the growth cones and to contribute to path finding decisions. Indeed, since a nerve process is proposed to grow by inserting new membrane materials into its growing tip [2], it is plausible that q-aga IVA-sensitive Ca2 + channels on growth cones may be involved in elongation of neurite tips by directed vesicle fusion during and/or following transmitter release.

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