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Attachment of growth cones on substrate observed by multi-mode light microscopy
Neuroscience Research 35 (1999) 197 – 206 www.elsevier.com/locate/neures
Attachment of growth cones on substrate observed by multi-mode light microscopy Hitoshi Tatsumi a,b,*, Yoshifumi Katayama c, Masahiro Sokabe a a
Department of Physiology, Nagoya Uni6ersity School of Medicine, 65 Tsuramai Showa-ku, Nagoya Aichi, 4668550 Japan b PRESTO, Japan Science and Technology Corporation, ‘The Intelligence and its Origin’, Tokyo, Japan c Department of Autonom. Physiology, Medical Research Institute, Tokyo Medical and Dentistry Uni6ersity, Tokyo, Japan Received 8 February 1999; accepted 30 August 1999
Abstract Evanescent light illumination was introduced into a multi-mode microscope to construct a new type of total internal reflection fluorescence microscope (TIRFM). This microscope, capable of TIRFM, high resolution video-enhanced differential interference contrast (DIC), epifluorescence, interference reflection (IR) imaging, was combined with an image acquisition system for time-lapse microscopy. Neuronal growth cones of a rat hippocampal neuron were stained with membrane labeling fluorescence dyes (DiI or octadecyl rohdamine B). Dynamic changes of the cell/substrate contact of the neuronal growth cone were observed using the multi-imaging capacities of this system. When growth cone regions were stimulated by pressure ejection of a high potassium solution, TIRFM intensity at the basal membrane of the growth cone increased, suggesting that basal membrane of growth cone approaches the glass substrate when excited. The approach of the ventral membrane to the substrate during excitatory stimulation was also observed with IR microscope. The functional importance of cell/substrate contact in growth cones is discussed. © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cell adhesion; Evanescent light microscope; Growth cone; Hippocampal neuron; Multi-imaging; Time lapse microscopy
1. Introduction Neurons protrude growth cones during development and also during nerve sprouting following neuronal damage (Brown et al., 1981). Previous studies (O’Connor et al., 1990; Meier et al., 1993) suggest that growth cones guide developing neurites along a precisely defined pathway and make synapses with their target cells. Thus, the growth cone plays an important role in pathway guidance. A number of possible mechanisms underlying the guidance of growth cones have been suggested. These include the ideas of selective adhesion (Hammarback et al., 1988), chemotaxis (Tessier-Lavigne and Goodman, 1996), and electric fields (Devenport and McCaig, 1993). Electrical activity in the growth cone region has been observed to affect the elongation of growth cone (Cohan and Kater, 1986), as * Corresponding author. Tel.: +81-52-7442054; fax: + 81-527442068. E-mail address: [email protected] (H. Tatsumi)
well as the adhesive contact between the growth cone and neuron (Tatsumi and Katayama, 1999). Furthermore, the existence of a traction force between the growth cone and contacting cell has been reported (Heidemann et al., 1990). Taken together, these studies strongly suggest that an adhesive contact between the growth cone membrane and substrate is necessary for the dynamic movement of growth cones. In order to fully understand the dynamic contact between the growth cone and a substrate and thus the mechanisms of movement, it is crucial to estimate the distance between the substrate and growth cone membrane of living neurons Interference reflection microscopy (IRM) (Gingell and Todd, 1979) and total internal reflection fluorescence microscopy (TIRFM) (Schwartz et al., 1980) have been found to be useful in the visualization of the cell membrane substrate contact region. In these techniques, the illumination of a water covered glass at angles greater than the critical angle results in an ‘evanescent field’ extending several hundred nanometers
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into the water. Specimen in the vicinity of the glass/water interface are illuminated by the evanescent field and thus can be observed. As TIRFM is very sensitive to the distance between neuron and substrate, more so than even IRM (Gingell et al., 1987). The cell-substrate contact region is selectively illuminated thus minimizing the exposure of the cell interior to the excitation light. As a result, fluorescence from fluorophores both in the bulk solution and inside the cells are suppressed, thus reducing background fluorescence. In addition, the healthy survival of cultured cells during imaging procedures is much enhanced relative to the standard epi-illumination case (Schwartz et al., 1980). Furthermore, TIRFM image interpretation is simpler than in IR contrast images, because light interference is a periodic function of wavelength in the latter case (Gingell and Todd, 1979). In the past, TIRFM images have been obtained in various types of cells including fibroblasts, endothelial cells (Burmeister et al., 1994), and muscle cells (Wang and Axelrod, 1994). TIRFM images have been analyzed qualitatively (Gingell and Todd, 1979; Lanni et al., 1985; Burmeister et al., 1994) and quantitatively (Truskey et al., 1992) by using a mathematical model to approximate the evanescent field. A recent study (Oheim et al., 1998) attempted a direct estimation of the penetration depth of the evanescent light by manipulating a small fluorescence bead in the evanescent field. The improvement of TIRFM will provide more definitive information on the distance between cell membrane and substrate. Since TIRFM visualizes only cell – substrate contact regions, observations of the same cell with epifluorescence and differential interference reflection (DIC) are crucial for correct interpretation of TIRFM images; DIC imaging gives information on cellular structures and epifluorescence imaging provides information of fluorescence dye distribution in the entire cell. In the present study, TIRFM, video-enhanced DIC, epifluorescence, and IR contrast optics, were combined in a single microscope. The addition of a nanometer precision positioning system allowed the evanescent field depth to be examined in situ and subcellular structures to be manipulated. Rat hippocampal neurons were cultured on a coverslip, and their neuronal process and growth cones observed with this system. Time-lapse TIRFM was used to image the region where neurons made close contact with the substrate and to analyze dynamic changes under high potassium stimulation.
brain was removed. A brain slice (400–500 mm thick) including the hippocampus region was made and dissected into smaller (200 mm3) lumps These pieces of brain tissue were treated with papain (Worthington biochemical Co., USA; 20.3 unit/ml) in low-Ca2 + and low-Mg2 + Kreb’s solution at 37°C for 20 min, and triturated using pipettes. Neurons were placed on a poly-L-lysine coated glass coverslip (0.22 mm thick soda lime glass, Matsunami glass, Japan) in Dulbecco’s modified Eagle’s minimum essential medium (DMEM) containing 10% fetal calf serum (FCS), 50 u/ml penicillin and 0.1 mg/ml streptomycin. Twenty to fifty neurons were observable at a single time in the 300 mm diameter detection region. Neurons protruded neuronal processes and growth cones after 3 h in culture. Neurons were observed in this culture solution or in the modified Kreb’s solution [117 mM NaCl; 4.7 KC1; 2.5 mM CaC12; 1.0 mM MgC12; 11 mM glucose; 25 mM 3-[N-Morpholino]propanesulfonic acid (MOPS); pH= 7.4 (adjusted by NaOH); (2.5 mM ethylenediaminetetrsacetic acid (EDTA) was added to this solution to make the low-Ca2 + and low-Mg2 + Kreb’s solution)].
2.2. Cell membrane labeling The cell membrane was fluorescently labeled by micro tube (0.1 mm diameter) pressure application of 1,1%dioctadecyl-3,3,3%,3%-tetramethylindocarbocyanine perchlorate (DiI18(3)) or FM™-DiI (water soluble derivative of DiI) in dimethylsulfoxide (DMSO, 10 mg/50 ml). For time lapse video microscopy, cells were labeled with octadecyl rhodamine B-chloride (0246, Molecular Probes, Inc., Eugene Oregon USA). 0246 (5 ml of stock solution 1 mg/ml N,N-dimethylformamide) was dissolved in the culture medium (5 ml) and applied to the specimen for 5 min. The specimen was then rinsed twice with culture medium. All of these dyes localize in the lipid bilayer of the cell membrane (Axelrod, 1979). Relatively weakly stained neurons were used for experiments. In some experiments, the cell membrane was labeled with concanavalin A conjugated with rhodamine (Molecular Probes Inc., USA), which labels membrane glycoproteins (Feldman et al., 1981). 1 mg/ml concanabalin-A (con-A), conjugated with rhodamine, was dissolved in culture medium and applied to neurons for 5 min and rinsed.
2.3. TIRFM and IR contrast microscope 2. Materials and methods
2.1. Cell culture Wistar rats (1–5 days after birth) were anaesthetized with Nembutal (pentobarbital sodium), and the whole
A TIRFM with a nanometer precision positioning system was constructed. TIRFM optics were incorporated into an inverted microscope (Nikon Diaphot TMD-300, Japan) along with DIC optics and epifluorescence illumination. Video-enhanced DIC imaging
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was used to examine the detail of cell structure, and epifluorescence imaging was used to monitor the distribution of florescence dye in the cell membrane. The basic microscope was equipped with a water immersion condenser (NA= 0.9) for DIC illumination and 60 times water immersion objective lens (NA= 1.2; Nikon, Japan) for DIC observation. This objective lens was also used for epifluorescence work. A custom built xyz translation stage (Narishige, Japan) positions the chamber in the focal plane of the microscope. This observation chamber consists of 0.22 mm thick soda lime glass coverslip and rectangular prism (Fig. 1). The flatness of cell substrate (glass coverslip) was estimated less than 1 nm in 10 mm square area using atomic force microscopy (SPI3800, Seiko, Japan)(Uma Maheswari et al., 1995). Neurons were cultured in this chamber before experiments for 3 to 12 h. During observation, the temperature of the chamber was kept between 28 and 34°C. For TIRFM work, 532 nm light (3 – 50 mW:) from an YAG laser (Coherent, DPSS 532, USA) was collimated by a lens (7 times beam expander; Meles Griot, England) to about 5 mm diameter beam and directed into the rectangular prism (2 mm height BK-7 glass prism, n= 1.522, Nihon Ryokyo Inc., Japan) by an achromatic lens ( f=200 mm) and a mirror (Fig. 1). The prism was optically coupled to the coverslip by adhesive glue (Optodyne, Daikin Inc., Japan). The inci-
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dent beam propagates in the glass coverslip toward the optical axis of the microscope via multiple internal reflections. TIR spots could be moved by slightly tilting the laser light with a mirror to place the spot at the center of the observation stage. The diameter of the each spot of laser light was about 500 mm. During x-axis movement, because the laser beam was parallel to the culture substrate, no shift occurs in the spot position. A 60 times water immersion objective was placed below the coverslip for observation. A 532 nm rejection filter (long wave length pass, 560 nm LP, Nikon) was used to eliminate the excitation light coming into the camera port. The fluorescence image was captured with either a cooled-CCD camera (Micromax, Princeton Instruments, USA) or a SIT camera (C2400, Hamamatsu Photonics, Japan). Images of neurons were processed with MetaMorph imaging system (Universal Imaging Co., USA). This setup is particularly well-adapted for viewing cells growing on the coverslips. This microscope arrangement is also extremely convenient, because samples can be manipulated from the upper free space. The incident angle (u) in our experimental condition was 66°. The depth (Dp) of the evanescent light penetration (l/e of surface light intensity) is estimated to be 106 nm for 532 nm light using (Burmeister et al., 1994; Zhu et al., 1985): Dp = l/(4pn1) (sin2u− (n2/n1)2),
(1)
where l is the wavelength of the laser light, n1 and n2 are the refractive indexes of water (1.33) and glass (1.52). As the actual experimental penetration depth depends on the optical index of medium, optical properties of the cell, shape of the approaching probe (microelectrode in this study) and smoothness of the reflection surface, the penetration depth of light was experimentally examined in this study (see results). In IR contrast imaging the epifluorescence mirror was simply replaced with a 50% mirror and sample illuminated with orange filtered (530 nm) light.
2.4. Confocal nucroscopy.
Fig. 1. Schematic of the multifunctional microscope used in this study. Top and side views of the setup are shown. A laser beam (frequency doubled Nd:YAG, 532 nm) was introduced into the glass-substrate plate with entrance prism. The entrance prism was painted with black enamel (pointed by arrow B) to prevent light scattering. Neurons were plated on a coverslip. The TIRF images of neurons were observed with a water immersion objective lens placed below the substrate. A glass capillary attached to piezo positioning equipment is to the right of the cell.
A confocal microscope (LSM-410, Carl Zeiss, Germany) was used to examine the distribution of the fluorescence dye in the cell membrane. Horizontal and vertical sectioning images were made with LSM-410 software (Carl Zeiss, Germany).
2.5. Nanometer precision positioning system. A xyz piezo positioner (Nano-positioner, Melles
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Fig. 2. Evanescent light intensity above the culture glass substrate. Evanescent light intensity was examined using the fine tip of a pipette containing fluorescent dye (FM-DiI) precisely placed above the total internal reflection surface in the experimental medium. Fluorescence from the tip appeared when it approached to less than 200 nm from the reflection surface (b). This shows that the evanescent illumination exciting the fluorescent dye in the tip extends less than 200 nm above the reflection surface. The intensity of the fluorescence was strongly depend on the probe-substrate separation (c). (a) shows the DIC image of the tip of pipette. Fluorescence intensity (60 counts) corresponds the background noise level of the recording system. Fine pipette for patch clamp recording was used as a probe. FM-DiI was attached at the tip of the electrode as well as inside. The pipette was bent along the substrate when they attached on the surface. The zero on the abscissa was determined when the fluorescence abruptly declined during upward movement of pipette from the surface.
Fig. 3. Images of a single neuron stained with fluorescence dye (Ii l-DiI) and observed under total internal reflection fluorescence (A), epifluorescence (B), and video-enhanced DIC (C) microscopes. The some, neurite, growth cone and filopodia (C) are clearly visible in the DIC image. The epifluorescence image shows staining of soma, neurite, growth cone and filopodia (B). In the TIRFM image, areas corresponding to the soma (arrow), growth cone (arrow head) and tip of filopodium (double arrow heads) are bright, indicating substantial cell/substrate contact at these regions (A).
Griot, UK) with built-in sensors to compensate for piezo hystersis was mounted on the microscope to perform high-precision positioning of a fine glass capillary above the TIR surface. The fluorescence intensity at the tip of DiI-filled glass capillary was used to estimate the depth of the evanescent light penetration (Fig. 2). When the tip of the capillary was moved downward to the substratum, only the tip of the capillary entered the evanescent field, and the fluorescence of DiI at the tip was observed (Fig. 2 b) although the entire capillary was filled with the fluorescence dye. The DiI-fluorescence quickly decreased as the tip was moved upward from the substratum and was disappeared at 200 nm, suggesting that the depth of the evanescent light penetration was around 100 nm (see discussion).
2.6. Drugs Drugs used were papain (Wothington, USA), 1,1%dioctadecyl-3,3,3%,3%-tetramethylindocarbocyanine perchlorate (DiICl3, Molecular Probes, USA), FM-DiI (Molecular Probes, USA) and octadecyl rhodamine B chloride (O246, Molecular Probes, USA). Other chemical compounds were from Sigma (USA).
3. Results
3.1. General obser6ations with TIRFM, epifluorescence and DIC microscopes Typical TIRFM, epifluorescence and DIC images of
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the same neuron are shown in Fig. 3. The DIC image shows the soma, neurite, flat growth cone, and extended filopodia (Fig. 3C). The epifluorescence image shows the bright soma region and uniform staining of processes, growth cone, and filopodia (Fig. 3B). The TIRFM image of the same neuron was very distinct from the DIC or epifluorescence images (Fig. 3A). In the TIRFM images the center of the growth cone and tip of filopodium were bright in the majority of cells. The tip of filopodium exhibited a bright fluorescence spot with the bright area sometimes extending the entire length of filopodium. Often neural processes were not illuminated with evanescent light. When varicositylike cell structures along the neurite were observed, varicosity-like structures were illuminated with evanescent light and observed with TIRFM. Soma regions were also bright and several bright spots (Fig. 3A) or large some area were observed. These TIRF images illuminate the regions of the closest contact to the substrate. Similar images of growth cones were observed when cells were stained with rhodamine-conjugated con-A which labels cell surface glicoproteins (Feldman et al., 1981) or with O246.
3.2. Confocal imaging of DiI stained neurons To examine the staining of cell, vertical sectioning of the cell membrane was made with a confocal microscope. The cell membrane was seen to be selectively
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stained with FM-DiI. The ventral membrane of soma and neuronal process located near the substrate (B2 mm).
3.3. Displacement of cell membrane of neurites with piezo positioning equipment In order to examine the distance between the neurite membrane and substrate, the neurite was pushed down to the substrate with a glass capillary tip attached to the piezo positioning equipment. The fluorescence intensity increased when the process was pushed down to the substrate (Fig. 4). A 100 nm downward displacement made the neurite slightly brighter, suggesting the neurite entered the evanescent field (approximately 200 nm from the substrate). An additional 100 nm downward displacement made the area much brighter (Fig. 4c). When a neurite was pressed against the substrate with a micro pipette, the cross sectional shape of the neurite might become flatter. When the capillary tip was withdrawn, fluorescence of the neurite decreased and disappeared, suggesting that the neurite returned to its original position above the evanescent field. When the downward displacement was maintained for a longer period of time (1–3 min), the fluorescence continued even after the tip was withdrawn in 4 cases out of 8. The bright part observed in Fig. 4e would come to evanescent region and stay within 100 nm from the substrate, suggesting the adhesive contact is formed. These results confirm the existence of a thin layer of evanescent field, and that TIRFM visualizes all the
Fig. 4. Downward displacement of neurite to the substrate. (a), control TIRFM image. The same neurite was positioned downward by 100 nm (b), 200 nm (c) and 400 nm (d) with a nano-positioning system. The fluorescence intensity of the neurite (arrow) was 0 (a), 22 (b) and 76 (a. u.) (c). (e), TIRF image after the neurite was held at 400 nm downward from control position for 3 min (d) and allowed to return to its original position. (f), the DIC image of a glass electrode and a neuron. Arrows in d and f show the glass capillary. (DiI, attached to the tip of the electrode, was excited by the evanescent light in d. The fluorescence of pipette overlaps the fluorescence image of neurite.)
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Fig. 5. Time lapse recordings of growth cone extension with TIRFM. Recordings of entire cell are shown (A). In B and C, the growth cone region (arrow and double arrow heads in A) of the same cell is shown in magnification. Time interval was 15 s. The region showing extension is indicated by the left most arrow and double arrow heads in A. In both cases the bright area extended from the growth cone. The shape of the growth cones are shown by broken lines in figures in B and C. Bar denotes 10 mm (A) and 3.6 mm (B and C).
Fig. 6. Topographical maps of fluorscence intensity of 5 × 5 mm section of the ventral membrane of extending growth cone shown in Fig. 5B under TIRF excitation. a, b, and c correspond to 15, 45, 75 s from the start of recording, respectively. A weak TIRF area extended from the growth cone (a) and a bright TIRF spot appeared in the center of the growth cone after 75 s of time lapse recording (indicated by arrowhead in c). Since TIR evanescent light preferentially illuminates the region of the membrane closest to the substrate surface, the bright spot corresponds to a cell/substrate contact point. Arrows on the right side of figures denote the back ground level of the TIRFM images.
membrane near (B ca. 200 nm) the substrate. These observations also show that the neurite is located between 200 nm and 2 mm from the substrate. When the soma and growth cone center were pushed to the substrate by the tip, the bright area in the TIRFM image below the soma became larger and brighter, and the area under the growth cone center became brighter.
3.4. Dynamic changes of the contact regions in the time lapse 6ideo microscopy Elongation of growth cone was observed with time lapse recordings of TIRFM images (Fig. 5). The time lapse recordings (Fig. 5 B and C) both show a small bright spot appearing beyond the tip of growth cone, the spot area increasing and finally merging to the growth cone. DIC observations and the time lapse recordings suggest that the tips of growth cone filopodia are attached on the substrate and the contact area
expands with time. According to the (Eq. (1)) and Fig. 2 of this study, the TIRFM image can be seen as an optical transformation of the cell substrate distance into a spatial pattern of image brightness. Fig. 6 shows topographical maps of TIRF intensity of 5×5 mm section of the ventral membrane of the extending growth cone shown in Fig. 5B. A moderately bright TIRFM area extended from the growth cone (Fig. 6a, arrow head) and a bright TIRFM area expanded in the center of growth cone during the 30 s of time lapse recordings (indicated by the arrow heads in Fig. 6a,b,c). Since TIR evanescent light preferentially illuminates the regions of the membrane closest to the substrate surface, a strong TIRF spot corresponds to a contact site (indicated by the arrow head in Fig. 6c). If homogeneous distribution of DiI is assumed, the change of the separation between the membrane and the substrate can be estimated as 73 nm (Dp = 106 nm assumed). This agrees with the DIC observation that growth cone
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region actively changes its form in culture (Tatsumi et al., 1993). When neurons were fixed with 4% formaldehyde and rinsed with phosphate buffered saline solution, the fluorescence intensity was not changed (less than 3%), indicating that the dynamic changes in the fluorescence intensity of living neurons was not the result of Brownian motion of the membrane.
3.5. Membrane approach to the substrate following excitatory stimulation to growth cone While high potassium solution was applied locally to the growth cone for 10 – 20 s to excite the neuron, TIRFM images were recorded at 5 s intervals. The high K+ stimulation induced a transient increase in the fluorescence intensity at the growth cone (Fig. 7) (n= 5). The maximum increase in fluorescence intensity was 64% of control after background subtraction (379 14%, n=12). This value (64%) corresponds to a 46 nm movement of the cell membrane in the direction of the substrate. Image subtraction (Fig. 7c,d,e) shows that the center of growth cone approached the substrate. The fluorescence intensity returned to the basal level 3 – 5 min after the end of high potassium stimulation. No significant increase of dynamic contact was observed in the growth cones when control modified Kreb’s solution was locally applied to the growth cone, excluding the possibility that the flow of medium pressed the neurite down to the substrate during
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ejection. IR contrast imaging has been used to visualize the cell/substrate contact area without fluorescence staining of cells (Gundersen, 1988). IR contrast images showed that center of the growth cone was darker comparing to the neurite, again suggesting contact between the growth cone and substrate. The images of TIRFM and IR contrast images are compared in Fig. 8E and F. Time-lapse IR contrast imaging of the growth cones was made under the same stimulation as above. The brightness of IR contrast image of the growth cone center transiently decreased and returned to the control level (Fig. 8) (n= 4). This again indicates that the growth cone membrane approaches to the substrate during the excitation. 4. Discussion The direct manipulation of the neuron’s membrane with a precision manipulator serves both to confirm the presence of evanescent field and estimate the separation between the cell and substrate. In this study the empirically estimated Dp value was slightly larger than the theoretical value. This may be a result of the volume of the dye in the glass capillary, which would result in an over estimation of the penetration depth of the evanescent field (Reddick et al., 1990; Salomon et al., 1991; Oheim et al., 1998). In addition, the shape of the tip could be critical for coupling between the evanescent
Fig. 7. The fluorescence intensity under TIR excitation increased in the ventral membrane of the growth cone following the excitatory stimulation. Excitatory simulation of the growth cone was made by pressure ejection of high potassium solution. (a) shows a TIRFM image of growth cone. Measurement of fluorescence intensities was made at regions denoted by 1 – 6. The changes in the fluorescence intensity were shown in (b). High potassium stimulation was applied during the period shown by horizontal bar. c to e images show the subtracted images (control fluorescence image captured just before the stimulation is subtracted from the stimulated image). The bright points correspond to the points where the TIRF intensity increased after the stimulation. c: 25 s (at end of high potassium stimulation). d: 40 s. e: 120 s. The shape of growth cone is shown by broken lines in figure e. Bar in e denotes 10 mm.
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Fig. 8. Time lapse images of the growth cone with IR microscopy and changes after high K stimulation. (A) DIC image of a growth cone and a pipette. (B) IR contract image of the same growth cone. The dark area in IR image indicates contact areas. (C) changes in the brightness of the area shown by a, b, c in B were plotted. (D) subtracted images at 55 s. Subtraction was made by subtracting control image captured just before the stimulation from the image at 55 s. TIRFM image (E) and IR contrast image (F) of the same neuron. Arrows show the same growth cone. Bar in B (5 mm) and F (10 mm).
field and the tip (Saiki et al., 1996). Theoretically, the curve in Fig. 2 should be exponential. But the coupling could deviate it from the exponential function as seen in Fig. 2. Theoretical model of the coupling is still under construction (Saiki et al., 1996). The TIRF and IR contrast images in this study showed that the ventral membrane of the growth cone and filopodia are attached to the substrate. The contact of the growth cone to the substrate in dorsal root ganglion neurons first reported using an IR contrast microscope (Gundersen, 1988) is thought to be the result of an adhesive molecule, integrin, located on the tips of the growth cone filopodia (Grabham and Goldberg, 1997). This adhesion and the subsequent production of tension by filopodia results in forward motion of the growth cone (Lamoureux et al., 1989; Heidemann et al., 1990). These observations, including present results, support the idea that tips of filopodia contact a target, and in some case initiate more wide contact of growth cone to the target, to produce dynamic movements. A bright area in TIRF image was also observed at the growth cone and varicosity-like structure. The growth cone and varicosity-like structure are known to release transmitters in culture (Sun and Poo, 1987; Allen and Brown, 1996; Soeda et al., 1997) and in vivo
as synapses en passant (Houser et al., 1983). In addition, cell–cell contact phenomenon after transmitter release from a growth cone has been reported (Buchanan et al., 1989; Tatsumi and Katayama, 1999). These suggest a close interrelation between exocytosis and cell/substrate contact. The TIRF images in this study showed that the vertical membrane of the growth cone approached to the substrate when excited. Although the mechanism of this approach is not known, neurotransmitter release from growth cone could insert new plasma membranes by exocytosis (Igarashi et al., 1996). This excess cell plasma membrane might allow the growth cone to approach the substrate (or target cells). Although this may decrease the density of the fluorescence molecule on the cell membrane, the approaching of the membrane to substrate would cause a much more increase in the fluorescence intensity, because the fluorescence intensity is steeply dependent on the distance between membrane and substrate under the TIR illumination. The volume expansion of neurons has been observed when excitatory stimulation was applied to axons (Tasaki and Byrne, 1990). The approach of the cell membrane toward the substrate might also be a result of the expansion of cell volume at growth cones. Our estimation of membrane approaching to substrate is
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about 5 times larger than that of single axon. The larger effect could be because of summation of multiple cellular processes; (1) the insertion of new plasma membranes at growth cone; (2) the large ion influx through the voltage-sensitive channels as shown in axons; and (3) K-depolarizanon causes cell volume increase (Tosteson and Foffman, 1960).Excitation of growth cones also affects the their movement. Excitatory transmitter or electrical stimulation of the growth cone inhibits elongation of growth cone (Cohan and Kater, 1986). The approach of the growth cone membrane to the substrate after excitation may lead to increased contact between them. This increased contact could temporarily stabilize the growth cone movement. Two types of contact, focal contact and matrix contact have been reported in various types of cells (Izzard and Lochner, 1976; Chen and Singer, 1982; Burridge et al., 1988). In focal contact, the membrane is separated from the substrate by 15 – 50 nm. In matrix contact, the cell membrane is separated by 100 nm or more from the substrate. In this study TIRFM was used to estimate the separation between the ventral membrane of growth cones and substrate. TIRFM images were obtained only when the membrane was located less than 200 nm from the substrate. The mean change of the separation during excitatory stimulation was estimated around 50 nm in this study. This means the growth cone membrane before stimulation was located between 50 and 200 nm from the substrate. Thus the contact could be classified as matrix contact. (In agreement with the previous indirect estimate of the cell/substratum contact using IR contrast microscope (Gundersen, 1988)). This study has shown that TIRFM is adequate for observing dynamic changes in membrane/substrate contact regions in living neurons. One important future question is the cellular and molecular mechanisms of the dynamic changes in the separation between the membrane and substrate.
Acknowledgements We thank Dr. J. D. White for cntical readings of an early version of the manuscript and comments. This study was supported by a Grant-in Aid for Scientific Research from The Ministry of Education, Science and Culture of Japan and Japan Science and Technology Corporation and Nakayama Foundation for Human Science.
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Attachment of growth cones on substrate observed by multi-mode light microscopy Hitoshi Tatsumi a,b,*, Yoshifumi Katayama c, Masahiro Sokabe a a
Department of Physiology, Nagoya Uni6ersity School of Medicine, 65 Tsuramai Showa-ku, Nagoya Aichi, 4668550 Japan b PRESTO, Japan Science and Technology Corporation, ‘The Intelligence and its Origin’, Tokyo, Japan c Department of Autonom. Physiology, Medical Research Institute, Tokyo Medical and Dentistry Uni6ersity, Tokyo, Japan Received 8 February 1999; accepted 30 August 1999
Abstract Evanescent light illumination was introduced into a multi-mode microscope to construct a new type of total internal reflection fluorescence microscope (TIRFM). This microscope, capable of TIRFM, high resolution video-enhanced differential interference contrast (DIC), epifluorescence, interference reflection (IR) imaging, was combined with an image acquisition system for time-lapse microscopy. Neuronal growth cones of a rat hippocampal neuron were stained with membrane labeling fluorescence dyes (DiI or octadecyl rohdamine B). Dynamic changes of the cell/substrate contact of the neuronal growth cone were observed using the multi-imaging capacities of this system. When growth cone regions were stimulated by pressure ejection of a high potassium solution, TIRFM intensity at the basal membrane of the growth cone increased, suggesting that basal membrane of growth cone approaches the glass substrate when excited. The approach of the ventral membrane to the substrate during excitatory stimulation was also observed with IR microscope. The functional importance of cell/substrate contact in growth cones is discussed. © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cell adhesion; Evanescent light microscope; Growth cone; Hippocampal neuron; Multi-imaging; Time lapse microscopy
1. Introduction Neurons protrude growth cones during development and also during nerve sprouting following neuronal damage (Brown et al., 1981). Previous studies (O’Connor et al., 1990; Meier et al., 1993) suggest that growth cones guide developing neurites along a precisely defined pathway and make synapses with their target cells. Thus, the growth cone plays an important role in pathway guidance. A number of possible mechanisms underlying the guidance of growth cones have been suggested. These include the ideas of selective adhesion (Hammarback et al., 1988), chemotaxis (Tessier-Lavigne and Goodman, 1996), and electric fields (Devenport and McCaig, 1993). Electrical activity in the growth cone region has been observed to affect the elongation of growth cone (Cohan and Kater, 1986), as * Corresponding author. Tel.: +81-52-7442054; fax: + 81-527442068. E-mail address: [email protected] (H. Tatsumi)
well as the adhesive contact between the growth cone and neuron (Tatsumi and Katayama, 1999). Furthermore, the existence of a traction force between the growth cone and contacting cell has been reported (Heidemann et al., 1990). Taken together, these studies strongly suggest that an adhesive contact between the growth cone membrane and substrate is necessary for the dynamic movement of growth cones. In order to fully understand the dynamic contact between the growth cone and a substrate and thus the mechanisms of movement, it is crucial to estimate the distance between the substrate and growth cone membrane of living neurons Interference reflection microscopy (IRM) (Gingell and Todd, 1979) and total internal reflection fluorescence microscopy (TIRFM) (Schwartz et al., 1980) have been found to be useful in the visualization of the cell membrane substrate contact region. In these techniques, the illumination of a water covered glass at angles greater than the critical angle results in an ‘evanescent field’ extending several hundred nanometers
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into the water. Specimen in the vicinity of the glass/water interface are illuminated by the evanescent field and thus can be observed. As TIRFM is very sensitive to the distance between neuron and substrate, more so than even IRM (Gingell et al., 1987). The cell-substrate contact region is selectively illuminated thus minimizing the exposure of the cell interior to the excitation light. As a result, fluorescence from fluorophores both in the bulk solution and inside the cells are suppressed, thus reducing background fluorescence. In addition, the healthy survival of cultured cells during imaging procedures is much enhanced relative to the standard epi-illumination case (Schwartz et al., 1980). Furthermore, TIRFM image interpretation is simpler than in IR contrast images, because light interference is a periodic function of wavelength in the latter case (Gingell and Todd, 1979). In the past, TIRFM images have been obtained in various types of cells including fibroblasts, endothelial cells (Burmeister et al., 1994), and muscle cells (Wang and Axelrod, 1994). TIRFM images have been analyzed qualitatively (Gingell and Todd, 1979; Lanni et al., 1985; Burmeister et al., 1994) and quantitatively (Truskey et al., 1992) by using a mathematical model to approximate the evanescent field. A recent study (Oheim et al., 1998) attempted a direct estimation of the penetration depth of the evanescent light by manipulating a small fluorescence bead in the evanescent field. The improvement of TIRFM will provide more definitive information on the distance between cell membrane and substrate. Since TIRFM visualizes only cell – substrate contact regions, observations of the same cell with epifluorescence and differential interference reflection (DIC) are crucial for correct interpretation of TIRFM images; DIC imaging gives information on cellular structures and epifluorescence imaging provides information of fluorescence dye distribution in the entire cell. In the present study, TIRFM, video-enhanced DIC, epifluorescence, and IR contrast optics, were combined in a single microscope. The addition of a nanometer precision positioning system allowed the evanescent field depth to be examined in situ and subcellular structures to be manipulated. Rat hippocampal neurons were cultured on a coverslip, and their neuronal process and growth cones observed with this system. Time-lapse TIRFM was used to image the region where neurons made close contact with the substrate and to analyze dynamic changes under high potassium stimulation.
brain was removed. A brain slice (400–500 mm thick) including the hippocampus region was made and dissected into smaller (200 mm3) lumps These pieces of brain tissue were treated with papain (Worthington biochemical Co., USA; 20.3 unit/ml) in low-Ca2 + and low-Mg2 + Kreb’s solution at 37°C for 20 min, and triturated using pipettes. Neurons were placed on a poly-L-lysine coated glass coverslip (0.22 mm thick soda lime glass, Matsunami glass, Japan) in Dulbecco’s modified Eagle’s minimum essential medium (DMEM) containing 10% fetal calf serum (FCS), 50 u/ml penicillin and 0.1 mg/ml streptomycin. Twenty to fifty neurons were observable at a single time in the 300 mm diameter detection region. Neurons protruded neuronal processes and growth cones after 3 h in culture. Neurons were observed in this culture solution or in the modified Kreb’s solution [117 mM NaCl; 4.7 KC1; 2.5 mM CaC12; 1.0 mM MgC12; 11 mM glucose; 25 mM 3-[N-Morpholino]propanesulfonic acid (MOPS); pH= 7.4 (adjusted by NaOH); (2.5 mM ethylenediaminetetrsacetic acid (EDTA) was added to this solution to make the low-Ca2 + and low-Mg2 + Kreb’s solution)].
2.2. Cell membrane labeling The cell membrane was fluorescently labeled by micro tube (0.1 mm diameter) pressure application of 1,1%dioctadecyl-3,3,3%,3%-tetramethylindocarbocyanine perchlorate (DiI18(3)) or FM™-DiI (water soluble derivative of DiI) in dimethylsulfoxide (DMSO, 10 mg/50 ml). For time lapse video microscopy, cells were labeled with octadecyl rhodamine B-chloride (0246, Molecular Probes, Inc., Eugene Oregon USA). 0246 (5 ml of stock solution 1 mg/ml N,N-dimethylformamide) was dissolved in the culture medium (5 ml) and applied to the specimen for 5 min. The specimen was then rinsed twice with culture medium. All of these dyes localize in the lipid bilayer of the cell membrane (Axelrod, 1979). Relatively weakly stained neurons were used for experiments. In some experiments, the cell membrane was labeled with concanavalin A conjugated with rhodamine (Molecular Probes Inc., USA), which labels membrane glycoproteins (Feldman et al., 1981). 1 mg/ml concanabalin-A (con-A), conjugated with rhodamine, was dissolved in culture medium and applied to neurons for 5 min and rinsed.
2.3. TIRFM and IR contrast microscope 2. Materials and methods
2.1. Cell culture Wistar rats (1–5 days after birth) were anaesthetized with Nembutal (pentobarbital sodium), and the whole
A TIRFM with a nanometer precision positioning system was constructed. TIRFM optics were incorporated into an inverted microscope (Nikon Diaphot TMD-300, Japan) along with DIC optics and epifluorescence illumination. Video-enhanced DIC imaging
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was used to examine the detail of cell structure, and epifluorescence imaging was used to monitor the distribution of florescence dye in the cell membrane. The basic microscope was equipped with a water immersion condenser (NA= 0.9) for DIC illumination and 60 times water immersion objective lens (NA= 1.2; Nikon, Japan) for DIC observation. This objective lens was also used for epifluorescence work. A custom built xyz translation stage (Narishige, Japan) positions the chamber in the focal plane of the microscope. This observation chamber consists of 0.22 mm thick soda lime glass coverslip and rectangular prism (Fig. 1). The flatness of cell substrate (glass coverslip) was estimated less than 1 nm in 10 mm square area using atomic force microscopy (SPI3800, Seiko, Japan)(Uma Maheswari et al., 1995). Neurons were cultured in this chamber before experiments for 3 to 12 h. During observation, the temperature of the chamber was kept between 28 and 34°C. For TIRFM work, 532 nm light (3 – 50 mW:) from an YAG laser (Coherent, DPSS 532, USA) was collimated by a lens (7 times beam expander; Meles Griot, England) to about 5 mm diameter beam and directed into the rectangular prism (2 mm height BK-7 glass prism, n= 1.522, Nihon Ryokyo Inc., Japan) by an achromatic lens ( f=200 mm) and a mirror (Fig. 1). The prism was optically coupled to the coverslip by adhesive glue (Optodyne, Daikin Inc., Japan). The inci-
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dent beam propagates in the glass coverslip toward the optical axis of the microscope via multiple internal reflections. TIR spots could be moved by slightly tilting the laser light with a mirror to place the spot at the center of the observation stage. The diameter of the each spot of laser light was about 500 mm. During x-axis movement, because the laser beam was parallel to the culture substrate, no shift occurs in the spot position. A 60 times water immersion objective was placed below the coverslip for observation. A 532 nm rejection filter (long wave length pass, 560 nm LP, Nikon) was used to eliminate the excitation light coming into the camera port. The fluorescence image was captured with either a cooled-CCD camera (Micromax, Princeton Instruments, USA) or a SIT camera (C2400, Hamamatsu Photonics, Japan). Images of neurons were processed with MetaMorph imaging system (Universal Imaging Co., USA). This setup is particularly well-adapted for viewing cells growing on the coverslips. This microscope arrangement is also extremely convenient, because samples can be manipulated from the upper free space. The incident angle (u) in our experimental condition was 66°. The depth (Dp) of the evanescent light penetration (l/e of surface light intensity) is estimated to be 106 nm for 532 nm light using (Burmeister et al., 1994; Zhu et al., 1985): Dp = l/(4pn1) (sin2u− (n2/n1)2),
(1)
where l is the wavelength of the laser light, n1 and n2 are the refractive indexes of water (1.33) and glass (1.52). As the actual experimental penetration depth depends on the optical index of medium, optical properties of the cell, shape of the approaching probe (microelectrode in this study) and smoothness of the reflection surface, the penetration depth of light was experimentally examined in this study (see results). In IR contrast imaging the epifluorescence mirror was simply replaced with a 50% mirror and sample illuminated with orange filtered (530 nm) light.
2.4. Confocal nucroscopy.
Fig. 1. Schematic of the multifunctional microscope used in this study. Top and side views of the setup are shown. A laser beam (frequency doubled Nd:YAG, 532 nm) was introduced into the glass-substrate plate with entrance prism. The entrance prism was painted with black enamel (pointed by arrow B) to prevent light scattering. Neurons were plated on a coverslip. The TIRF images of neurons were observed with a water immersion objective lens placed below the substrate. A glass capillary attached to piezo positioning equipment is to the right of the cell.
A confocal microscope (LSM-410, Carl Zeiss, Germany) was used to examine the distribution of the fluorescence dye in the cell membrane. Horizontal and vertical sectioning images were made with LSM-410 software (Carl Zeiss, Germany).
2.5. Nanometer precision positioning system. A xyz piezo positioner (Nano-positioner, Melles
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Fig. 2. Evanescent light intensity above the culture glass substrate. Evanescent light intensity was examined using the fine tip of a pipette containing fluorescent dye (FM-DiI) precisely placed above the total internal reflection surface in the experimental medium. Fluorescence from the tip appeared when it approached to less than 200 nm from the reflection surface (b). This shows that the evanescent illumination exciting the fluorescent dye in the tip extends less than 200 nm above the reflection surface. The intensity of the fluorescence was strongly depend on the probe-substrate separation (c). (a) shows the DIC image of the tip of pipette. Fluorescence intensity (60 counts) corresponds the background noise level of the recording system. Fine pipette for patch clamp recording was used as a probe. FM-DiI was attached at the tip of the electrode as well as inside. The pipette was bent along the substrate when they attached on the surface. The zero on the abscissa was determined when the fluorescence abruptly declined during upward movement of pipette from the surface.
Fig. 3. Images of a single neuron stained with fluorescence dye (Ii l-DiI) and observed under total internal reflection fluorescence (A), epifluorescence (B), and video-enhanced DIC (C) microscopes. The some, neurite, growth cone and filopodia (C) are clearly visible in the DIC image. The epifluorescence image shows staining of soma, neurite, growth cone and filopodia (B). In the TIRFM image, areas corresponding to the soma (arrow), growth cone (arrow head) and tip of filopodium (double arrow heads) are bright, indicating substantial cell/substrate contact at these regions (A).
Griot, UK) with built-in sensors to compensate for piezo hystersis was mounted on the microscope to perform high-precision positioning of a fine glass capillary above the TIR surface. The fluorescence intensity at the tip of DiI-filled glass capillary was used to estimate the depth of the evanescent light penetration (Fig. 2). When the tip of the capillary was moved downward to the substratum, only the tip of the capillary entered the evanescent field, and the fluorescence of DiI at the tip was observed (Fig. 2 b) although the entire capillary was filled with the fluorescence dye. The DiI-fluorescence quickly decreased as the tip was moved upward from the substratum and was disappeared at 200 nm, suggesting that the depth of the evanescent light penetration was around 100 nm (see discussion).
2.6. Drugs Drugs used were papain (Wothington, USA), 1,1%dioctadecyl-3,3,3%,3%-tetramethylindocarbocyanine perchlorate (DiICl3, Molecular Probes, USA), FM-DiI (Molecular Probes, USA) and octadecyl rhodamine B chloride (O246, Molecular Probes, USA). Other chemical compounds were from Sigma (USA).
3. Results
3.1. General obser6ations with TIRFM, epifluorescence and DIC microscopes Typical TIRFM, epifluorescence and DIC images of
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the same neuron are shown in Fig. 3. The DIC image shows the soma, neurite, flat growth cone, and extended filopodia (Fig. 3C). The epifluorescence image shows the bright soma region and uniform staining of processes, growth cone, and filopodia (Fig. 3B). The TIRFM image of the same neuron was very distinct from the DIC or epifluorescence images (Fig. 3A). In the TIRFM images the center of the growth cone and tip of filopodium were bright in the majority of cells. The tip of filopodium exhibited a bright fluorescence spot with the bright area sometimes extending the entire length of filopodium. Often neural processes were not illuminated with evanescent light. When varicositylike cell structures along the neurite were observed, varicosity-like structures were illuminated with evanescent light and observed with TIRFM. Soma regions were also bright and several bright spots (Fig. 3A) or large some area were observed. These TIRF images illuminate the regions of the closest contact to the substrate. Similar images of growth cones were observed when cells were stained with rhodamine-conjugated con-A which labels cell surface glicoproteins (Feldman et al., 1981) or with O246.
3.2. Confocal imaging of DiI stained neurons To examine the staining of cell, vertical sectioning of the cell membrane was made with a confocal microscope. The cell membrane was seen to be selectively
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stained with FM-DiI. The ventral membrane of soma and neuronal process located near the substrate (B2 mm).
3.3. Displacement of cell membrane of neurites with piezo positioning equipment In order to examine the distance between the neurite membrane and substrate, the neurite was pushed down to the substrate with a glass capillary tip attached to the piezo positioning equipment. The fluorescence intensity increased when the process was pushed down to the substrate (Fig. 4). A 100 nm downward displacement made the neurite slightly brighter, suggesting the neurite entered the evanescent field (approximately 200 nm from the substrate). An additional 100 nm downward displacement made the area much brighter (Fig. 4c). When a neurite was pressed against the substrate with a micro pipette, the cross sectional shape of the neurite might become flatter. When the capillary tip was withdrawn, fluorescence of the neurite decreased and disappeared, suggesting that the neurite returned to its original position above the evanescent field. When the downward displacement was maintained for a longer period of time (1–3 min), the fluorescence continued even after the tip was withdrawn in 4 cases out of 8. The bright part observed in Fig. 4e would come to evanescent region and stay within 100 nm from the substrate, suggesting the adhesive contact is formed. These results confirm the existence of a thin layer of evanescent field, and that TIRFM visualizes all the
Fig. 4. Downward displacement of neurite to the substrate. (a), control TIRFM image. The same neurite was positioned downward by 100 nm (b), 200 nm (c) and 400 nm (d) with a nano-positioning system. The fluorescence intensity of the neurite (arrow) was 0 (a), 22 (b) and 76 (a. u.) (c). (e), TIRF image after the neurite was held at 400 nm downward from control position for 3 min (d) and allowed to return to its original position. (f), the DIC image of a glass electrode and a neuron. Arrows in d and f show the glass capillary. (DiI, attached to the tip of the electrode, was excited by the evanescent light in d. The fluorescence of pipette overlaps the fluorescence image of neurite.)
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Fig. 5. Time lapse recordings of growth cone extension with TIRFM. Recordings of entire cell are shown (A). In B and C, the growth cone region (arrow and double arrow heads in A) of the same cell is shown in magnification. Time interval was 15 s. The region showing extension is indicated by the left most arrow and double arrow heads in A. In both cases the bright area extended from the growth cone. The shape of the growth cones are shown by broken lines in figures in B and C. Bar denotes 10 mm (A) and 3.6 mm (B and C).
Fig. 6. Topographical maps of fluorscence intensity of 5 × 5 mm section of the ventral membrane of extending growth cone shown in Fig. 5B under TIRF excitation. a, b, and c correspond to 15, 45, 75 s from the start of recording, respectively. A weak TIRF area extended from the growth cone (a) and a bright TIRF spot appeared in the center of the growth cone after 75 s of time lapse recording (indicated by arrowhead in c). Since TIR evanescent light preferentially illuminates the region of the membrane closest to the substrate surface, the bright spot corresponds to a cell/substrate contact point. Arrows on the right side of figures denote the back ground level of the TIRFM images.
membrane near (B ca. 200 nm) the substrate. These observations also show that the neurite is located between 200 nm and 2 mm from the substrate. When the soma and growth cone center were pushed to the substrate by the tip, the bright area in the TIRFM image below the soma became larger and brighter, and the area under the growth cone center became brighter.
3.4. Dynamic changes of the contact regions in the time lapse 6ideo microscopy Elongation of growth cone was observed with time lapse recordings of TIRFM images (Fig. 5). The time lapse recordings (Fig. 5 B and C) both show a small bright spot appearing beyond the tip of growth cone, the spot area increasing and finally merging to the growth cone. DIC observations and the time lapse recordings suggest that the tips of growth cone filopodia are attached on the substrate and the contact area
expands with time. According to the (Eq. (1)) and Fig. 2 of this study, the TIRFM image can be seen as an optical transformation of the cell substrate distance into a spatial pattern of image brightness. Fig. 6 shows topographical maps of TIRF intensity of 5×5 mm section of the ventral membrane of the extending growth cone shown in Fig. 5B. A moderately bright TIRFM area extended from the growth cone (Fig. 6a, arrow head) and a bright TIRFM area expanded in the center of growth cone during the 30 s of time lapse recordings (indicated by the arrow heads in Fig. 6a,b,c). Since TIR evanescent light preferentially illuminates the regions of the membrane closest to the substrate surface, a strong TIRF spot corresponds to a contact site (indicated by the arrow head in Fig. 6c). If homogeneous distribution of DiI is assumed, the change of the separation between the membrane and the substrate can be estimated as 73 nm (Dp = 106 nm assumed). This agrees with the DIC observation that growth cone
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region actively changes its form in culture (Tatsumi et al., 1993). When neurons were fixed with 4% formaldehyde and rinsed with phosphate buffered saline solution, the fluorescence intensity was not changed (less than 3%), indicating that the dynamic changes in the fluorescence intensity of living neurons was not the result of Brownian motion of the membrane.
3.5. Membrane approach to the substrate following excitatory stimulation to growth cone While high potassium solution was applied locally to the growth cone for 10 – 20 s to excite the neuron, TIRFM images were recorded at 5 s intervals. The high K+ stimulation induced a transient increase in the fluorescence intensity at the growth cone (Fig. 7) (n= 5). The maximum increase in fluorescence intensity was 64% of control after background subtraction (379 14%, n=12). This value (64%) corresponds to a 46 nm movement of the cell membrane in the direction of the substrate. Image subtraction (Fig. 7c,d,e) shows that the center of growth cone approached the substrate. The fluorescence intensity returned to the basal level 3 – 5 min after the end of high potassium stimulation. No significant increase of dynamic contact was observed in the growth cones when control modified Kreb’s solution was locally applied to the growth cone, excluding the possibility that the flow of medium pressed the neurite down to the substrate during
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ejection. IR contrast imaging has been used to visualize the cell/substrate contact area without fluorescence staining of cells (Gundersen, 1988). IR contrast images showed that center of the growth cone was darker comparing to the neurite, again suggesting contact between the growth cone and substrate. The images of TIRFM and IR contrast images are compared in Fig. 8E and F. Time-lapse IR contrast imaging of the growth cones was made under the same stimulation as above. The brightness of IR contrast image of the growth cone center transiently decreased and returned to the control level (Fig. 8) (n= 4). This again indicates that the growth cone membrane approaches to the substrate during the excitation. 4. Discussion The direct manipulation of the neuron’s membrane with a precision manipulator serves both to confirm the presence of evanescent field and estimate the separation between the cell and substrate. In this study the empirically estimated Dp value was slightly larger than the theoretical value. This may be a result of the volume of the dye in the glass capillary, which would result in an over estimation of the penetration depth of the evanescent field (Reddick et al., 1990; Salomon et al., 1991; Oheim et al., 1998). In addition, the shape of the tip could be critical for coupling between the evanescent
Fig. 7. The fluorescence intensity under TIR excitation increased in the ventral membrane of the growth cone following the excitatory stimulation. Excitatory simulation of the growth cone was made by pressure ejection of high potassium solution. (a) shows a TIRFM image of growth cone. Measurement of fluorescence intensities was made at regions denoted by 1 – 6. The changes in the fluorescence intensity were shown in (b). High potassium stimulation was applied during the period shown by horizontal bar. c to e images show the subtracted images (control fluorescence image captured just before the stimulation is subtracted from the stimulated image). The bright points correspond to the points where the TIRF intensity increased after the stimulation. c: 25 s (at end of high potassium stimulation). d: 40 s. e: 120 s. The shape of growth cone is shown by broken lines in figure e. Bar in e denotes 10 mm.
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Fig. 8. Time lapse images of the growth cone with IR microscopy and changes after high K stimulation. (A) DIC image of a growth cone and a pipette. (B) IR contract image of the same growth cone. The dark area in IR image indicates contact areas. (C) changes in the brightness of the area shown by a, b, c in B were plotted. (D) subtracted images at 55 s. Subtraction was made by subtracting control image captured just before the stimulation from the image at 55 s. TIRFM image (E) and IR contrast image (F) of the same neuron. Arrows show the same growth cone. Bar in B (5 mm) and F (10 mm).
field and the tip (Saiki et al., 1996). Theoretically, the curve in Fig. 2 should be exponential. But the coupling could deviate it from the exponential function as seen in Fig. 2. Theoretical model of the coupling is still under construction (Saiki et al., 1996). The TIRF and IR contrast images in this study showed that the ventral membrane of the growth cone and filopodia are attached to the substrate. The contact of the growth cone to the substrate in dorsal root ganglion neurons first reported using an IR contrast microscope (Gundersen, 1988) is thought to be the result of an adhesive molecule, integrin, located on the tips of the growth cone filopodia (Grabham and Goldberg, 1997). This adhesion and the subsequent production of tension by filopodia results in forward motion of the growth cone (Lamoureux et al., 1989; Heidemann et al., 1990). These observations, including present results, support the idea that tips of filopodia contact a target, and in some case initiate more wide contact of growth cone to the target, to produce dynamic movements. A bright area in TIRF image was also observed at the growth cone and varicosity-like structure. The growth cone and varicosity-like structure are known to release transmitters in culture (Sun and Poo, 1987; Allen and Brown, 1996; Soeda et al., 1997) and in vivo
as synapses en passant (Houser et al., 1983). In addition, cell–cell contact phenomenon after transmitter release from a growth cone has been reported (Buchanan et al., 1989; Tatsumi and Katayama, 1999). These suggest a close interrelation between exocytosis and cell/substrate contact. The TIRF images in this study showed that the vertical membrane of the growth cone approached to the substrate when excited. Although the mechanism of this approach is not known, neurotransmitter release from growth cone could insert new plasma membranes by exocytosis (Igarashi et al., 1996). This excess cell plasma membrane might allow the growth cone to approach the substrate (or target cells). Although this may decrease the density of the fluorescence molecule on the cell membrane, the approaching of the membrane to substrate would cause a much more increase in the fluorescence intensity, because the fluorescence intensity is steeply dependent on the distance between membrane and substrate under the TIR illumination. The volume expansion of neurons has been observed when excitatory stimulation was applied to axons (Tasaki and Byrne, 1990). The approach of the cell membrane toward the substrate might also be a result of the expansion of cell volume at growth cones. Our estimation of membrane approaching to substrate is
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about 5 times larger than that of single axon. The larger effect could be because of summation of multiple cellular processes; (1) the insertion of new plasma membranes at growth cone; (2) the large ion influx through the voltage-sensitive channels as shown in axons; and (3) K-depolarizanon causes cell volume increase (Tosteson and Foffman, 1960).Excitation of growth cones also affects the their movement. Excitatory transmitter or electrical stimulation of the growth cone inhibits elongation of growth cone (Cohan and Kater, 1986). The approach of the growth cone membrane to the substrate after excitation may lead to increased contact between them. This increased contact could temporarily stabilize the growth cone movement. Two types of contact, focal contact and matrix contact have been reported in various types of cells (Izzard and Lochner, 1976; Chen and Singer, 1982; Burridge et al., 1988). In focal contact, the membrane is separated from the substrate by 15 – 50 nm. In matrix contact, the cell membrane is separated by 100 nm or more from the substrate. In this study TIRFM was used to estimate the separation between the ventral membrane of growth cones and substrate. TIRFM images were obtained only when the membrane was located less than 200 nm from the substrate. The mean change of the separation during excitatory stimulation was estimated around 50 nm in this study. This means the growth cone membrane before stimulation was located between 50 and 200 nm from the substrate. Thus the contact could be classified as matrix contact. (In agreement with the previous indirect estimate of the cell/substratum contact using IR contrast microscope (Gundersen, 1988)). This study has shown that TIRFM is adequate for observing dynamic changes in membrane/substrate contact regions in living neurons. One important future question is the cellular and molecular mechanisms of the dynamic changes in the separation between the membrane and substrate.
Acknowledgements We thank Dr. J. D. White for cntical readings of an early version of the manuscript and comments. This study was supported by a Grant-in Aid for Scientific Research from The Ministry of Education, Science and Culture of Japan and Japan Science and Technology Corporation and Nakayama Foundation for Human Science.
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