Calcium imaging in live rat optic nerve myelinated axons in vitro using confocal laser microscopy

Journal of Neuroscience Methods 102 (2000) 165 – 176 www.elsevier.com/locate/jneumeth

Calcium imaging in live rat optic nerve myelinated axons in vitro using confocal laser microscopy Yubo Ren, Andrew Ridsdale, Elaine Coderre, Peter K. Stys * Loeb Health Research Institute, Di6ision of Neuroscience, 725 Parkdale A6enue, Ottawa, Ontario, Canada K1Y 4K9 Received 6 April 2000; received in revised form 14 July 2000; accepted 20 July 2000

Abstract Intracellular Ca2 + plays a major role in the physiological responses of excitable cells, and excessive accumulation of internal Ca2 + is a key determinant of cell injury and death. Many studies have been carried out on the internal Ca2 + dynamics in neurons. In constrast, there is virtually no such information for mammalian central myelinated axons, due in large part to technical difficulty with dye loading and imaging such fine myelinated structures. We developed a technique to allow imaging of ionized Ca2 + in live rat optic nerve axons with simultaneous electrophysiological recording in vitro at 37°C using confocal microscopy. The K+ salt of the Ca2 + -sensitive indicator Oregon Green 488 BAPTA-2 and the Ca2 + -insensitive reference dye Sulforhodamine 101 were loaded together into rat optic nerves using a low-Ca2 + /low-Na+ solution. Axonal profiles, confirmed immunohistochemically by double staining with neurofilament-160 antibodies, were clearly visualized by S101 fluorescence up to 800 mm from the cut ends. The Ca2 + signal was very low at rest, just above the background fluorescence intensity, indicating healthy tissue, and increased significantly after caffeine (20 mM) exposure designed to release internal Ca2 + stores. The health of imaged regions was further confirmed by a virtual absence of spectrin breakdown, which is induced by calpain activation in damaged CNS tissue. Red and green fluorescence decayed to no less than 70% of control after 60 min of recording at 37°C, with the green:red fluorescence ratio increasing slightly by 21% after 60 min. Electrophysiological responses recorded simultaneously with confocal images remained largely stable as well. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Ca2 + ; Caffeine; Axon; Myelin; Confocal microscopy; Oregon Green 488 BAPTA-2; Sulforhodamine 101; Electrophysiology; Fluorescence

1. Introduction Intracellular Ca2 + accumulation is thought to play a key role in mediating neuronal damage from a variety of conditions, such as anoxia/ischemia or trauma (Sweeney et al., 1995; Kristia´n and Siesjo¨, 1996; Stys, 1998). The in vitro rat optic nerve is a popular model for the study of central white matter injury (Stys et al., 1991; Fern et al., 1993; Brown et al., 1998; Stys and Lopachin, 1998; Garthwaite et al., 1999a,b) and has been used to confirm the role of cellular Ca2 + overload as a key step in anoxic and ischemic white matter damage (Stys et al., 1991; Stys and Steffensen, 1996). Elevation of intracellular Ca2 + may originate from * Corresponding author. Tel.: +1-613-7615444; fax: + 1-6137615330. E-mail address: [email protected] (P.K. Stys).

internal compartments such as mitochondria or endoplasmic reticulum (Kostyuk and Verkhratsky, 1994; Babcock et al., 1997; Golovina and Blaustein, 1997; Brustovetsky and Dubinsky, 2000) or result from flux across the cell membrane through channels or transporters (Clapham, 1995; Ghosh and Greenberg, 1995; Berridge, 1998). In optic nerves, there is evidence that injurious Ca2 + overload may occur from influx of extracellular Ca2 + (Stys et al., 1991, 1992; Fern et al., 1995; Stys and Steffensen, 1996; Brown et al., 1998; Stys and Lopachin, 1998) and possibly also from release of intracellular stores (Steffensen and Stys, 1996). Although subcellular Ca2 + changes have been measured in central white matter using electron probe microanalysis (LoPachin and Stys, 1995; Stys et al., 1997), this technique has limited sensitivity for Ca2 + detection and can only measure total (free+bound) elemental Ca2 + . This cation is heavily bound and

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sequestered in cells, and because free Ca2 + is the important agent in cell signaling and injury, the ability to measure free [Ca2 + ] in myelinated axons is a powerful tool for studying physiological and injury responses in central axons. To date, because of the technical difficulties with loading fluorescent Ca2 + indicators into CNS myelinated axons, most Ca2 + imaging studies have been performed on neonatal optic nerves or unmyelinated axons (Kriegler and Chiu, 1993; Callewaert et al., 1996; Lu¨scher et al., 1996; Fern, 1998; Wa¨chtler et al., 1998; Mayer et al., 1999; Edwards and Cline, 1999). To our knowledge, there have been no reports of fluorescent ion imaging in mature mammalian, central myelinated axons at physiological temperature. In the present study, we describe a technique to allow imaging of ionized Ca2 + in live rat optic nerve axons in vitro with simultaneous electrophysiological recording at 37°C using confocal microscopy. By using a Ca2 + -sensitive and a Ca2 + -insensitive dye pair, we are able to generate Ca2 + signals that show reliable relative changes in axoplasmic [Ca2 + ]. Our data provide the first example of direct confocal axonal Ca2 + imaging in live rat optic nerves which will be a powerful technique for studying subcellular Ca2 + dynamics in central mammalian axons under physiological and pathophysiological conditions.

2. Methods

2.1. Materials The green-emitting Ca2 + -sensitive fluorescent probe Oregon Green 488 BAPTA-2 (OGB-2) octapotassium salt, the red-emitting Ca2 + -insensitive reference indicator sulforhodamine-101 (S101) and Alexa-488 and -594 labeled secondary antibodies were purchased from Molecular Probes (Eugene, OR). Stock solutions of OGB-2 and S101 were prepared using double distilled water. Caffeine, N-methyl-D-glucamine, 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC), and antineurofilament 160 (NF160) mouse monoclonal antibody were purchased from Sigma (St Louis, MO, USA). Rabbit antiserum against spectrin breakdown products (SBP) was a generous gift from Dr Robert Siman (University of Pennsylvania).

2.2. Buffers Artificial CSF (aCSF) contained (in mM) NaCl 126, NaHCO3 26, KCl 3.0, NaH2PO4 1.25, Mg2SO4 2.0, CaCl2 2.0, dextrose 10. For the low Ca2 + /low Na + buffer (low CaCSF), designed to reflect axoplasmic Na+ and Ca2 + concentrations (see Section 4), NaCl and CaCl2 were omitted from the above solution, and

126 mM N-methyl-D-glucamine was added instead. The low CaCSF buffer was prepared in a plastic bottle 30 min prior to the experiment. Low CaCSF was then adjusted to pH  7.45 with concentrated HCl. The pH/Ca2 + buffer, which included (in mM): NaCl 100, Tris base 50, EGTA 5.0, CaCl2 3.0, was adjusted to pH 7.4 with concentrated HCl. The free Ca2 + concentration in the pH/Ca2 + buffer was calculated to be 105 nM using Patcher’s Power Tools (written by Francisco Mendez; http://www.wavemetrics.com/TechZone/ User – ThirdParty/ppt.html).

2.3. Tissue preparation Adult Long Evans rats (50–70 days) were anesthetized with 80% CO2/20% O2 and decapitated. The optic nerves, approximately 10 mm in length, were dissected out and an additional 1 mm was cut off from each end after immersion in low CaCSF buffer at 4°C. The nerve was incubated in oxygenated low CaCSF buffer with 40 mM OGB-2 and 100 mM S101 at room temperature for 2 h and then washed in aCSF at 10°C for 1 h to re-establish normal extracellular ion concentrations before being mounted in the perfusion chamber where all recording was performed at 37°C.

2.4. Axonal Ca 2 + imaging and electrophysiology The chamber was mounted on an inverted microscope (Olympus IX 70, 60X, 1.4 NA oil objective) with a confocal laser-scanning system (BioRad 1024). The dye-loaded nerve was placed on the bottom of the chamber with a glass bottom made of a c 0 coverslip, and perfused at 3 ml/min at 37°C with oxygenated aCSF. One end of the nerve was drawn into a suction electrode to evoke compound action potentials with 100 ms constant voltage pulses, while a bipolar surface electrode was gently applied to the opposite end to allow simultaneous recording and confocal imaging (Fig. 1); the electrodes also provided adequate mechanical stabilization of the tissue to prevent movement during image acquisitions. OGB-2 was excited at 488 nm and collected through a 5229 16 nm bandpass filter while S101 was excited at 568 nm and collected through a 630 nm longpass filter. Time-lapse images of live optic nerve axons loaded with OGB-2 and S101 were collected at 1 min intervals in aCSF or with 20 mM caffeine application to induce a rise in axoplasmic [Ca2 + ] by releasing internal Ca2 + stores (Fig. 5A–F). Data analysis was conducted using NIH Image 1.61 (http://rsb.info.nih.gov/nih-image/default.html) and Microsoft Excel on a Macintosh Power PC. The intensities of red and green fluorescence were normalized independently as F/F0 where F is the raw pixel intensity value obtained during the experiment and F0 is averaged baseline fluorescence collected dur-

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ing the first 5 min. The ratio of normalized green to red fluorescence provided an estimate of relative axoplasmic [Ca2 + ] changes, at least partially corrected for photobleaching, dye loss and cell volume changes. The fluorescence signal was not calibrated for absolute [Ca2 + ].

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floating sections of optic nerve were incubated with conjugated anti-NF 160 in Tris buffer for 3 h at room temperature. Sections were mounted and examined on the confocal microscope. OGB-2 in rat optic nerve was excited at 488 nm with images collected at 522 916 nm and Alexa Fluor 594 was excited at 568 nm with fluorescence collected at \ 585 nm.

2.5. OGB-2 fixation in situ and immunohistochemistry EDC was used to fix OGB-2 in situ in previously loaded nerves using a modification of the method by Tymianski et al. (1997). EDC buffer (10 mg/ml in aCSF) was freshly prepared 10 min prior to fixation. OGB-2 loaded optic nerves were equilibrated in the recording chamber at 37°C for 30 min and then immersed in EDC buffer at 4°C for 4 h. The nerves were then washed 3 times in 0.1 M glycine PBS, three times in 0.2% triton X-100 0.1M PBS before being transferred to 20% sucrose solution at 4°C overnight. Anti-NF160 was directly conjugated with Alexa Fluor 594 (Alexa Fluor 594 Protein Labeling Kit, Molecular Probes) and used at a dilution of 1:50. Thirty-micrometer thick

2.6. Immunostaining for spectrin breakdown products and NF160 Nerves were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer for 24 h, transferred into 20% sucrose in 0.1 M phosphate buffer before 30 mm cryostat sections were made. The sections were rinsed three times for 30 min each in Tris buffer, pretreated with cold methanol for 20 min, then rinsed 3×10 min again in Tris buffer, and incubated with 10% Tris–Triton for 30 min followed by 1 h incubation in 4% normal goat serum and 0.1% Tris–Triton. After overnight incubation with primary antibodies (rabbit anti-SBP, 1:5000; mouse anti-NF160, 1:10 000) in 0.1% Tris-Triton+2%NGS, sections were rinsed 3×10 min in Tris buffer, then incubated for 1 h with goat antirabbit Alexa Fluor 488 (1:1600) and goat anti-mouse Alexa Fluor 594 (1:100). The sections were rinsed 3× 10 min in Tris buffer and mounted with Prolong antifade agent (Molecular Probes).

2.7. Caffeine interaction and Ca 2 + sensiti6ity of OGB-2 and S101 To exclude any direct interaction between caffeine and the fluorescent dyes, a 50 ml drop of pH/Ca2 + buffer containing 40 mM OGB-2 and 100 mM S101 was first placed on a coverslip and several control images were acquired on the confocal microscope using the same settings as those for optic nerve studies. A second 50 ml drop of dye-containing pH/Ca2 + buffer and with 40 mM caffeine was added to the first drop while images were collected. The final images represent changes in fluorescence of the dyes induced by stepping caffeine concentration from 0 to 20 mM. To confirm that S101 is relatively insensitive to [Ca2 + ] changes, the same experiment was performed except that 2 ml of 51 mM CaCl2 solution was added instead, producing a step change in [Ca2 + ] from 100 nM to 48 mM (Fig. 6).

3. Results Fig. 1. Diagram illustrating simultaneous optical and electrical recording in live rat optic nerve axon in vitro. (A) Compound action potential recorded from rat optic nerve in vitro using a bipolar surface electrode. (B) Schematic diagram illustrating perfusion chamber, models of surface and suction electrode and laser path.

3.1. Loading of adult optic ner6e axons with OGB-2 salt and S101 A series of image pairs was collected simultaneously from green (522916 nm) and red (\ 630 nm) channels

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at 1 min intervals in a normal control nerve. Fig. 2A shows representative optic nerve axons double stained with OGB-2 and S101, imaged at 600 mm from the cut end. Axons appeared largely red because of very low green fluorescence in healthy control specimens (see Section 4). Tissue imaged more than 800 mm from the

end showed poor fluorescence in both channels because of inadequate dye concentrations. We therefore chose to study areas as far from the cut ends as possible, yet where fluorescence emission was easily detectable, typically between 400 and 800 mm from the end. Capillaries were easily identified by their large size and frequent presence of red blood cells (Fig. 2A ’Cap’), and were typically surrounded by glial processes strongly stained with OGB-2 (Fig. 2A ’G’). The remaining long red linear profiles were taken to be axon cylinders, and this was confirmed immunohistochemically (see below). Fig. 2B shows a graph of normalized fluorescence changes in the red and green channels obtained from normal live optic nerve axons in aCSF at 37°C during 1 h. One image was acquired every 60 s. Although not apparent in the normalized graph, green emission was much weaker than red fluorescence, because of the low resting axoplasmic [Ca2 + ] in healthy fibers. Both channels exhibited a slow decay to no less than 70% of control values at the end of 1 h, with the red channel decreasing slightly more than the green. As a result, the ratio of normalized green/red fluorescence increased by 219 18% (SD; n= 9) after 1 h (Fig. 2B, bottom graph). Compound action potentials were simultaneously recorded by surface electrode during image collection. After a 10–20 min equilibration period, the shape of the compound action potential remained stable for at least 60 min at 37°C in aCSF. The amplitude of peak 1 (see Fig. 2C) increased to 1519 37% of control after 60 min, whereas peak 2 was more stable at 92935% (n=7). The third peak was very variable and often absent altogether.

3.2. Immunohistochemical localization of OGB-2 Because optic nerve contains not only axons, but glial cells (oligodendrocytes, astrocytes, O-2A glial progenitor cells) and capillaries, dyes might have been loaded into glial processes from the cut end as well. To confirm that linear profiles were indeed axons, nerves

Fig. 2.

Fig. 2. Confocal image of live rat optic nerve axons double stained with OGB-2 and S101, and dynamic fluorescence changes in green and red channels. (A) optic nerve double stained with OGB-2 and S101, imaged at 600 mm from the cut end. Glial processes (G) are seen wrapping around capillaries (Cap). The long red linear profiles (arrows) were immunohistochemically confirmed to be axon cylinders (see Section 2). The axons appear largely red because of a virtual absence of green fluorescence at physiologically low [Ca2 + ] (see Section 4). (B) Top graph shows time course of red and green fluorescence changes as normalized mean fluorescence changes (F/F0) in normal live rat optic nerve axons (eleven axons from three nerves). Both channels exhibited a slow fluorescence decay to no less than 70% of control values at the end of 1 h, with the red channel decreasing slightly more than the green. Bottom graph shows the ratio of normalized green/red fluorescence which increased by 21% after 1 h. (C) Representative compound action potentials simultaneously recorded with bipolar surface electrodes.

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Fig. 3. Colocalization of neurofilament and OGB-2 in rat optic nerves in situ. OGB-2 was fixed in dye-loaded optic nerves with the watersoluble reagent EDC, and the nerve was subsequently processed for neurofilament staining. Neurofilament stain was found throughout the optic nerve while fixed OGB-2 was seen only within approximately 800 mm of the cut end because of limited dye diffusion. The majority of fixed OGB-2 (top panel) colocalized with neurofilament staining (bottom panel), indicating that OGB-2 was selectively loaded into axons.

loaded with OGB-2 (but not with S101 to avoid interference with NF160 label) were immunostained with anti-NF160 antiserum. OGB-2 was fixed with the water-soluble reagent EDC (Tymianski et al., 1997) and subsequently processed for NF160 staining. NF160 staining was found throughout the optic nerve as linear profiles, whereas EDC-fixed OGB-2 appeared only within 800 mm of the nerve ends, in agreement with the observations in live unfixed nerves. As shown in Fig. 3, the majority of fixed OGB-2 colocalized with neurofilament staining indicating that broad linear structures loaded with this indicator were axon cylinders. To examine whether the dye loading procedure or mechanical transection of the nerve ends might have damaged the areas selected for imaging, we immunolabeled processed nerves with antiserum specific for calpain-cleaved spectrin breakdown products, a reliable indicator of damaged axons and neurons (RobertsLewis et al., 1994; Bu¨ki et al., 1999). NF160 was used to identify axon cylinders as above. In nerves normally loaded with OGB-2 and S101, then processed for immunohistochemistry but omitting the primary and secondary antibodies, some faint green stain persisted in an area within 30–40 mm from the cut end, which was likely residual OGB-2 dye (Fig. 4A,B). Secondary antibody controls (omitting primary anti-NF160 and spectrin breakdown product antiserum) was free of nonspecific binding (Fig. 4C,D). The green fluorescence near the end (Fig. 4C) is likely residual OGB-2 (compare with Fig. 4A). In Fig. 4E and F, nerves were loaded with OGB-2 and S101 then processed for immunohistochemistry, now including labeling steps for both NF160 (red) and spectrin breakdown products (green). These samples showed well-stained axon cylin-

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ders with NF160 but only very faint spectrin breakdown. The exception was again within 30–40 mm of the nerve end, which could represent staining of spectrin breakdown products, residual OGB-2 fluorescence, or both. As a control, in order to ensure that the spectrin breakdown product antiserum could adequately detect structural damage in our tissue, nerves were subjected to chemical ‘ischemia’ by a 60 min exposure to 2 mM NaCN and 1 mM iodoacetate, agents known to block oxidative phosphorlyation and glycolysis, respectively. Fig. 4G and H confirm that ischemic axons exhibited marked spectrin breakdown throughout the length of the nerve. The red NF160 signal was also noticeably reduced in these nerves compared to non-ischemic controls.

3.3. Caffeine-induced axonal Ca 2 + increase Before analyzing caffeine-induced fluorescence changes, it was necessary to explore the Ca2 + sensitivities of both OGB-2 and S101. Fig. 6A shows that OGB-2 fluorescence increased more than six-fold as [Ca2 + ] was stepped from 100 nM to 48 mM, whereas red emission from S101 remained largely unchanged. The low green fluorescence in control nerves may have been due to either low resting [Ca2 + ] or inadequate loading with OGB-2. To test whether our Ca2 + indicator was adequately loaded into axons and could report axoplasmic [Ca2 + ] changes, 20 mM caffeine was bath applied to release internal Ca2 + stores (Usachev et al., 1993; Tsai and Barish, 1995). S101 fluorescence remained very stable following caffeine treatment (Fig. 5A,B), whereas the Ca2 + -sensitive OGB-2 signal increased to more than twice control. This green fluorescence, reflecting axonal [Ca2 + ] (Fig. 5C,D), began to increase after  3 min of caffeine application and plateaued after 25–30 min (Fig. 5G–J). Wash with aCSF only partially reversed the increase in axonal [Ca2 + ]. The ratio of green to red channels yields a reliable measure of [Ca2 + ] changes (Fig. 5H,J). Given a possible direct interaction between caffeine and fluorescent dyes (Muschol et al., 1999), we tested the effects of 20 mM caffeine on OGB-2 and S101 fluorescence in isolation. Fig. 6B shows that 20 mM caffeine caused a reduction in green fluorescence by about 13%. Red fluorescence rose by about 15%. These changes are consistent with those reported for a related green calcium indicator, fluo-3 (decrease in fluorescence), and S101 by Muschol et al. (1999). Compared to the caffeine-induced fluorescence changes seen in optic axons, the effects of direct interactions between caffeine and fluorescent dyes appear negligible. Indeed, the lack of observed response by S101 to caffeine in loaded nerves (Fig. 5G,I) suggests that this effect is less important in situ.

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Fig. 4. Confocal images of dye-loaded rat optic nerve without or with immunohistochemical staining using antibodies against spectrin breakdown products and neurofilament-160. Left column of images was taken at the end of the nerve section and the right column is 600 mm away from the cut end. (A and B) OGB-2 and S101-loaded nerve was subjected to the entire immunostaining process without primary or secondary antibodies. Some faint green stain was noted within 30–40 mm from the cut end, likely residual OGB-2 dye. (C and D) Secondary antibody controls (omitting primary anti-NF160 and anti-spectrin breakdown product antibodies) showed no nonspecific signal beyond 30 – 40 mm from the end. (E and F) Dye-loaded nerve double-stained for NF160 (red) and spectrin breakdown products (green) showed well stained axon cylinders with NF160 but only very faint spectrin breakdown. This indicates that the dye-loaded nerve was healthy in that very little calpain-mediated spectrin cleavage was detected, other than possibly at the end of the nerve. (G and H) As a positive control, chemical ischemia produced a strong increase in spectrin breakdown and loss of neurofilament antigenicity throughout the length of the nerve, confirming that these antisera are capable of detecting damage to the axonal cytoskeleton.

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Fig. 5. Caffeine-induced axonal Ca2 + imaging in live optic nerves. Single optical sections showing baseline red (A) and green fluorescence (C). The green channel is weak because the fibers are healthy with a low resting [Ca2 + ]. After addition of caffeine to release internal Ca2 + stores, the red channel appears unchanged (B) but green fluorescence more than doubled (D). The Ca2 + -sensitive channel is shown in pseudocolor to better emphasized the Ca2 + rise in panels E and F. Graphs G (twelve axons from three nerves) and I (twelve axons from four nerves) show changes in red and green fluorescence more quantitatively. Several regions of interest within axons (e.g. white arrows, panel A) were selected and fluorescence calculated, showing normalized mean fluorescence changes over time as a function of caffeine exposure. The red channel remains very stable while there is a marked increase in Ca2 + -sensitive green fluorescence, which is only partially reversible after caffeine removal (G). The ratio of green to red channels (H, J) is used to yield a reliable measure of relative changes in axoplasmic [Ca2 + ].

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Fig. 6. Direct effects of Ca2 + or caffeine on OGB-2 and S101. Dyes were dissolved in pH- and Ca2 + -buffered solution which was imaged by confocal microscopy. (A) [Ca2 + ] was stepped from 100 nM to 48 mM (arrow), causing the Ca2 + -sensitive green fluorescence to rise more than six-fold and then saturate. Red fluorescence remained largely unchanged indicating that S101 is Ca2 + -insensitive. (B) Caffeine (20 mM) was added at the arrow, causing minor but sustained effects on the emissions of both dyes. These changes would tend to underestimate Ca2 + changes when green/red fluorescence ratios are considered (see text).

4. Discussion The development of ion-sensitive fluorescent probes has stimulated a large number of studies examining fundamental physiological and pathophysiological processes in living cells (Johnson, 1998; Stricker and Whitaker, 1999; Takahashi et al., 1999). In particular, Ca2 + dyes have allowed very detailed examination of spatio-temporal dynamics of this key ion in neurons and glia (Lev-Ram and Ellisman, 1995; Fern, 1998; Pozzo-Miller et al., 1999; Takahashi et al., 1999). A common difficulty with using Ca2 + indicators is the need to adequately load the cytoplasmic compartment with a sufficient amount of dye to generate detectable fluorescence. This problem was partially solved by the development of acetoxymethyl ester derivatives where the charged groups are temporarily masked, rendering the molecule lipophilic and membrane-permeable; the free indicator is then reconstituted and trapped inside the cell when cleaved by intracellular esterases (Tsien, 1981; Hayashi and Miyata, 1994). This technique can be very effective for cultured or dissociated cells, but

more intact and mature tissue such as brain slices is more difficult to load, often requiring that indicators be introduced through a patch pipette. Adult myelinated axons, particularly small diameter fibers from the mammalian CNS, pose a unique challenge because of their dense and highly lipophilic myelin. Axonal Ca2 + transients have been recorded in non-myelinated fibers (Kriegler and Chiu, 1993; Callewaert et al., 1996; Lu¨scher et al., 1996; Wa¨chtler et al., 1998; Mayer et al., 1999; Edwards and Cline, 1999). Although minute activity-induced changes in fura-2 fluorescence have been described in myelinated optic nerve axons by Lev-Ram and Grinvald (1987), to our knowledge there have been no reports of reliable fluorescent ion measurements in myelinated central axons from a mammalian species. In our earlier experiments with acetoxymethyl dye derivatives, we found that what little dye penetrated the outer nerve sheath was taken up by the myelin, which in turn presented a barrier preventing access of the indicator to the axon itself (results not shown). Alternatively, impaling fine myelinated axons with a sharp electrode is extremely difficult, and applying a patch pipette, as is done for neuronal somata or glia, is impossible in the presence of an intact myelin sheath. For these reasons we elected to use the salt form of the indicators and devised a technique to load the axons through their cut ends. A key requirement therefore is to prevent resealing of the fibers to allow time for the indicator to diffuse into and down the axon cylinder. Resealing of transected myelinated axons is a Ca2 + -dependent process involving the activation of calpain (Xie and Barrett, 1991; Shi and Blight, 1996; Howard et al., 1999). Axotomy causes an increase in axoplasmic [Ca2 + ] and [Na+] at the cut end (van Egeraat et al., 1993; Ziv and Spira, 1995; David et al., 1997); the Ca2 + rise would promote resealing and potentially contribute to structural injury whereas high internal Na+ would disrupt the transmembrane Na+ gradient, in turn disturbing the operation of important Na+-dependent transporters such as Na+/Ca2 + and Na+/H+ exchange, and Na+-dependent glutamate transport (Stys et al., 1991, 1992; Agrawal and Fehlings, 1996; Stys and Steffensen, 1996; Choi and Chiu, 1997; Storozhevykh et al., 1998; Domercq et al., 1999; Khodorov et al., 1999). In order to prevent axon resealing while at the same time minimizing intracellular ionic perturbations, we prepared the loading solution (low CaCSF) with [Ca2 + ] and [Na+] similar to axoplasm. We reasoned that lowering [Ca2 + ] alone would be suboptimal; in a high Na+ loading solution, once axons accumulate excess Na+ ions, they may suffer Ca2 + overload after being returned to normal Ca2 + -containing aCSF. In our low CaCSF solution residual Ca2 + was measured to be :5 mM with no added Ca2 + . With addition of 40 mM OGB-2 (Kd : 580 nm), the final free [Ca2 + ] was calculated to be : 100 nM, a level that approximates [Ca2 + ]

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in neurons and glia (DeCoster, 1995; Stys et al., 1995), and which is low enough to prevent resealing of the transected ends (Howard et al., 1999). Note that for dye loading the nerve ends were always recut in cold low CaCSF to discard portions that were initially cut during dissection and thus may have suffered damage and begun to reseal. Returning the loaded nerves to a high Ca2 + (2 mM) aCSF solution will then promote resealing of the ends thus trapping the indicators inside the axon cylinder. The Oregon Green 488 BAPTA dyes are new indicators designed for use with the 488 nm line of the argon laser. Compared to the older Calcium Green dyes, for example, they exhibit significant advantages in 488 nm extinction coefficient as well as improved photostability. Moreover, the relatively low affinity of OGB-2 for Ca2 + (Kd :580 nm) makes it a good choice for recording large Ca2 + transients, with a good dynamic range of emitted fluorescence. We elected to use a dual-indicator technique for several reasons. OGB-2 emits virtually no fluorescence at free [Ca2 + ] below 100 nM, therefore absence of green fluorescence, in an axon that later responded to a manipulation that increased Ca2 + , indicates an initially healthy fiber with a low resting [Ca2 + ] (e.g. Fig. 5C,D). In this case, a second indicator is required to identify appropriate regions of interest. S101 is an inexpensive water-soluble red-fluorescent polar tracer with strong absorbance and good photostability. Its emission spectrum is well separated from that of OGB-2 eliminating crosstalk between the Ca2 + -sensitive and -insensitive fluorophores. The Ca2 + -insensitive red fluorescence is also useful to partially correct for dye concentration changes due to cell volume changes, and rundown from photobleaching, dye diffusion down the axon, and possible removal from the fiber by anion transporters (Mitsui et al., 1993; Munsch and Deitmer, 1995). This assumes that the rates of loss of OGB-2 and S101 are similar, and in practice the decays are comparable as can be seen near the end of the green and red plots in Fig. 5I, after the caffeine-induced Ca2 + increase has plateaued (the drift in the green channel of control nerves as shown in Fig. 2B cannot be used because the very low absolute signal level, close to background fluorescence intensity, largely represents drift in the black level of the photomultiplier tube rather than loss of green fluorescence, therefore high-Ca2 + images with a brighter green emission must be used). Using a higher affinity dye such as Oregon Green 488 BAPTA-1 made it difficult to select healthy fibers because of the greater baseline fluorescence; with OGB-2, control fibers with strong red but absent green emissions are likely to be healthy and well loaded with both indicators. While the linear profiles that loaded well with both indicators strongly resembled myelinated axons, with brightly stained axoplasm surrounded by a dark rim of

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unlabeled myelin (e.g. Fig. 5A and D), it was necessary to unequivocally confirm the identity of these structures given that optic nerves also contain glia cells (oligodendrocytes, astrocytes, O-2A glial progenitor cells) and capillaries. Indeed, oligodendrocytic processes extend axially parallel with axons (Butt and Ransom, 1989; Ransom et al., 1991; Butt et al., 1994) and could be mistaken for the latter. After standard loading, OGB-2 was fixed in situ and the tissue double stained with antibodies against NF160, which selectively labels axon cylinders (Iwanaga et al., 1989). As shown in Fig. 3, signals from the fixed OGB-2 co-localize very well with NF160-positive axonal profiles, particularly in the larger fibers. This coupled with the fact that oligodendroglial processes do not extend more than 200 mm along the length of the nerve, indicates that elongated profiles imaged 400–800 mm from the end must be axons. Capillaries could be easily identified by their size, frequent red blood cells in their lumens, and green-stained fine glial processes surrounding the vessels (Fig. 2A). Why glial processes readily accumulated only OGB-2 which, in contrast to axons, emitted brightly in control tissue, is unknown. Either resting [Ca2 + ] in glial processes was high, or OGB-2 concentrations were high from robust uptake, or both. Because of the short distance that the indicators could diffuse into the axons, we were limited to studying fibers within 800 mm from the cut ends. It was therefore important to establish that the region being studied was not injured during dissection or by the loading solution. Spectrin is a structural protein found in most cells (Hayes et al., 1995; Beck and Nelson, 1996), and is broken down by calpain in tissue injured by ischemia or trauma in a Ca2 + -dependent manner (Roberts-Lewis et al., 1994; Morimoto et al., 1997; Newcomb et al., 1997; Bu¨ki et al., 1999). Immunostaining for calpain-cleaved spectrin breakdown fragments is therefore a reliable method for detecting structural injury in cells. It was important to duplicate exactly the dissection and loading conditions, including the presence of the Ca2 + -sensitive dye used as a Ca2 + buffer to clamp the trace Ca2 + at physiological concentrations. Before fluorescence immunohistochemistry could be performed, we needed to see how much fluorescence would remain from the S101 and OGB-2 after the loading procedure followed by processing for immunohistochemistry. As shown in Fig. 4A and B, the red S101 signal was completely eliminated by the processing steps, and only a small amount of green fluorescence remained confined to within 30–40 mm of the end. Therefore any signal from immunohistochemical staining would originate from secondary antibodies, except at the nerve end. Normal nerve subjected to dye loading and incubation in the imaging chamber showed robust NF160 staining and very little detectable spectrin damage several hundred microns from the end

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(Fig. 4F). The green label near the nerve end (Fig. 4E) is probably residual OGB-2 and some spectrin breakdown. We did not do additional experiments to distinguish these. The lack of labeling by the spectrin breakdown antibody was a true negative, as this antiserum strongly stained nerves intentionally damaged by in vitro ischemia, a procedure known to cause substantial accumulation of spectrin breakdown products in optic nerve (Jiang and Stys, 2000). The final step was to demonstrate that the Ca2 + probe is able to report changes in axoplasmic [Ca2 + ]. Caffeine is widely used to induce intracellular Ca2 + release from internal stores (Ehrlich et al., 1994; Verkhratsky and Petersen, 1998). Twenty millimoles of caffeine induced a rise in axoplasmic Ca2 + that began within 3 min of application and plateaued after 30 min at a green fluorescence level that was more than twice control. The majority of axons visible in each image was responsive to caffeine administration, and the effect was only partially reversible after 30 min wash, possibly related to slow entry and egress from heavily myelinated fibers. Some axons failed to respond to caffeine. This may have been due to the following reasons: (1) axons may differ in their sensitivity to caffeine or be devoid of caffeine-sensitive Ca2 + pools altogether; (2) the caffeine response might be brief, lasting a minute or two (Schoppe et al., 1997) and therefore not captured by our image acquisition rate; (3) dyes are not loaded evenly in all axons; (4) some axons fail to respond due to damage in the preparation process, though we found little evidence of injury as discussed above. Fig. 6A confirms that OGB-2 exhibits a robust response to changes in [Ca2 + ] whereas S101 is largely Ca2 + -insensitive. It has been reported that caffeine interacts directly with S101 and fluorescent Ca2 + indicator dyes such as Calcium Green (Muschol et al., 1999). It was therefore necessary to exclude a direct effect of caffeine on emitted fluorescence of S101 and OGB-2. As illustrated in Fig. 6B, these interactions were minor, and are consistent with those reported for a related green calcium indicator, fluo-3, and S101 by Muschol et al. (1999). Indeed the small caffeine-induced drop in OGB-2 emission and rise in S101 fluorescence would tend to underestimate any true Ca2 + increases when these two signals are ratioed. Interestingly, the effect of caffeine on S101 fluorescence was not apparent in situ (Fig. 5G and I). This may have been due to a lower effective caffeine concentration in the axoplasm compared to the perfusate, or differences in the behavior of fluorophores in a cytosolic environment compared to an artificial buffering solution, by virtue of differences in polarity, viscosity, ionic strength and protein concentration (Roe et al., 1990; Harkins et al., 1993). In conclusion, we have developed a technique to allow imaging of ionized Ca2 + in live rat optic nerve

axons with simultaneous electrophysiological recording in vitro at 37°C using confocal microscopy. The use of dual indicators yields data that can compensate for dye concentration changes, allowing recordings for at least 1 h at physiological temperature. Although image collection is limited to within 800 mm of the cut ends, this region of the excised optic nerve appears healthy with no evidence of structural damage. This is the first report of confocal ion imaging of mature central myelinated fibers from a mammalian species and will permit detailed analysis of physiological and pathological ionic responses in central axons.

Acknowledgements We would like to thank Mr Victor Boyko from the National Research Council of Canada for performing trace Ca2 + analyses on our solutions. This work was supported by Ontario Neurotrauma Foundation grant c ONAO-99107. PKS is supported by a Career Investigator Award from the Heart and Stroke Foundation of Ontario.

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