1Department of Neurology and 2Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington 98195
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ABSTRACT |
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Brown, Angus M.,
Ruth E. Westenbroek,
William A. Catterall, and
Bruce R. Ransom.
Axonal L-Type Ca2+ Channels and Anoxic Injury in
Rat CNS White Matter.
J. Neurophysiol. 85: 900-911, 2001.
We studied the magnitude and route(s) of
Ca2+ flux from extra- to intracellular
compartments during anoxia in adult rat optic nerve (RON), a central
white matter tract, using Ca2+-sensitive
microelectrodes to monitor extracellular [Ca2+]
([Ca2+]o). One hour of
anoxia caused a rapid loss of the stimulus-evoked compound action
potential (CAP), which partially recovered following re-oxygenation,
indicating that irreversible injury had occurred. After an initial
increase caused by extracellular space shrinkage, anoxia produced a
sustained decrease of 0.42 mM (29%) in
[Ca2+]o. We quantified
the [Ca2+]o decrease as
the area below baseline
[Ca2+]o during anoxia and
used this as a qualitative index of suspected Ca2+ influx. The degree of RON injury was
predicted by the amount of Ca2+ leaving the
extracellular space. Bepridil, 0 Na+ artificial
cerebrospinal fluid or tetrodotoxin reduced suspected Ca2+ influx during anoxia implicating reversal of
the Na+-Ca2+ exchanger as a
route of Ca2+ influx. Diltiazem reduced suspected
Ca2+ influx during anoxia, suggesting that
Ca2+ influx via L-type Ca2+
channels is a route of toxic Ca2+ influx into
axons during anoxia. Immunocytochemical staining was used to
demonstrate and localize high-threshold Ca2+
channels. Only 1C and
1D subunits were detected, indicating that
only L-type Ca2+ channels were present. Double
labeling with anti-neurofilament antibodies or anti-glial fibrillary
acidic protein antibodies, localized L-type Ca2+
channels to axons and astrocytes.
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INTRODUCTION |
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In the adult rat optic nerve
(RON), a CNS white matter (WM) tract, extracellular
Ca2+ is required for anoxic injury (Ransom
et al. 1997; Stys et al. 1990b
). Previous
studies have used indirect techniques to measure the relationship
between Ca2+ and injury in WM and to determine
routes of Ca2+ entry. While increased
[Ca2+]i has been shown in
anoxic WM, total elemental Ca2+ (i.e., bound and
unbound) was reported (LoPachin and Stys 1995
). It is
ionized [Ca2+], however, that is of greatest
significance in pathophysiological Ca2+-mediated
injury (Choi 1988
), and this has not been previously studied.
We used ion-sensitive microelectrodes to measure ionized
[Ca2+] in the RON during anoxia. We also used
pharmacological agents and ion channel immunocytochemistry to
critically evaluate mechanisms of Ca2+ entry
during anoxia. In the absence of glutamatergic synapses (Ransom
et al. 1990), Ca2+ influx in WM appears
to proceed via reverse
Na+-Ca2+ exchange, as
blockers of Na+-Ca2+
exchange are protective against anoxic injury (Stys et al.
1992
). Preventing intracellular Na+
accumulation either by replacing Na+ in the
bathing solution or blocking voltage-gated Na+
channels with tetrodotoxin (TTX) is also protective against anoxic injury, suggesting that during anoxia in WM, accumulation of
intracellular Na+ and depolarization cause the
electrogenic Na+-Ca2+
exchanger to run in reverse (Stys et al. 1992
). This
theory was reinforced by immunocytochemical data, suggesting that the
Na+-Ca2+ exchanger was
present at axonal nodes (Steffensen et al. 1997
).
Data showing that Ca2+ channel blockers reduced
anoxic injury (Fern et al. 1995a) provided indirect
evidence that a possible role for Ca2+ channels
in anoxic injury may have been overlooked (Stys et al. 1990a
). The Fern et al. study (1995a)
, however,
did not attempt to localize Ca2+ channels, and
the results have been questioned on methodological grounds (Stys
1998
). While Ca2+ channels have been
shown to be located on peripheral (Elliott et al. 1989
;
Wachtler et al. 1998
) and central (Callewaert et al. 1996
; Scholfield 1988
; Sun and Chiu
1999
) unmyelinated axons, no study has convincingly shown
Ca2+ channels on adult central myelinated axons.
To establish the role of Ca2+ channels in anoxic WM injury, we monitored anoxia-induced changes in [Ca2+]o using ion-sensitive microelectrodes. This allowed us to directly assess the hypothesis that ionized Ca2+ leaves the extracellular space during WM anoxia via Ca2+ channels. We also used electrophysiological and immunocytochemical techniques to determine if Ca2+ channels were present on RON axons. Our results indicated that Ca2+ channels were involved in mediating the toxic suspected Ca2+ influx during anoxia in WM. We also present evidence suggesting that L-type Ca2+ channels, but not other high-threshold Ca2+ channels, are present on RON axons (and astrocytes), implying therefore that suspected anoxic Ca2+ influx may occur directly into axons via L-type Ca2+ channels.
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METHODS |
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Preparation
All experiments were carried out in accordance with the
guidelines for Animal Care of the University of Washington. Long Evans rats, aged 45+ days, were deeply anesthetized with 80%
CO2-20% O2 in an enclosed
chamber and then decapitated. The optic nerves were removed, gently
freed from their dural sheaths, and placed in an interface perfusion
chamber (Medical Systems, Greenvale, NY) (see also Stys et al.
1990b). RONs were maintained at 37°C and perfused with
artificial cerebrospinal fluid (ACSF) bubbled with 95%
N2-5% CO2 (pH 7.45), which
contained (in mM) 153 Na+, 3 K+, 2 Mg2+, 2 Ca2+, 143 Cl
, 26 HCO3
, 1.25 HPO42
,
and 10 glucose. Nerves were allowed to equilibrate for 60 min before
recording commenced. Ca2+-free ACSF was made by
omitting CaCl2 and adding 5 mM ethylene glycol-bis(
-aminoethylether)-N, N,N',N'-tetraacetic acid
(EGTA). Introduction of test ACSF containing 1 mM or 0.5 mM
Ca2+ or 0 Ca2+ occurred 20 min prior to the 1 h period of anoxia to allow the extracellular
space (ECS) and bath solution to equilibrate. In low-Ca2+ solutions containing 0.05 mM
Ca2+, additional MgCl2 was
added to maintain a total divalent ion concentration of 4 mM; 0 Na+ ACSF was made by substituting equimolar
choline chloride for NaCl. A stock solution of 10 mM nifedipine was
dissolved in 100% ethanol and added to ACSF to give a final
concentration of 10 µM. All experiments using nifedipine were carried
out in reduced ambient light.
Nerves were constantly aerated with humidified gas mixture supplied at
2 l/min. Anoxia was induced by switching from the control gas mixture
of 95% O2-5% CO2 to a
mixture containing 95% N2-5% CO2. The chamber was modified (Ransom and
Philbin 1992) to ensure rapid exchange of the ambient air
mixture in the chamber.
Electrodes
Ion-sensitive microelectrodes were made with double-barreled piggyback glass (WPI, PB150F-6). Electrodes were pulled on an upright puller producing tips of about 1 µm in diameter (Narashige, East Meadow, NY; PP83). These were subsequently beveled to a tip diameter of 2-5 µm (Sutter, Novato, CA; BV-10). The tip of the ion-sensitive barrel was filled with hexamethyldisilazine (Fluka, Ronkonkoma, NY; 52619) and baked at 160°C for 1 h. The indifferent barrel was filled with 150 mM NaCl and the ion-sensitive barrel was back-filled with (in mM) 120 NaCl, 3 KCl, 20 HEPES, and 1 CaCl2 adjusted to pH 7.2 with 1 M HCl. The ion-sensitive barrel was filled at the tip by back injection to create a short (100-400 µm) column of Ca2+-sensitive liquid ion sensor (Fluka, Cocktail A 21098). Electrodes were calibrated in a solution containing (in mM) 120 NaCl, 3 KCl, and 20 HEPES and Ca2+ concentrations of 20 µM, 200 µM, and 2 mM. All electrodes were individually calibrated, and only those showing stable, near Nernstian responses (i.e., 25-30 mV) to decade changes in [Ca2+] were used for experimental measurements. Electrodes were re-calibrated after each experiment, and data from electrodes with greater than a 5 mV deviation in response to decade changes in [Ca2+] were discarded. The average between the initial and final calibration responses was used to evaluate the experimental measurement. Ion-sensitive microelectrodes were connected via chlorided wires to an amplifier (Axon Instruments, Foster City, CA; Axoclamp 2A). The ion-sensitive barrel was connected via a high-impedance headstage (HS-2 × 0.0001 M) and the indifferent signal was subtracted from the ion-sensitive signal using a differential amplifier (Stanford Research Systems, Sunnyvale, CA; SRS 560). The signal was amplified 100 times, filtered at 1 Hz, and acquired at 1 Hz. Ca2+-sensitive microelectrodes were inserted into RONs under direct visual guidance. The electrode was adjusted to a position well within the nerve by moving the electrode until the largest possible compound action potential (CAP) was recorded from the indifferent barrel.
A suction electrode back filled with ACSF was attached to the distal end of the nerve and stimulated every 30 s (WPI, Sarasota, FL; Isostim 130). A recording suction electrode filled with ACSF was attached to the proximal end of the nerve to record the CAP, which was evoked by a 125% supramaximal stimulus (50 µs duration). CAPs were recorded from a suction electrode connected to an Axoclamp 2A amplifier, and the signal was amplified 500 times, filtered at 30 kHz, and acquired at 25 kHz. Data were acquired on-line (Axon Instruments, Digidata 1200A) using proprietary software (Axon Instruments, Axotape). CAP area was calculated using pClamp (Axon Instruments), and the ion-sensitive signal was converted to [Ca2+]o using a template created in Excel (Microsoft, Redmond, WA) based on the Nernst equation. Data are presented as means and standard error. Significance was determined by ANOVA using Tukey's post test.
ECS measurements
Changes in ECS volume were measured using
K+-sensitive microelectrodes as previously
described (Ransom et al. 1985). The ion-sensitive electrode was manufactured as described in the preceding text, the only
exception being that K+-sensitive resin (Corning,
San Francisco, CA; 477317) was substituted for the
Ca2+-sensitive resin and the filling solution in
the ion-sensitive barrel was 100 mM tetramethylammonium chloride
(TMA+Cl
). The perfusion
solution was control ACSF containing 1.5 mM TMA+.
Because of its size and charge, TMA+ is
restricted to the ECS (Nicholson and Phillips 1981
).
Corning 477317 resin is much more sensitive to
TMA+ than to K+, thus
voltage deflections of the ion-sensitive electrode reflect changes in
[TMA+]o exclusively
(Ransom et al. 1985
). Variations in ECS volume were
calculated using the following expression
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Immunocytochemistry
Antibodies that specifically recognize the
Ca2+ channel 1 subunits
of class A (anti-CNA1), class B (anti-CNB1), class C (anti-CNC1), class
D (anti-CND1), and class E (anti-CNE1) were used in this study. Their
generation, purification, and characterization have been reported
previously (Hell et al. 1993
; Sakurai et al.
1995
; Westenbroek et al. 1992
; Yokoyama
et al. 1995
). Adult rats were anesthetized with pentobarbital
sodium (Nembutal) and perfused intracardially with 4% paraformaldehyde
in 0.1 M phosphate buffer. The optic nerves were removed immediately
and postfixed for 2 h. The tissue was then cryoprotected in 10%
(wt/vol) sucrose for 12 h and 30% sucrose for 48 h. Sections
(20 µm) were cut on a cryostat, thaw-mounted onto superfrost slides
(Fischer), and stored at
80°C. Sections were then warmed at room
temperature or in a 37°C oven for 30 min before being processed for
immunocytochemistry. Tissue sections were rinsed in 0.1 M phosphate
buffer for 5 min, fixed in 4% paraformaldehyde for 20 min, rinsed in
0.1 M phosphate buffer for 5 min, rinsed in 0.1 M Tris buffer (TB) for
15 min, rinsed in 0.1 M Tris buffered saline (TBS) for 15 min, and then blocked in TBS containing 10% nonfat milk for 30 min. The tissue sections were then incubated in affinity-purified anti-CNA1, anti-CNB1, anti-CNC1, anti-CND1, or anti-CNE2 antibodies (all were diluted 1:15)
overnight at 4°C. All antibodies were diluted in a solution containing 0.02% Triton X-100 and 10% nonfat milk in 0.1 M TBS. The
tissue sections were rinsed for 30 min in TBS and incubated in
biotinylated goat anti-rabbit IgG (Vector, Burlingame, CA) diluted
1:300 for 2 h at room temperature. The tissue sections were then
rinsed with TBS for 30 min and then incubated in avidin D-fluorescein (Vector, Burlingame, CA) diluted 1:300 for
2 h at room temperature. The sections were rinsed in TBS for 10 min, rinsed in TB for 20 min, coverslipped with Vectashield, sealed with nail polish, and viewed with a Bio-Rad MRC 600 confocal microscope located in the W. M. Keck Imaging Facility at the University of Washington. Double-labeling studies were carried out as described in
the preceding text except the tissue sections were incubated in
anti-CNC1 or anti-CND1 antibodies and anti-neurofilament antibodies (diluted 1:100; Dako, Carpinteria, CA) simultaneously. Other tissue sections were incubated simultaneously with anti-CNC1 or anti-CND1 antibodies and anti-glial fibrillary acidic protein (GFAP) antibodies (diluted 1:300; Chemicon, Temecula, CA). For double labeling, the
anti-CNC1 and anti-CND1 antibodies were detected as described in the
preceding text. The anti-neurofilament and anti-GFAP antibodies were
detected using anti-mouse IgG labeled with Texas Red (diluted 1:75).
The Texas-Red-labeled antibodies were incubated simultaneously with the
biotinylated goat anti-rabbit IgG or the avidin
D-fluorescein. Control sections were incubated in normal
rabbit serum or the primary antibody was omitted or the anti-CND1 and
anti-CNC1 antibodies were preincubated with their respective peptide
(20 µM). In all cases, no specific staining was observed.
Image collection
Confocal sections with an optical thickness of 1 µm were collected using a Biorad MRC 600 confocal microscope. For single labeling, the images were collected using a single channel, which excites at 488 nm. Double-labeled images were collected using dual excitation at 488 and 548 nm. The images were captured using a Kalman filter. The images were processed (i.e., cropping, image size, merging of images etc.) in Adobe Photoshop (5.0) and then placed into Canvas (5.0) to create composite pictures.
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RESULTS |
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[Ca2+]o in RON
The standard insult used in these experiments was a 1-h period of anoxia. The extracellular Ca2+ concentration ([Ca2+]o) was recorded for up to 30 min prior to the anoxic insult to ensure a stable baseline and for 1 h of re-oxygenation after the insult, the recovery period. The CAP was also recorded over this period of time by a suction electrode.
The initial [Ca2+]o
recorded in the optic nerve perfused with control ACSF containing 2 mM
Ca2+ was 1.45 ± 0.15 (SE) mM
(n = 11), a concentration that remained stable for
several hours. The fact that ionized [Ca2+] was
less than total [Ca2+] was due to the presence
of HCO3. Ionized
[Ca2+] was the same as total
[Ca2+] in
HCO3
-free, HEPES-buffered solution. It has
been reported that HCO3
buffers about 25% of
free Ca2+ (Schaer 1974
), agreeing
with our measurements of
[Ca2+]o in
HCO3
-buffered calibration solution and in
the HCO3
-buffered nerve. Anoxia causes an acid
shift of ECS pH (to approximate pH 6.9) (Ransom et al.
1992
). As this pH change would affect the amount of free
HCO3
, it could possibly affect the buffering
capacity of the ACSF during anoxia. We monitored
[Ca2+]o continually in
ACSF-buffered to pH 7.45 and then introduced ACSF that was buffered to
pH 6.9. There was no effect on
[Ca2+]o, indicating that
changes in [Ca2+]o during
anoxia were independent of pH changes within this range (data not
shown). This is consistent with the fact that pH changes between 6.9 and 7.45 should only produce nanomolar changes in [HCO3
].
[Ca2+]o falls during anoxia
The effects of 1 h of anoxia on [Ca2+]o and CAP area are shown in Fig. 1. Soon after the onset of anoxia, [Ca2+]o transiently increased followed by a sustained decrease that lasted the duration of the anoxic insult. CAP function declined within the first 5 min of anoxia and was completely lost within 10 min. In control ACSF containing 2 mM [Ca2+], [Ca2+]o increased to an average peak of 1.89 ± 0.12 mM from a baseline of 1.45 ± 0.15 mM during the first 10 min of anoxia, followed by a decrease to a stable level of 1.03 ± 0.25 mM. On return to normoxia, [Ca2+]o increased toward the preanoxia baseline level (1.43 ± 0.18 mM, n = 11, Fig. 1A). A 1-h recovery period restored CAP function to 31.8 ± 10.6% (n = 6) of control, indicating that irreversible injury had occurred (Fig. 1B). The same pattern of [Ca2+]o and CAP changes was seen in nerves perfused with lower bath [Ca2+] (i.e., 0.5 mM or 1 mM). Baseline [Ca2+]o during perfusion with 1 mM [Ca2+] was 0.77 ± 0.02 mM. Anoxia induced an increase in [Ca2+]o to 0.98 ± 0.08 mM followed by a decrease to 0.53 ± 0.06 mM, which increased to 0.72 ± 0.08 mM on return to normoxia (n = 4, Fig. 1C). CAP recovery was significantly greater than seen in control ACSF (53.7 ± 1.5 vs. 31.8 ± 10.6%: P < 0.05: n = 4, Fig. 1D). RONs perfused with bath Ca2+ of 0.5 mM registered [Ca2+]o of 0.37 ± 0.02 mM. A 1-h period of anoxia produced an initial increase to 0.47 ± 0.06 mM followed by a sustained decrease to 0.21 ± 0.05 mM (n = 4, Fig. 1E). CAP function recovered to 62.3 ± 3.2% (n = 4, Fig. 1F), significantly greater (P < 0.01) than recovery in control ACSF. RONs perfused with Ca2+-free ACSF (5 mM EGTA, no Ca2+ added) for 20 min prior to the introduction of anoxia had [Ca2+]o of between 1 and 10 µM. This drop in [Ca2+]o was accompanied by a reversible fall in CAP area to 74.0 ± 3.2% (n = 5) of control CAP area in 2 mM bath Ca2+ (Fig. 1H), and this was maintained at this level for up to 1 h of exposure to Ca2+-free solution (data not shown). Anoxia in the presence of the Ca2+-free ACSF resulted in a minor fluctuation in [Ca2+]o (Fig. 1G). At the end of the anoxic insult, control ACSF was reintroduced and [Ca2+]o rapidly rose to 1.40 ± 0.17 mM (n = 5) and CAP area recovered to 96.6 ± 10.5% (n = 5, Fig. 1H) of control, indicating that anoxia-induced injury to the adult RON requires the presence of extracellular Ca2+.
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The drop in [Ca2+]o during anoxia indicated movement of Ca2+ into an intracellular compartment. We used the time-magnitude integral of [Ca2+]o decrease below baseline during anoxia as a qualitative index of net suspected Ca2+ influx (Fig. 1, A, C, and E, shaded area). The integral of [Ca2+]o decrease during anoxia declined as the bath [Ca2+] declined (0.02 ± 0.02, 3.89 ± 0.69, 7.46 ± 2.35, or 18.67 ± 1.30 mM/s in 0, 0.5, 1, or 2 mM Ca2+, respectively). The integral of [Ca2+]o decrease, our proxy measurement for net Ca2+ influx during anoxia, was inversely related to the degree of CAP recovery (Fig. 3C). In other words, the degree of CAP recovery could be predicted by the amount of suspected Ca2+ influx during anoxia.
ECS changes during anoxia
To determine whether the initial increase in [Ca2+]o during anoxia was due to ECS volume changes, experiments were carried out to directly measure ECS changes during anoxia. Anoxia (10 min) increased [Ca2+]o from 1.51 ± 0.01 to 1.98 ± 0.08 mM followed by a decrease to 1.52 ± 0.04 mM on return to normoxia (Fig. 2A, n = 4). To measure changes in ECS volume, nerves were bathed in ACSF containing 1.5 mM TMA+. Baseline [TMA+]o concentration was 1.51 ± 0.02 mM. Anoxia (10 min) produced an increase in [TMA+]o to 2.02 ± 0.04 mM, which fell to 1.57 ± 0.04 mM on return to normoxia (Fig. 2A, n = 4). This corresponded to a 25% reduction in ECS volume, and this anoxia-induced ECS shrinkage had a time course that mirrored that of the [Ca2+]o increase. Presuming that the apparent increase in [Ca2+]o was a result of ECS shrinkage, the calculated ECS shrinkage, 24%, was nearly the same as determined from TMA+ measurements.
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The true onset of [Ca2+]o decrease was obscured by ECS shrinkage as discussed in the preceding text. We estimated the "real" time course of [Ca2+]o change by compensating for the time course of ECS shrinkage. The observed change in [Ca2+]o (n = 11) and the change in [TMA+]o (n = 4) are shown in Fig. 2B. The adjusted [Ca2+]o trace is shown in Fig. 2C. The transient [Ca2+]o fluctuations in the adjusted time course at the onset and conclusion of anoxia are probably artifacts related to the limitations of this estimation technique. The adjusted [Ca2+]o time course relative to CAP area illustrates that CAP fell slightly earlier than the initiation point of [Ca2+]o decrease.
[Ca2+]o and blockade of Na+-Ca2+ exchange during anoxia
If blockade of reverse
Na+-Ca2+ exchange protects
WM by blocking Ca2+ uptake (Stys et al.
1992), we would predict a reduction in suspected Ca2+ influx during anoxia. To test this
prediction, we used bepridil (50 µM), a compound known to inhibit
both forward and reverse Na+-Ca2+ exchange
(Stys et al. 1992
), which has been used extensively to
investigate the neuroprotective effects of inhibiting the
Na+-Ca2+ exchanger
(Fern 1998
; Lehning et al. 1996
;
Stys and Lopachin 1998
; Stys et al.
1992
). With bepridil present throughout the experiment, we
found no significant effect on the initial increase in
[Ca2+]o (from 1.56 ± 0.07 to 1.98 ± 0.28 mM), but the maximum decrease in
[Ca2+]o was significantly
smaller than under control conditions (1.27 ± 0.14 mM;
P < 0.05; n = 6, compared with
1.03 ± 0.25 mM) as was the integral of
[Ca2+]o decrease
(11.03 ± 2.27 compared with 18.67 ± 1.30 mM/s, Fig. 3A). Bepridil also
significantly improved CAP recovery to 47.6 ± 6.5%
(n = 6: P < 0.05; Fig. 3B).
To further study the role of reverse
Na+-Ca2+ exchange in
anoxia-induced changes in
[Ca2+]o, experiments were
carried out in ACSF that contained no Na+; 0 Na+ ACSF was present throughout the entire
experiment. This prevents the buildup of intracellular
Na+ necessary for the exchanger to operate in
reverse mode (Stys et al. 1992
) and should, therefore
attenuate the anoxia-induced [Ca2+]o fall compared
with control. In ACSF containing 0 Na+, anoxia
produced no increase in
[Ca2+]o (1.60 ± 0.05 mM), and there was no significant fall in
[Ca2+]o below the
baseline level of 1.60 ± 0.07 mM. On return to normoxia, there
was a rapid transient increase in
[Ca2+]o to 2.18 ± 0.14 mM, which gradually returned to baseline level, 1.48 ± 0.07 mM, after 1 h of recovery (n = 4). The integral of [Ca2+]o decrease in the
absence of Na+ was minimal compared to the
control situation (0.70 ± 0.32 compared with 18.67 ± 1.30 mM/s; Fig. 3A). Experiments to determine if perfusing 0 Na+ ACSF during anoxia affected CAP recovery,
employed a slightly different protocol than described for
[Ca2+]o measurements.
Twenty minutes prior to anoxia, 0 Na+ ACSF was
perfused and was replaced by control ACSF 15 min after the end of the
1-h period of anoxia. Under these conditions, CAP recovery was
significantly increased to 82.4 ± 7.1% compared with control
(n = 6, P < 0.0005, Fig.
3B).
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Na+ loading should also be prevented by
Na+ channel block. Tetrodotoxin (TTX; 100 nM) was
added to the ACSF to prevent Na+ entry via
voltage-gated Na+ channels. A 1-h period of
anoxia did not significantly affect the initial increase in
[Ca2+]o (from 1.49 ± 0.03 to 1.87 ± 0.05 mM), but the maximum decrease in
[Ca2+]o was significantly
smaller than under control conditions (1.23 ± 0.05 mM;
P < 0.05; n = 4, compared with
1.03 ± 0.25 mM), as was the integral of
[Ca2+]o decrease
(3.99 ± 0.64 compared with 18.67 ± 1.30 mM/s). Due to the
long washout period for TTX (Stys et al. 1992), no CAPs were recorded in this condition.
[Ca2+]o and blockade of L-type Ca2+ channels during anoxia
It has previously been shown that the benzothiazepine class L-type
calcium channel blocker diltiazem protects against anoxic injury in RON
(Fern et al. 1995a). We used diltiazem in preference to
nifedipine due to the concentration dependence of the neuroprotective effects of nifedipine (Fern et al. 1995a
). Diltiazem (50 µM) had no significant effect on baseline
[Ca2+]o (1.49 ± 0.07 mM). The presence of diltiazem during anoxia did not significantly
affect the initial rise in
[Ca2+]o (1.90 ± 0.11 mM), but the sustained fall in
[Ca2+]o was significantly
attenuated compared with control (1.30 ± 0.17 mM,
n = 6, vs. 1.03 ± 0.25 mM in control conditions,
P < 0.05, Fig. 3A). The integral of
[Ca2+]o decrease was also
significantly less than seen under control conditions (7.75 ± 2.79 vs. 18.67 ± 1.30 mM/s, respectively, P < 0.01). CAP recovery in the presence of diltiazem was also significantly greater than control recovery (58.3 ± 8.8 vs. 31.8 ± 10.6%; P < 0.01, n = 6, Fig.
3B).
As neither diltiazem nor bepridil fully protected the RON from anoxic injury, we applied both diltiazem and bepridil together. Perfusion with ACSF containing 50 µM bepridil and 50 µM diltiazem throughout the entire experiment provided greater protection than seen with either drug alone. In combination, diltiazem and bepridil reduced the sustained fall in [Ca2+]o to 1.42 ± 0.11 mM from a baseline of 1.46 ± 0.07 mM (n = 4), a larger attenuation of the fall in [Ca2+]o than seen with either drug alone and significantly higher than in control (P < 0.01) or in the presence of diltiazem (P < 0.05) or bepridil (P < 0.05; Fig. 3A). The integral of [Ca2+]o decrease was significantly less than in control conditions indicated by the shaded green area in Fig. 3A (5.50 ± 2.23 vs. 18.67 ± 1.30 mM/s, respectively, P < 0.01). CAP recovery was significantly increased to 69.9 ± 3.7% compared with control (n = 4: P < 0.005; Fig. 3B). The relationship between the integral of [Ca2+]o decrease and CAP recovery in the presence of blockers of Ca2+ influx was roughly linear (Fig. 3C). The data from experiments where ACSF [Ca2+] was varied during anoxia (Fig. 1) are also included in Fig. 3C. These data reinforced our hypothesis that it was net influx of Ca2+ into intracellular compartments that predicted the extent of RON injury as judged by the CAP.
Are Ca2+ channels present on axons?
The previous results imply that L-type Ca2+
channels play a role in toxic suspected Ca2+
influx during anoxia. To determine whether
Ca2+ channels, which would act as
a direct route of Ca2+ influx, were present on
the axons of the adult RON, electrophysiological and immunocytochemical
techniques were employed. In the RON perfused with control ACSF, the
evoked CAP had the characteristic three peaks previously described
(Fig. 4A) (Foster et
al. 1982). The CAP area was unchanged by switching to low
[Ca2+] (0.05 mM; Fig. 4, A and
B). Addition of 100 nM TTX abolished the CAP, indicating
that the majority of inward current is carried by
Na+ ions through TTX-sensitive
Na+ channels and that Ca2+
channels, if they are expressed at all, are not expressed in sufficient
density to conduct a propagated action potential (Fig. 4C).
As anticipated, 10 µM nifedipine, an L-type
Ca2+ channel antagonist, had no effect on the CAP
(Fig. 4D). Ca2+ influx, therefore has
a negligible role in axonal conduction under normal conditions.
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In several mammalian unmyelinated preparations, blockade of a delayed
rectifier K+ current is required before the
Ca2+ contribution to an action potential is
revealed (Scholfield 1988; Wachtler et al.
1998
). Addition of 1 mM 4-aminopyridine (4-AP) increased CAP
duration, the effect being greater on the slower third peak than on the
faster, first peaks (Fig. 5A)
(see Foster et al. 1982
). The large increase in CAP area
caused by decreasing the repolarizing effect of
IK is illustrated in Fig.
5B. Experiments were conducted to determine if inhibition of
IK revealed a
Ca2+ contribution to the CAP. Introducing a
low-[Ca2+] ACSF (50 µM) reversibly reduced
the repolarizing phase of the CAP (Fig. 5, C and
D). In experiments designed to test whether high-threshold
Ca2+ channels contributed to the CAP area, 100 µM cadmium (Cd2+) was added to the ACSF. This
resulted in a significant and reversible decrease in the repolarizing
phase of the CAP (data not shown). To test which subtype(s) of
high-threshold Ca2+ channel was contributing to
the CAP, 10 µM nifedipine was added to the ACSF (Fig. 5, E
and F). This produced a very similar pattern of CAP
reduction to that seen in the low Ca2+ ACSF. We
examined the relationship between nifedipine concentration and
reduction in CAP area, and these data are illustrated in Fig. 5G. We found that increasing concentrations of nifedipine
from 1 to 10 µM produced increased reduction in CAP area. The pattern of inhibition illustrated in Fig. 5G is similar to that seen
for L-type Ca2+ channels in isolated neurons
(Brown et al. 1994
; Regan et al. 1991
).
|
A histogram showing the quantitative decrease in CAP area produced by low-[Ca2+] ACSF, Cd2+, or nifedipine is illustrated in Fig. 5H. Reducing Ca2+ in the 4-AP-containing ACSF decreased CAP area to 80.3 ± 1.3% compared with control (n = 6, P < 0.001). Cadmium (100 µM), which blocks all high-threshold Ca2+ currents, reduced CAP area to 85.2 ± 1.4% compared with control (n = 6, P < 0.001). The specific L-type Ca2+ channel blocker nifedipine reduced CAP area to 83.7 ± 1.5% compared with control (n = 6, P < 0.001). Tukey's post test revealed that there was no significant difference in the reduction of CAP area among the three conditions (low-Ca2+ ACSF vs. cadmium, P > 0.05: low-Ca2+ ACSF vs. nifedipine, P > 0.05: cadmium vs. nifedipine, P > 0.05). These results suggested that because nifedipine, which is specific for L-type Ca2+ channels, produced the same reduction in CAP area as cadmium, which blocks all high-threshold Ca2+ currents, all of the Ca2+ current contributing to the CAP occurred via nifedipine-sensitive L-type Ca2+ channels.
Localization of L-type Ca2+ channels
Immunocytochemical studies were carried out to confirm the
presence of L-type Ca2+ channels in axons located
in the optic nerve. Tissue sections were immunoreactive along the
length of the axons using both anti-CNC1 and anti-CND1 antibodies (Fig.
6, C and D) but no
immunoreactivity was seen using anti-CNA1, anti-CNB1, or anti-CNE1
(Fig. 6, A, B, and E, respectively).
The staining was diffuse along the length of the axon, suggesting that
the channels were not specifically localized at nodes (compared with
staining of Na+ channels) (Rasband and
Shrager 2000). When tissue sections were incubated with
anti-CNC1 antibodies preabsorbed with CNC1 peptide or with anti-CND1
antibodies preabsorbed with CND1 peptide, no labeling was observed
(Fig. 6, F and G, respectively). To confirm that
the antibodies to L-type Ca2+ channels were
actually labeling axons, sections were double labeled with
anti-neurofilament antibodies to demonstrate the pattern of axonal
distribution within the optic nerve. Sections labeled with anti-CNC1
(Fig. 7A) or anti-CND1 (Fig.
7D) antibodies are represented in green, and regions labeled
with anti-neurofilament antibodies are red (Fig. 7, B and
E). The merged images are shown in Fig. 7, C and
F, where regions of co-localization are yellow or orange in
these two images. Intensity of staining with anti-neurofilament was so
intense in large axons that on merging the images with the
Ca2+ channel staining, the disparity between the
intensity in large and small axons resulted in loss of observable
staining in some of the small axons. These studies illustrating the
pattern of co-localization of neurofilament and anti-CNC1 or anti-CND1
antibodies suggest that the L-type Ca2+ channels
containing
1C and
1D
subunits are present in the axons of the RON.
|
|
Because not all of the labeling with antibodies to L-type
Ca2+ co-localized with neurofilament protein,
double-labeling was done with anti-GFAP antibodies to determine if
these channels were also present in astrocytes. In these images the
anti-CNC1 (Fig. 8A) or
anti-CND1 (Fig. 8D) antibodies are represented in green, and
regions labeled by anti-GFAP antibodies are red (Fig. 8, B
and E). Areas of co-localization are depicted in the merged images shown in Fig. 8, C and F. These results
show that anti-CNC1 strongly labeled cell bodies and processes of
GFAP-positive glial cells in RON. In contrast to the results for
anti-CNC1, immunoreactivity for anti-CND1 directed against
1D subunits does not appreciably overlap with
GFAP (Fig. 8, D-F). These results show that
1D is expressed at much lower levels than
1C in the processes of GFAP-positive glial
cells. These findings indicate that L-type Ca2+
channels are differentially expressed in the glia of the RON. The
anti-CND1 antibodies, in contrast with the anti-CNC1 antibodies, minimally labeled the processes of GFAP positive glial cells. These
findings suggest that L-type Ca2+ channels were
also present in astrocytes of the RON.
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DISCUSSION |
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Our results indicated that during anoxia in WM,
Ca2+ from the ECS entered intracellular
compartment(s). Moreover, the magnitude of this movement of
Ca2+ from the ECS was related to the extent of
injury. Two mechanisms of Ca2+ entry were
verified: reverse Na+-Ca2+
exchange and L-type Ca2+ channels. These findings
extend and complement prior studies on the pathophysiology of WM anoxic
injury. Stys et al. (1990b) showed that WM injury
depends on the presence of extracellular Ca2+.
The pattern of ultrastructural changes seen during anoxia are consistent with toxic Ca2+ overload, and these
changes are absent when Ca2+ is omitted from the
perfusate during anoxia (Waxman et al. 1993
). Analysis
of elemental Ca2+ deposition using electron
microprobe techniques shows Ca2+ accumulation in
axons and glia during anoxia (Lehning et al. 1995
). The
technique employed here to measure Ca2+ movements
has two important advantages: it measures ionized
[Ca2+] concentration and it measures
[Ca2+]o changes
continuously in real time. Ionized Ca2+ is of
greatest significance in the pathophysiology of
Ca2+-mediated cell death (Choi
1988
), and the time course of Ca2+
movements is vital in understanding the role of this ion in
anoxia-induced cell destruction.
[Ca2+]o during anoxia
The time course of anoxia-induced Ca2+
movement from the ECS is complicated by an additional anoxia-induced
effect, namely shrinkage of the ECS. This is a phenomenon that has been
reported both in vitro and in vivo (Ransom and Philbin
1992; Syková 1997
). The effect of ECS
shrinkage is to effectively concentrate ions in the ECS, and this
accounted for the increase in
[Ca2+]o that occurred at
the onset of anoxia.
We have estimated the degree of Ca2+ flux from the ECS to intracellular compartment(s) as the time-dependent integral of [Ca2+]o decrease below baseline during anoxia. This qualitative estimate does not take into account the distortion in [Ca2+]o introduced by ECS shrinkage, as discussed in the preceding text, that would tend to underestimate the amount of suspected Ca2+ influx. It is important to emphasize that the ECS is not a closed compartment but is in slow diffusional equilibrium with the bath. As Ca2+ leaves the ECS to enter cells, [Ca2+]o falls, creating a diffusion gradient for Ca2+ from the bath to the ECS. The diffusional entry of Ca2+ from the bath to the ECS will attenuate the fall in [Ca2+]o and also leads to an underestimation of the integral measure employed here. Fortunately, these errors should be uniform among the various conditions tested here. The strong correlation between the integral of [Ca2+]o decrease and CAP recovery indicated, as predicted, that the magnitude of suspected Ca2+ influx was associated with the degree of axonal injury. This correlation held for various bathing concentrations of Ca2+ and in the presence of Ca2+ influx inhibitors, validating the integral of [Ca2+]o decrease as an estimate of suspected Ca2+ influx. Measurable decreases in [Ca2+]o were never seen during periods of physiological activity; for example, 50-Hz stimulation for 60 s produced only transient increases in [Ca2+]o that were due to ECS shrinkage (Brown and Ransom, unpublished data). In WM, therefore [Ca2+]o decreases strongly suggest a pathological influx of Ca2+ into axons and/or glia.
Routes of suspected Ca2+ influx during anoxia
Our data confirmed that
Na+-Ca2+ exchange and
L-type Ca2+ channels participated in
Ca2+ entry. Under normal ionic conditions, the
Na+-Ca2+ exchanger works to
extrude Ca2+ in exchange for
Na+ influx. The electrogenic nature of the
exchanger confers voltage sensitivity such that the direction of
exchange is under the influence of both the trans-membrane
ionic gradients and membrane potential (Stys et al.
1992). During anoxia there is a breakdown in
trans-membrane ionic gradients
([Na+]i and
[K+]o rise), leading to
membrane depolarization (Leppanen and Stys 1997
). These
conditions cause the
Na+-Ca2+ exchanger to
reverse direction leading to Ca2+ influx.
Blockade of Na+-Ca2+
exchange during anoxia should reduce Ca2+ influx
and attenuate the fall in
[Ca2+]o, which was
observed. Immunohistochemical data suggest that the
Na+-Ca2+ exchanger is
located at internodal regions of axons (Steffensen et al.
1997
), but EM studies are required to conclusively demonstrate this.
The involvement of Ca2+ channels in
Ca2+ loading during anoxia in WM has been
controversial (Fern et al. 1995a; Stys
1998
; Stys et al. 1990a
) although the membrane
depolarization necessary to activate these channels occurs in RON
during anoxia (Leppanen and Stys 1997
; Stys et
al. 1993
). Ca2+ channels do not
contribute directly to action potential conduction in RON axons under
control conditions and were thus assumed to be absent. In addition,
initial studies in the adult RON showed that there was no protective
effect of nifedipine or nimodipine during anoxia (Stys et al.
1990a
). However, subsequent data suggested that these drugs
were neuroprotective but that the effects of nifedipine were
concentration dependent (Fern et al. 1995a
). Nifedipine protected against anoxic injury, but at higher concentrations, this
effect decreased. A possible explanation is that nifedipine blocked
neuroprotective anoxia-induced adenosine release (Fern et al.
1995b
). It has been argued that alternative actions of Ca2+ channel blocking drugs explain their
protective effects against anoxia in WM (Stys 1998
). To
assess the involvement of Ca2+ channels in anoxic
injury of RON more critically, we sought evidence of
Ca2+ channels on RON.
Localization of Ca2+ channels
Imaging studies have shown increases in intracellular
[Ca2+] evoked by RON stimulation
(Kriegler and Chiu 1993; Lev-Ram and Grinvald
1987
; Sun and Chiu 1999
). It is difficult to
state with certainty that the Ca2+ transients
originate in the axons rather than the surrounding glial cells due to
both the difficulties of selectively loading axons with dye and
convincingly resolving the cellular components. Our
electrophysiological data indicated that Ca2+
channels are not expressed in sufficient density to propagate an action
potential, which agreed with previous studies (Fern et al.
1995a
; Foster et al. 1982
). We used blockade of
K+ channels to reveal a subtle contribution of
Ca2+ channels to the CAP, a technique used
successfully elsewhere (Elliott et al. 1989
;
Scholfield 1988
; Sun and Chiu 1999
).
Intracellular single axon recordings from adult RON demonstrated the
spike broadening effects of 4-AP, increasing action potential duration
up to 25 ms (Gordon et al. 1988
). We found that
inhibiting suspected Ca2+ influx reduced the
repolarizing phase of the CAP in 4-AP-treated RONs, indicating that
Ca2+ channels do contribute to the currents
producing the CAP, albeit after pharmacological manipulation. Cadmium
is used routinely to completely inhibit all high-threshold
Ca2+ channels. Because cadmium and nifedipine
reduced the CAP by the same amount, we concluded that only L-type
Ca2+ channels were present. The role of axonal
Ca2+ channels is unknown, but
Ca2+ entry could act as an indicator of axonal
activity and play a regulatory role in axonal transport.
Our results indicated that only L-type Ca2+
channels were present in the adult RON; but previous studies in both
adult RON (Fern et al. 1995a) and rat spinal cord
(Imaizumi et al. 1999
) have suggested that
Ca2+ channels other than L-type are present.
These results can be explained as nonspecific inhibitory effects of
SNX-124 or SNX-230 [synthetic
-conotoxin (CgTX) GVIA or synthetic
-CgTX MVIIC, respectively]. It has been demonstrated that at the
high concentrations (1 µM) of the substances used in these studies,
synthetic conotoxins inhibit the
1D subunit of
nifedipine sensitive L-type Ca2+ channels
(Williams et al. 1992
).
The presence of -CTx-GVIA-sensitive N-type
Ca2+ channels in neonatal RON axons has been
suggested (Sun and Chiu 1999
). The axons of the neonate
are unmyelinated at this stage of development, one of the many features
that distinguish neonatal and adult axons (Waxman and Ritchie
1993
). As our data indicate that N-type
Ca2+ channels are not expressed in the adult,
this suggested developmental alterations in Ca2+
channel expression. In retinal ganglion cells, the cell bodies of
origin of optic nerve axons, studies have shown that L-type Ca2+ currents decrease and N-type
Ca2+ currents increase during development
(Schmid and Guenther 1999
). Our results suggest that
reciprocal developmental changes may take place in axons.
The immunocytochemical data demonstrated that only antibodies that
specifically recognize the 1 subunits of classes C (anti-CNC1) and D
(anti-CND1) channels stained the adult RON. The absence of staining for
antibodies that specifically recognize the
1 subunits of classes A,
B, and E showed that P/Q, N, and R Ca2+ channels
containing these
1 subunits, respectively, are absent in this
tissue. Double-labeling studies using anti-neurofilament antibodies,
which labeled only axons, showed a high degree of co-localization,
illustrating that
1C and
1D subunits are located along the length of
the axons. Double-labeling studies also provide evidence that anti-CNC1
antibodies, but not anti-CND1 antibodies, are expressed in the cell
bodies and processes of some GFAP positive astrocytes, suggesting
different functional roles for the two isoforms of L-type
Ca2+ channels in RON.
It is well established that cultured astrocytes from different regions
of the brain express voltage-gated Ca2+ channels
(MacVicar 1984; MacVicar and Tse 1988
)
although expression may depend on the presence of neurons
(Corvalan et al. 1990
) or on factors that increase
intracellular cAMP levels (MacVicar and Tse 1988
).
Regardless, the expression of Ca2+ channels in
cultured astrocytes is fairly well established. Controversy remains,
however, about the presence of voltage-gated Ca2+
channels in vivo. Astrocytes from acute brain slices or acutely dissociated astrocytes from the hippocampus increased their
Ca2+ current following K+
stimulation, and this response was reduced or inhibited by verapamil (Duffy and MacVicar 1996
; Porter and McCarthy
1995
). Other investigators, however, have suggested that
Ca2+ channel antagonists reduce
[Ca2+]i increases in
astrocytes by blocking neuronal release of glutamate, which acts on
astrocytic metabotropic receptors (Carmignoto et al.
1998
). Astrocytes in vivo also upregulate expression of the
1C subunit of L-type
Ca2+ channels after injury (Westenbroek et
al. 1998
). Our study provides the first immunocytochemical
evidence of L-type Ca2+ channels on uninjured
astrocytes in situ.
We have shown that anoxic injury to the adult RON not only requires the presence of extracellular Ca2+ but that the degree of injury can be predicted by the net amount of suspected Ca2+ influx into intracellular compartments. In addition, inhibitors of Ca2+ influx suggest that Ca2+ enters via two separate routes, the Na+-Ca2+ exchanger and L-type Ca2+ channels. Our evidence indicated that L-type Ca2+ channels are present on axons and astrocytes of the adult RON. While axonal Ca2+ channels do not appear to participate in the action potential conduction, they appear to be important during anoxic injury. Inhibition of Ca2+ influx by application of Ca2+ channel blockers may be an effective means of protection against WM injury associated with stroke and spinal cord trauma.
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ACKNOWLEDGMENTS |
---|
We thank S. Agulian for the gift of Corning resin 477317, T. Möller for helpful discussion, and R. Wender for critically reading the manuscript.
This research was supported by National Institute of Neurological Disorders and Stroke Grants NS-15589 (B. R. Ransom) and NS-22625 (W. A. Catterall) and a research grant from the Muscular Dystrophy Association (R. E. Westenbroek) and the Eastern Paralyzed Veterans Association (A. M. Brown and B. R. Ransom).
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FOOTNOTES |
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Address for reprint requests: A. M. Brown, Dept. of Neurology, Box 356465, University of Washington School of Medicine, Seattle, WA 98195 (E-mail: ambrown{at}u.washington.edu).
Received 24 July 2000; accepted in final form 18 October 2000.
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REFERENCES |
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