Calcium was elevated by photolysis of caged calcium compounds (DM-nitrophen from Cal-Biochem; nitrophenyl [NP]1-EGTA from Molecular Probes; Nitr-5 from Cal-Biochem). It was not possible to measure calcium levels with Fura-based ratio-imaging in these experiments, because the excitation wavelengths for Fura compounds overlap extensively with the caged calcium compound photolysis wavelengths. Consequently, resting calcium levels, and levels after photolysis, were measured by fluorescence of Calcium-Green-1 (CG-1; Molecular Probes). Initially, the relationship between CG-1 fluorescence and calcium ion concentration in the presence of caged calcium was examined.
In Vitro Calibration of CG-1 Fluorescence versus Calcium Ion Concentration.
In vitro calibration of the Ca2+
-sensitive dye CG-1 fluorescence was carried out under conditions matching, as much as possible, the subsequent photolysis experiments on cultured neurons (see below). Five solutions with different Ca2+
-free, three intermediate Ca2+
levels, and saturated Ca2+
) were prepared by mixing different amounts of Ca-EGTA and K2
-EGTA, each with a total EGTA concentration of 20 mM. These solutions all contained 1.2 mM NP-EGTA and 0.06 mM CG-1 and all were adjusted to pH 7.2 and ionic strength 175 mM by addition of K-MOPS and K-gluconate. Free [Ca2+
] in each solution was calculated using a Kd
for EGTA adjusted for an ionic strength of 175 mM, obtained by linear interpolation of the hydrogen- and calcium-binding constants of EGTA at 100 and 250 mM, and calculating the resultant Kd
at pH 7.2 using the equations of Tsien and Pozzan (1989)
. Free [Ca2+
] was calculated from numerical solution of the equilibrium buffer equations for a mixture of 20 mM EGTA, 1.2 mM NP-EGTA, and 0.06 mM CG-1, using Kd
s of 179, 90, and 190 nM, respectively. These calculations were checked by measuring [Ca2+
] in solutions without NP-EGTA and CG-1, using Ca2+
-selective electrodes (Microelectrodes Inc.), and confirming that the measured [Ca2+
] levels were close to those calculated for such solutions. The volumes of our final calibration solutions were not large enough to permit the use of Ca2+
For calibration measurements, solutions were placed in 20-μm path-length microcuvettes (Vitro Dynamics). CG-1 fluorescence was converted to Ca2+
concentration using the following equation:
where Kd is the dissociation constant for CG-1, and Fmax is the fluorescence level for saturating Ca2+ and Fmin for zero Ca2+. Kd and ratio of Fmax/Fmin were obtained from the calibration curve (Fig. a).
Figure 1 (a) Calibration of CG-1 at pH (7.2) and ionic strength (175 mM) appropriate for grasshopper Ringer and in the presence of 1.2 mM NP-EGTA. Fluorescence intensities are normalized to Fmin. (b) Computed effect of 200-ms photolysis on [Ca2+]i in (more ...)
We also tried to produce a calibration curve for the CG-1/NP-EGTA mixture after photolysis of NP-EGTA by amounts similar to those in our experiments. To do this, we exposed the microcuvettes to large-field illumination using the same optical arrangements as for photolysis in cells, calculating the amount of NP-EGTA photolysis and the expected effect on the free [Ca2+] in each calibration solution, and measuring the CG-1 fluorescence in each Ca2+-buffer solution. Wide-field illumination was used to minimize the rapid reduction in [Ca2+] that would occur after photolysis due to rapid diffusion of uncaged Ca2+ out of the photolysis spot into the large volume of the cuvette.
] should have increased after photolysis, fluorescence dropped in all solutions, apparently due to bleaching of CG-1 by the photolysis illumination. This invalidated our attempt to obtain a CG-1 Ca2+
-calibration curve in the presence of partially photolyzed NP-EGTA. In contrast to these observations, in cells filled with NP-EGTA and CG-1, localized photolysis always led to an increase in CG-1 fluorescence. This is probably because, in nerve axons, unbleached CG-1 would quickly replace locally bleached CG-1 by diffusion, whereas Ca2+
itself would diffuse much more slowly because it is bound to nondiffusible native buffers in cytoplasm. Therefore, the local CG-1 fluorescence would quickly recover to the level appropriate for nonbleached CG-1 and Ca2+
released from NP-EGTA photolysis. There was no easy way to replicate this situation of rapid CG-1 diffusion but restricted Ca2+
diffusion in our calibration experiments. However, the effects of NP-EGTA photolysis on the Ca2+
sensitivity of CG-1 are likely to be modest (Neher and Zucker, 1993
), so we may use the calibration curve without photolysis to get a reasonable idea of the approximate levels of [Ca2+
reached in our experiments.
[Ca2+]i Measurements in Cultured CNS Neurons.
To examine [Ca2+]i elevation induced by photolysis, we next loaded cultured embryonic CNS neurons with CG-1 and caged calcium compounds. The cells were first exposed to membrane-permeable NP-EGTA (2 μM), CG-1 (2 μM), and 0.02% Pluronic (Molecular Probes) for 40 min at room temperature. Then, after washing three times, cells were rested for ~1 h before experiments.
For photolysis, UV light from a 100-W mercury lamp was passed through a 505 dichroic mirror (74100 BS&M; Chroma) and a 60×, NA 1.4 Nikon objective lens, or through a Nikon UV-2A filter block and a Nikon 40×, NA 1.3 objective lens (UV light passage through these combinations was similar). Flash duration, ranging from 100 to 400 ms, was controlled by an electronic shutter (Ludl Electronic Products Ltd.).
After photolysis, Ludl filter wheels were repositioned to place in the light path a ND filter (1–4) to reduce photo damage to cells, and an appropriate excitation filter set (74100 BS&M; Chroma). Fluorescent images were collected by a CCD camera (SenSys; Photometrics), and transferred to imaging software (Metamorph; Universal Imaging). CG-1 fluorescence initially was measured 2–3 s after photolysis, and at 2–3 s or longer intervals thereafter.
The CG-1 concentration loaded into axons of cultured neurons was estimated to be ~60 μM by comparing the resting fluorescence of filled axons to that of a micropipette shank of similar diameter filled with various test concentrations of CG-1 and with [Ca2+
] buffered to ~100 nM with 20 mM of a Ca-EGTA mixture. Acetoxymethyl ester compounds load into cells at rates highly dependent on their molecular weights, which are 1,147 and 654 D for esterified CG-1 and NP-EGTA, respectively. Cytoplasmic NP-EGTA should accumulate ~10–20 times faster than CG-1 (Zhao et al., 1997
), to a level of ~1.2 mM.
To estimate how high [Ca2+
might rise under our experimental conditions, we measured the photolysis efficiency of our illumination spot by opening the field stop iris and exposing a 20-μm path length microcuvette (VitroCom) containing 5 mM DM-nitrophen and 2.5 mM Ca2+
plus 0.1 mM Fluo-3 (Molecular Probes) in 100 mM KCl and 10 mM MOPS, pH 7.2. Exposure duration was varied until a duration was found that just caused a large increase in Fluo-3 fluorescence, measured shortly after photolysis. This provides an estimate of the time needed to photolyze half the DM-nitrophen (Zucker, 1993
), which we found to be 80 ms when a 10% neutral density filter was used to attenuate photolysis light intensity. Assuming equal quantum efficiencies for Ca-free and Ca-bound DM-nitrophen (Ellis-Davies et al., 1996
), we converted the calculated photolysis rate for DM-nitrophen (87/s without the neutral density filter) to a photolysis rate for NP-EGTA (21.6/s) from their relative quantum efficiencies and ultraviolet absorbances (Kaplan and Ellis-Davies, 1988
; Ellis-Davies and Kaplan, 1994
The effect of a 200-ms exposure to this light on the [Ca2+
in a cell was simulated computationally by solving differential equations representing competing buffer reactions, NP-EGTA, and a Ca2+
extrusion process (Fig. b). We included 1.2 mM NP-EGTA and 0.06 mM CG-1 plus 3 mM endogenous Ca2+
buffer (buffer ratio 150:1, Kd
= 20 μM, kon
= 2 × 108
) and a first-order calcium removal process with pump rate 10−4
. Total [Ca2+
] in the mixture was set to produce a resting [Ca2+
of 60 nM. Association and dissociation rates for NP-EGTA were 1.91 × 107
and 1.36 s−1
(from Ellis-Davies et al., 1996
; modified for 175 mM ionic strength). CG-1 had a Ca2+
association rate of 6 × 108
of 119 nM (Fig. a). Fig. b shows the expected elevation in [Ca2+
in a neuronal process exposed to our Hg lamp through our microscope optics for 200 ms. [Ca2+
should rise to ~4 μM at the end of the light pulse, then drop within 3 s to 750 nM, and then gradually back to baseline within 15 s. Since the exact loading of NP-EGTA is uncertain, we also performed calculations assuming a cytoplasmic concentration of 0.6 mM NP-EGTA. In that case, [Ca2+
should rise to nearly 2 μM, and drop within 3 s to 400 nM.
We also estimated the [Ca2+
in cultured CNS neurons using the values of Kd
and ratio of Fmax
from the CG-1 calibration curve (Fig. a) and the measurements of CG-1 fluorescence inside the cultured neurons. The peak Ca2+
level after a 200-ms photolysis was obtained using the following equation:
is the CG-1 fluorescent intensity of the axon at peak [Ca2+
after photolysis, and Fr
is the CG-1 fluorescence at resting [Ca2+
before photolysis. From our measurements of CG-1 fluorescent intensity inside axons in culture after photolysis, the average Fp
was ~1.743 ± 0.075 (± SEM, n
= 3). Minimal CG-1 fluorescence was measured from cells bathed in Ca2+
-free grasshopper saline with addition of 5 mM EGTA and 5 mM ionomycin (Molecular Probes). The average Fmin
was ~0.423 ± 0.066 (± SEM, n
= 3). Fmax
can then be calculated using the ratio of Fmax
from the in vitro calibration. Using Eq. 2
and all the values we obtained for each parameter, [Ca2+
inside cultured axons was ~360 nM.
Similarly, the resting [Ca2+
was estimated using the following equation:
Again, using the values of Kd
from our in vitro calibration and the measurements of minimal CG-1 fluorescence inside actual axons in zero calcium solution, [Ca2+
was ~60 nM from Eq. 3
. Our procedure for estimating Fmin
may overestimate this parameter, so our estimates for [Ca2+
may be somewhat lower than actual values.
[Ca2+]i Analysis in Ti1 Afferent Neurons In Situ.
Ti1 afferent neurons on limb fillets were pressure injected (Narishige USA Inc.) with caged Ca2+ injection solution (50 mM Nitr-5 with 20 mM CaCl2 or 20 mM NP-EGTA with 10 mM CaCl2, 140 mM K Hepes, pH 7.3). To estimate the amount that was injected, solutions also contained 2 mM rhodamine-dextran (Molecular Probes). Rhodamine fluorescence in injected neurons was compared with that of micropipettes of similar diameter containing various known concentrations of rhodamine-dextran. We found that injection with 3–5 pulses (1.0 PSI; 300 ms duration) gave a dilution of ~1/40 in the cell regions selected for photolysis, with an expected Nitr-5 concentration of ~1.25 mM.
From the relative absorbances and quantum efficiencies of DM-nitrophen and Nitr-5, we calculated a photolysis rate of 21.8 s−1 for Ca2+-loaded Nitr-5, and 7.9 s−1 for Ca2+-free Nitr-5. Assuming a Nitr-5 concentration of 1.25 mM and a Ca2+ loading that leaves the resting [Ca2+]i at 60 nM, UV exposures of 100 and 400 ms are predicted to elevate [Ca2+]i to 300 and 900 nM, respectively (Fig. c).
Successfully injected neurons were labeled with 1,1′-dihexadecyl-3,3, 3′,3′-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes) by gently touching the cell body membrane with DiI crystals on an electrode tip (O'Connor et al., 1990
). Within ~10–15 min, the lipophilic dye diffused to the growth cone and labeled all filopodia. For control experiments, Ti1 neurons were either labeled with DiI alone or injected with BAPTA injection solution (50 mM Nitr-5, 20 mM CaCl2
, 140 mM K Hepes, 50 mM K2
BAPTA, pH 7.3) and labeled with DiI. Photolysis for 100, 150, and 200 ms was calculated to elevate [Ca2+
to 98, 111, and 120 nM, respectively (Fig. c). The distribution of flash durations for control experiments, between 100 and 200 ms, was matched to the distribution of flash durations that had induced filopodial extension.
Using a diaphragm to select a small area (20–30 μm in diameter) on the growth cone, nascent axon, or on filopodia, injected cells were illuminated either through the 505 dichroic/60× objective, or the UV-2A filter block/ 40× objective. Starting with a 100-ms flash, photolysis time was increased in 50-ms increments until a morphological response was observed or until a maximum flash length of 400 ms was reached.
Using the CCD camera with a ND1 or ND2 neutral density filter and a Chroma phycoerythrin filter block (41003; Chroma), DiI labeled processes were imaged for 50–200 ms. For growth cones and nascent axons, sets of 3–5 focal planes were taken: the axons are 1–2 μm in diameter; for 3-focal plane sets, one plane was taken through the middle of the axons, and one 1–1.5 μm above and below the axons; for 5-focal plane sets, the additional planes were at least 2 μm away from the axon. Measurement of filopodial length was done using the MetaMorph Imaging System or NIH Image.
To confirm that similar results would be observed in culture, plated neurons were loaded with NP-EGTA (as above), rinsed, and then incubated in 6 μg/ml DiI solution for 15 min. Cells were rested for 1 h before experiments.