Highly Localized Ca²⁺ Accumulation Revealed by Multiphoton Microscopy in an Identified Motoneuron and Its Modulation by Dopamine

Measurement of voltage-induced Ca²⁺ influx in PD neurons

Of the Ca²⁺ indicators tested (Fluo-3, Indo-1, fura-2, and Calcium Green-1) we found Calcium Green-1 to have the most suitable properties for Ca²⁺ imaging in the STG, and we used it in all experiments in which voltage-dependent Ca²⁺ accumulation was measured. It could be loaded reasonably rapidly into the large neurons of the STG, did not leak out over the course of the experiment, and had a sufficient fluorescence intensity change during a single voltage pulse to produce high signal-to-noise data. All dyes examined exhibited binding to internal proteins and membranes, reducing the maximum change in fluorescence possible with a given calcium influx. However, Calcium Green-1-loaded (and fura-2) cells appeared to have a lower fraction of bound, inactive dye. The parameters for dye injection were optimized empirically to provide good signals with minimal effect on the immediate physiological properties of the PD neurons, which were monitored during loading in a rhythmically active preparation. The Calcium Green-1-labeled PD neuron was isolated from chemical synaptic input by bath application of PTX (5 × 10⁻⁶m). To improve the voltage clamp by reduction of electrotonic decay (Yuste et al., 1994), all identified conductances other than Ca²⁺ were blocked by bathing the preparation in TTX (10⁻⁷m), CsCl (5 × 10⁻³m), TEA (2–10 × 10⁻²m), and in some recordings 4-AP (4 × 10⁻³m). Imaging was not started until ∼1 hr after loading to allow for a uniform distribution of dye within the neuron and time for the bath-applied inhibitors to take effect.

By spectrofluorometric measurement, we determined that ∼120 fmol of Calcium Green-1 is iontophoresed out of the electrode during 20 min at −10 nA (our standard dye loading protocol). Although PD cell volumes vary from ganglion to ganglion, we estimated an average volume of ∼8 × 10⁵μm³ (800 nl) based on volumetric analysis of several different image stacks of dye-filled PD neurons. From the amount of dye injected and the cell volume, we estimated that the total concentration of Calcium Green-1 was ∼150 μmin our experiments. Measurements of the fluorescence intensity of iontophoretically loaded PD neurons before and after the addition of a calcium ionophore showed that ∼70% of the total dye concentration was available to dynamically bind Ca²⁺(i.e., not bound to internal membranes or proteins), relative to the expected fluorescence change from resting level to saturation in vitro. From this measurement, we estimated the concentration of “active” Calcium Green-1 in the PD neurons to be ∼100 μm. All preparations were loaded in the same manner, and we assumed that any measured differences in Ca²⁺ influx, kinetics, and accumulation between different experiments was not attributable to variations in indicator concentration.

The resting Ca²⁺ concentration of PD neurons was measured by iontophoretic injection of the emission ratiometric calcium indicator Indo-1. Although internal binding of Indo-1 was severe, limiting the in vivo response of the indicator, we calculated a resting level of 97 nmat a holding potential of −45 mV, assuming ak_d of 250 nm(Grynkiewiecz et al., 1985). The actual in vivo k_d is not known in this system and is a potential source of error in this measurement. Internal binding of the dye to cellular components reduced the measurable emission ratio change during the in vivo calibration procedure (0 Ca²⁺ to saturated dye) to ∼20%, which introduces further error from a reduced dynamic range. Using Calcium Green-1 fluorescence, only relative changes in [Ca²⁺] (usuallyF/F₀) are reported; however, the peak values of Ca²⁺ reached during the depolarizing voltage pulses can be estimated. Using a resting level of ∼100 nmCa²⁺, the in vivo minimum (0 Ca²⁺) and maximum (saturated dye) fluorescence intensities of a typical Calcium Green-1-loaded cell and ak_d of 190 nm, the peak [Ca²⁺] reached during a voltage pulse to 0 mV was ∼400 nm. As with the Indo-1 measurements, the in vivo k_d of Calcium Green-1 in this system is not known and is a potential source of error in this estimate.

The three dimensional structure of the Calcium Green-1-loaded PD neuron was visualized with high spatial resolution to a depth of several hundred micrometers in the living ganglion. Sites of Ca²⁺ influx imaged in this study were usually between 150 and 250 μm below the surface of the preparation, with occasional regions of interest deeper than 350 μm (see Fig.5A). To reconstruct the cell morphology, a full z-series of each investigated neuron was usually collected after the physiological measurements (Fig. 1). We could recognize all the typical morphological features of the PD neuron that were originally described by King (1976b). A single primary process leaves the soma and extends into the neuropil, where it eventually becomes the axon. Within the neuropil, a number of secondary processes branch from the primary process and then branch into higher order processes. Localized on these finer processes are irregularly shaped, enlarged varicosities, which have been demonstrated by electron microscopic studies to be either presynaptic or postsynaptic sites (King, 1976a; see Discussion).

To stimulate Ca²⁺ accumulation, the soma membrane potential was typically voltage clamped from a holding potential of −45 to 0 mV for 200 msec. The data acquisition and analysis is demonstrated in Figure 2. To maximize temporal resolution, we used a line scan mode in which a single line is scanned successively at 2 or 4 msec intervals. The line scans were displayed sequentially to produce a spatiotemporal image (Fig. 2D). The membrane potential was simultaneously recorded on the second channel of scanning microscope to provide an accurate temporal synchronization of the membrane potential (Fig. 2C) and voltage-induced calcium transients (Fig. 2D). Intensity change over time was extracted from regions of the line scan images and fit to Equation 1 given in Materials and Methods (Fig. 2E).

Voltage dependence of the induced Ca²⁺ influx

Considering its size and complex morphology, perfect voltage control of the entire PD neuron is not expected when voltage clamped from the soma. However, using a combination of ion channel blockers (Yuste et al., 1994), we were able to sufficiently reduce the electronic decay from the cell body to very distal arborizations so that we could demonstrate a clear, reproducible voltage dependence of the Ca²⁺ signal. This is shown in Figure4 in which 200 msec voltage pulses of increasing amplitude were applied from a holding potential of −45 mV. Ca²⁺ accumulation can be detected starting at voltages more positive than −40 mV, and the maximum is reached ∼0 to +10 mV. More depolarized voltage steps often led to irreversible loss of signal and were not applied. However, the voltage dependence of the Ca²⁺ accumulation in Figure 4 is similar to the voltage dependence of Ca²⁺currents measured from the PD soma (Johnson et al., 1999) and other stomatogastric neurons (Hurley and Graubard, 1998). Although the absolute voltage might differ somewhat from the measured voltage because of imperfect space clamp of the distal neuritic compartments, these results demonstrate that we had reasonable voltage control of distal regions of the neuron. Hyperpolarizing prepulses (up to 1 sec at −100 mV) did not increase the Ca²⁺signal.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 4.

Voltage dependence of the induced Ca²⁺ influx in a PD neuron. A, Thethree traces show the kinetics of the influx and decay with three different voltage pulse amplitudes marked on the plots (dashed lines represent the voltage pulse). The holding potential is −45 mV. The top trace (no change in voltage) is also shown to demonstrate that photobleaching of indicator dye did not occur during line scan acquisition. B, The maximum Ca²⁺ signal as a function of the voltage pulse amplitude. Voltage pulses (200 msec) of varying amplitude were applied to the soma, and the Ca²⁺ influx was monitored in active regions in the neuropil. Data from four different PD neurons are shown (open and closed circles and triangles).

The voltage-evoked Ca²⁺ accumulation was completely dependent on the extracellular Ca²⁺. Extracellular solutions with low [Ca²⁺] (but not <10–20%) reduced the voltage activated Ca²⁺ accumulation reversibly (n = 3). When Ca²⁺-free solution was applied, the Ca²⁺ response was completely abolished. However, the response usually did not recover, even after prolonged (>1 hr) return to normal saline. Extracellular application of ≥200 μm CdCl₂ also blocked the Ca²⁺ signal.

Localization of points of Ca²⁺ influx

An initial series of experiments was undertaken to determine the spatial distribution of voltage-evoked Ca²⁺ accumulation. Line scans were used to measure voltage-induced Ca²⁺ accumulation at various regions of the neuron, including the cell body and primary, secondary, and terminal arborizations. The maximal amplitude and time course of the voltage-evoked Ca²⁺ signal was not homogenous within the neuron. In general, the amount of voltage-stimulated Ca²⁺ accumulation decreased from distal to proximal with only small and slow voltage-induced Ca²⁺ accumulation in the soma and major primary processes, unless arborizations were located near the soma as in the cell shown in Figure5C. Analysis of the time course and amplitude at different locations in the PD neuron indicated that the Ca²⁺ signal originates mostly from small, spatially restricted sites on neuritic arborizations. Our data suggest that voltage-activated Ca²⁺influx is restricted to morphologically distinct regions of the neurite, which typically have the shape of enlarged varicosities along or protruding from the neurite (Fig.5A,C; see Fig.7A,B) or smaller “bulbs” clustered in regions of small branches (Figs. 2B,5B). Our results correlate well with earlier findings in which regional variations of [Ca²⁺] in rhythmically active stomatogastric neurons were found (Graubard and Ross, 1985; Ross and Graubard, 1989). Using electron microscopy, King (1976a) showed that similar varicosities are specialized regions of chemical presynaptic or postsynaptic contacts usually containing more than one synapse. He did not detect presynaptic and postsynaptic sites in the same varicosity. The varicosities had diameters of up to 10 μm on neuritic processes otherwise only 1–3 μm in diameter. Our hypothesis that the presence of voltage-gated Ca²⁺ channels is primarily restricted to these regions is demonstrated in Figures 2 and 5. In both figures, localization is evidenced by differences in the delay in the onset of Ca²⁺ accumulation on neuritic arborizations measured by separate line scans taken a few micrometers apart along the neurite (Fig. 5B) or from the same line scan taken along a process (Figs. 2B–E, 5A). In Figure 2E, the rise time gets slower and the maximal Ca²⁺ level decreases with increasing distance from the region in which Ca²⁺ influx is assumed (Fig.2E, compare 1, 2), indicating localized Ca²⁺ influx at site 2 (Fig.2B,D). This is demonstrated again in Figure 5, A and B, in which only the rising phase of the Ca²⁺ signal is plotted. At regions along the neurite, but away from varicosities, a delay in the Ca²⁺ rise is clearly seen relative to the start of the voltage pulse (Fig.5A,B). The delay in the rise was phenomenologically quantified using Equation 1 (see Materials and Methods), and the delay time increases with the distance of the line scan from the region of influx. Overall, diffusion in vivois anomalous (Brown et al., 1999). Factors such as morphology and differences in intrinsic Ca²⁺ buffer concentrations will affect the observed kinetics. At short distances (<2 μm) from the influx point, diffusion appears very slow, perhaps because of localized buffering and spatial restriction (Svoboda et al., 1997) (see also Fig. 5). However, over distances more than a few micrometers from the assumed influx point, the delay time becomes a coarse, but useful, indicator of the distance from the actual Ca²⁺ source (Fig.6A).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 5.

Voltage-induced Ca²⁺accumulation is highly localized in PD neurites. A, Image of a neuritic varicosity similar to those described by King (1976a) as a swelling on a process. The image in A is a single optical section of the region under investigation. A line scan image from the region is shown below thex–y image. The bars mark the corresponding regions. The extracted data from these regions during the voltage pulse are shown in A1 and A2illustrating the delay that is seen at regions away (A2) from the site at which Ca²⁺ influx seems to occur (A1). The voltage pulse (0 mV; 200 msec; holding potential of −45 mV) starts at 0 msec. B, Process from a different PD neuron with varicosities branching off the neurite (similar to those shown in Fig. 2). The Ca²⁺ rise during the voltage pulse is shown from three different positions on the neurite and its attached synaptic structures (marked by the color-coded bars 1, 2, and 3). The Ca²⁺ accumulation during a 200 msec voltage pulse is plotted in B1 –B3. The pulse starts at 0 msec. In B1, the plotted data are the average from the two regions indicated by the black bars at position1 in B; both areas had similar rise kinetics and were averaged to reduce the noise. Voltage-gated Ca²⁺ channels seem to be located on the two bulbous varicosities in B (region 1), as evidenced by the fast rise without delay. Further away along the dendrite (sites B2 and B3), the delay-to-rise markedly increased. C, A large varicosity near the soma of a PD neuron. The low-magnification (20× 0.5 NA) image is a projection of 100 sections. Pixel values of the cell body were processed off-line (scaled to low values) to show the connection to the primary neurite. The inserted image is a high-magnification (40× 0.8 NA) view of the varicosity. The plot below the images show the maximal value ofF/F₀ attained during the voltage pulse as a function of distance (color-coded 1.8 micrometer steps) from the tip of the structure.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 6.

Correlation between the delay time and other experimental parameters. A, Delay time as a function of the distance from the presumed point of Ca²⁺ influx (defined by ∼0 delay time) from four PD neurons that had regions of Ca²⁺ accumulation that were demonstrably isolated from other active varicosities along the same neurite.B, Correlation of the rise time with the delay time for 438 measurements taken from 11 different PD neurons. The rise time increases with the distance from the region in which Ca²⁺ influx is assumed as measured by the increased delay time (as expected from diffusion of Ca²⁺).C, Correlation of the decay time with the delay time from 438 measurements taken from 11 PD neurons. Points near the region of fastest Ca²⁺ accumulation consistently exhibit the fastest apparent decay time. Decay rates are highly variable at longer distances from the presumed influx point.

Rise-time time constants obtained by fitting the Ca²⁺ accumulation during the voltage pulse to Equation 1 ranged between 10 and 20 msec at points of presumed influx (defined by a delay parameter of <20 msec) (Fig.6B). The rise times become slower as the distance from the point of presumed Ca²⁺ influx increases (Fig. 6B). Only data taken from line scans at the actual region of the Ca²⁺ source could be fit as a simple exponential rise [1 − exp(−t/τ)]; most line scans required a sigmoidal function with a delay to onset for an adequate description (Fig.5A,B).

The apparent rate at which the Ca²⁺ signal dissipates after a voltage pulse in the STG can depend on several factors, including the rate of extrusion out of the neurite, uptake into internal stores, the internal buffering capacity, the relative fractions of mobile and immobile buffers, and the distance from the region of influx (Sala and Hernandez-Cruz, 1990; Neher and Augustine, 1992; Nowycky-Martha and Pinter, 1993). With discrete regions of influx separated by tens of micrometers, such as found in the STG neuropil, the “blurring” of the induced Ca²⁺pulse caused by diffusion is a primary factor in the slowing of the decay of the Ca²⁺ signal at points further away from the point of influx. This produces an increase in the decay time of the Ca²⁺ signal, the extent of which is further affected by the in vivo buffering capacity of the neurite. In the STG neuropil, the measured decay constants correlate with the distance of the measurement site from influx point. Figure 6C shows a plot of the decay constant against the delay time from 438 line scans performed in 11 different PD neurons. Although the linear correlation between these two parameters is weak, it is clear that, near the varicosity (at which the delay time is minimal), the decay rate of Ca²⁺ is fastest. The decay time at a distance from the assumed Ca²⁺ influx point varied greatly, depending on the particular morphology. Ca²⁺ influx from multiple distant varicosities could also add complexity. This is shown in Figure2E in which the Ca²⁺signal decays much slower from the neurite region marked 1compared with region 2, and the level of Ca²⁺ remains higher at the end of the trace (F/F₀ = 1.4 compared with ∼1.2). This particular region of the PD neuritic tree contained several sites of Ca²⁺ influx (the numerous bulbous structures shown in Fig. 2B), which all contribute to the Ca²⁺ kinetics observed in the neurite at region 1.

Large arborizations were occasionally found at the primary neurite close to the soma (Fig. 5C). Localization of calcium channels to these structures and a reduction of Ca²⁺ diffusion into the neurite is evident, as indicated by both differences in rise kinetics (data not shown) and the spatial dependence of the maximal fluorescence signal (F_max) along the structure and into the neurite (Fig. 5C).

Although voltage-activated fast Ca²⁺accumulation was always restricted to the described varicosities, we found that only 30–50% of these investigated varicosities show voltage-dependent Ca²⁺ accumulation under our voltage-clamp conditions. Many regions that appeared to have the appropriate morphology did not respond to voltage stimulation, although other varicosities within the same neuron, or even on the same neurite, would (see Discussion).

Effective diffusion coefficient of Ca²⁺ in the neuropil estimated from the delay of Ca²⁺ signal rise

In regions in which only a single arborization or a very closely spaced group of arborizations was found, the in vivodiffusion coefficient of free calcium could be estimated. The following assumptions were made. (1) The measured Ca²⁺ influx occurs only from a single region small enough to be considered a point source from several micrometers away. (2) The diffusion of Calcium Green-1 (both calcium-bound and -unbound) is too slow to account for the observed kinetics of fluorescence change. (3) A Fickian 1-D diffusion model applies: D_eff = <r²>/2t, whereD_eff is the effective diffusion coefficient for Ca²⁺, and r is the mean distance traveled in time t. We also assume that the delay time, as defined in Materials and Methods, provides a reasonable estimate of the time (t) in the above equation. Assumption 1 has been demonstrated in the previous section; there are clearly isolated, discrete regions in which Ca²⁺ influx occurs from a small area. We validated assumption 2 by measuring the diffusion of the dye using multiphoton fluorescence photobleaching recovery (Brown et al., 1999). Using this technique, it is possible to measure the diffusion coefficient of the dye in thick samples in vivo. In eight independent measurements in various regions of the neuron, the diffusion coefficient for Calcium Green-1 was on the order of 1–5 μm²/sec, assuming Fickian diffusion and a single diffusion coefficient model. Slow diffusion of Calcium Green-1 was expected in this system based on the relatively long time required for dye equilibration after iontophoresis. Based on these measurements of dye diffusion and the much more rapid time scales observed for spreading of the Ca²⁺ signal, we can neglect the diffusion of Ca-bound dye and assume that the observed delay is attributable entirely to Ca²⁺movement along the neurite.

The calculated values of D_eff for Ca²⁺ 2–10 μm from the presumed Ca²⁺ influx point average 237 ± 24 μm²/sec (n = 6). This is similar to values reported by others for Ca²⁺ in cytosol (Allbritton et al., 1992;Gabso et al., 1997). Calculation of diffusion coefficients from data taken further away from the point of influx (>10 μm) yield larger values of D_eff. The most likely reason for this is that the simple Fickian diffusion model is too simple to explain the complex dynamics for Ca²⁺diffusion in a real neuron. It is unlikely thatD_eff is a constant in the cytoplasm; rather, it can vary with the strength of fixed and mobile buffering capacity, distance from nearby sites of Ca²⁺ entry, etc. (W. Zipfel and P. Kloppenburg, unpublished observations).

Effect of dopamine on Ca²⁺ signals

DA has two, potentially separate, effects on the PD neuron: (1) it decreases the excitability of the PD neuron by increasing two K⁺ currents,I_A andI_O(Ca) (Kloppenburg et al., 1999); and (2) it greatly reduces and sometimes abolishes synaptic transmission from PD output synapses (Johnson and Harris-Warrick, 1990). Whereas the modulation of K⁺ currents may contribute to reducing release, Ca²⁺ currents could be selectively modulated at synaptic nerve terminals in a way that is not easily detectable by voltage clamp from the soma (Johnson et al., 1999). To test this hypothesis, we determined whether DA could modulate the voltage-dependent neuritic Ca²⁺accumulation. We first identified active regions in which rapid Ca²⁺ accumulation occurred and made sure that this voltage-activated Ca²⁺ response was stable over time by applying voltage pulses (of 1 per min) for 5 min. If the response was reproducible over this period of time, we found that the signal amplitude would remain stable for long periods (>1 hr); such regions were then used for studying the effects of 10⁻⁴m bath-applied DA. The principal effect of DA was a reversible decrease in magnitude of the Ca²⁺ signal. An example is shown in Figure 7A–D in which the voltage-induced Ca²⁺ accumulation is shown before, during, and after DA application. The effect was fully developed after ∼10 min of DA application. The Ca²⁺ signals returned slowly to control levels upon return to normal saline, and full recovery was typically observed after ∼30 min of wash.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 7.

Dopamine effect on peak Ca²⁺accumulation. A is an image of the region in the STG neuropil from which the data were obtained. B–Ddemonstrate a reduction of Ca²⁺ accumulation during bath application of 10⁻⁴m dopamine. The red bar in A indicates the area of the line scan that was analyzed in B–D.C shows three representative line scan images before, during, and after application of DA (10⁻⁴m). D, Extracted fluorescence data and fit lines from the line scans of C (for details, see Fig.2). B shows the time course of dopamine-induced reduction of peak Ca²⁺ accumulation. Eachpoint represents the maximal value ofF/F₀ (determined from the fit line) during a 200 msec voltage pulse as shown in C andD. The gray rectangle indicates the time of DA superfusion. E, A region in which voltage-activated Ca²⁺ influx could be reproducibly measured for a long period (>1 hr) but which showed no statistically significant response to dopamine as shown in F.F, Time course of peak Ca²⁺accumulation from the region shown in E. Data are plotted as in B.

To quantify the effects of DA on the voltage-evoked Ca²⁺ signals, we analyzed the peak amplitude, rise time, and decay time of the signal. In 4 of 11 experiments (neurons), we found statistically significant and reversible reductions in Ca²⁺ accumulation (p < 0.025, t test). In these varicosities that had a dopamine response, DA decreased the average peak amplitude of Ca²⁺ accumulation by 16.5 ± 8% [decreases were 22% (p < 0.00002), 22% (p < 0.0000003), 18% (p < 0.00008), and 4% (p < 0.002)]. No statistically significant change in the average time constant for rise and decay was observed. In three of the remaining experiments, DA decreased the voltage-activated Ca²⁺ accumulation, but the effect did not reverse. In three experiments, there was no significant change in the voltage-induced Ca²⁺ accumulation during DA application (Fig. 7F). Finally, in one neuron in which we measured two different sites sequentially, Ca²⁺ accumulation was significantly decreased by DA at one site and unaltered at the other. Based on these results, we postulate that there is a spatially differential effect of DA on the varicosities within a single PD neuron; that is, DA modulates Ca²⁺ entry at certain sites, although others are not affected.

During the DA experiments, we also monitored the cell input resistance with small hyperpolarizing (10 mV) voltage steps or ramps. Under our experimental conditions in which most conductances other than Ca²⁺ were blocked, DA had no measurable effect on the cell input resistance (data not shown). This suggests that the DA effect on Ca²⁺ accumulation was not attributable to a simple increase in electrotonic decay with consequent loss of voltage control of the neurites.