Activity-Dependent [Ca2+]i Changes in Guinea Pig Vagal Motoneurons: Relationship to the Slow Afterhyperpolarization

Nechama Lasser-Ross1, William N. Ross1, and Yosef Yarom2

1 Department of Physiology, New York Medical College, Valhalla, New York 10595; and 2 Department of Neurobiology, Life Sciences Institute, Hebrew University, Jerusalem 91904, Israel

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Lasser-Ross, Nechama, William N. Ross, and Yosef Yarom. Activity-dependent [Ca2+]i changes in guinea pig vagal motoneurons: relationship to the slow afterhyperpolarization. J. Neurophysiol. 78: 825-834, 1997. Vagal motoneurons in slices from the guinea-pig brain stem were injected with the fluorescent [Ca2+]i indicators fura-2, furaptra, or Calcium Green-1. Spike-induced fluorescence changes were measured in the soma and dendrites and simultaneously the long-lasting afterhyperpolarization was recorded with a sharp microelectrode in the soma. Na+ spikes or Ca2+ spikes increased [Ca2+]i (measured as a change in indicator fluorescence) in all locations in the soma and dendrites. Each spike in a train of action potentials caused a step increase in fluorescence of about equal amplitude when nonsaturating indicators were used. Peak changes at all locations occurred at the time of the last action potential. Transients measured with low concentrations of Calcium Green-1 or furaptra had a recovery time constant of ~500-1,500 ms in the cell body. The recovery time course was faster in the dendrites than in the soma. The norepinephrine-sensitive, slow afterhyperpolarization (sAHP) had a time to peak of ~800 ms and a recovery time constant of 2-5 s, much longer than the recovery time course of the fluorescence changes. Some of these experiments were repeated on pyramidal neurons from the CA1 region of the rat hippocampus with similar results. In both cell types, the data suggest that the time course of neither the rising phase nor the falling phase of the sAHP, nor the underlying conductance, directly reflects the time course of the [Ca2+]i change. The mechanism connecting the parameters remains unclear. One possibility is that an additional second messenger system is involved.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

An activity-dependent afterhyperpolarization (AHP) can be detected in many neurons. In the mammalian CNS, at least three pharmacologically distinct components have been described (for review see Storm 1993). The longest lasting of these potentials, dependent on a rise in [Ca2+]i, blockable by neuromodulators like norepinephrine, and insensitive to apamin, is prominent in neurons from the dorsal vagal motor nucleus (Hocherman et al. 1992; Sah 1992, 1993; Sah and McLachlan 1991; Yarom et al. 1985), hippocampus (Gustafsson and Wigstrom 1981; Lancaster and Adams 1986; Madison and Nicoll 1982), olfactory cortex (Constanti and Sim 1987), and sensorimotor cortex (Schwindt et al. 1988). In guinea pig vagal motoneurons, this potential has a slow time to peak of ~0.6-1.2 s and decays in 3-8 s (Hocherman et al. 1992; Sah 1993). Although the Ca2+ dependence of the K+ conductance underlying this potential has been well established (Hocherman et al. 1992; Schwartzkroin and Stafstrom 1980; Sah 1992; Zhang et al. 1995), the relationship between the time courses of the [Ca2+]i change and the conductance has been examined only cursorily (Knopfel and Gahwiler 1992; Knopfel et al. 1990). These measurements showed that in CA3 pyramidal neurons somatic [Ca2+]i, detected with fura-2, peaked at the time of the depolarizing potential and recovered with a time course that paralleled the recovery phase of the slow AHP (sAHP). Although these are the only published observations comparing the kinetics, the parallel recovery time courses observed in these early experiments have been accepted as generally true (e.g., Lancaster and Zucker 1994; Sah 1996; Zhang et al. 1995). Consequently, the most common model for the sAHP assumes that the underlying conductance directly depends on [Ca2+]i.

One problem with this simple model is that there is a delayed time to peak of the sAHP compared with the time of maximum [Ca2+]i change. Four theories have been developed to explain this delay. Hocherman et al. (1992) suggested that the process that links the [Ca2+]i with the AHP conductance depends on the square of the [Ca2+]i and has at least one slow rate constant. Sah and McLachlan (1991) suggested that the slow onset of the AHP was due to a comparably slow release of Ca2+ from intracellular stores. Lancaster and Zucker (1994) and Zhang et al. (1995) proposed that the delay was due to the time for diffusion of Ca2+ from the site of entry to the K+ channels underlying the sAHP. Schwindt, Spain, and Crill (1992a) suggested that the dependence of the sAHP on Ca2+ is likely to be indirect, mediated by a Ca2+-dependent enzyme. Although evidence has been presented in support and against each one (see DISCUSSION), they are somewhat contradictory and no clear explanation has emerged.

One kind of experiment that could help clarify these issues is simultaneous measurement of the sAHP and spatial and temporal characteristics of the associated [Ca2+]i changes under conditions where the measuring process does not distort the underlying [Ca2+]i dynamics. To this end, we have performed experiments using several different [Ca2+]i indicators on vagal motoneurons in the guinea pig slice preparation. Our main result is that neither the rising phase nor the falling phase of the sAHP match the dynamics of the [Ca2+]i transient in any part of the cell, suggesting that there is some additional factor regulating the underlying conductance. Similar results were found in pyramidal neurons from the CA1 region of the rat hippocampus, where the sAHP also has been studied. Some of these results have been reported in abstract form (Lasser-Ross et al. 1994).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

For most experiments, guinea pigs (200-400 g) were anesthetized with sodium barbitol, decapitated, and slices (250-300-µm thick) from the brain stem were prepared as previously described (Hocherman et al. 1992). Slices were maintained in oxygenated Krebs solution at room temperature and transferred to a submerged chamber on either a Nikon or Zeiss upright microscope. The composition of the solution was (in mM) 124 NaCl, 5 KCl, 1.2 MgSO4, 1.2 K2PO4, 26 NaHCO3, 2.3 CaCl2, and 10 glucose. In some experiments, we used 300-µm-thick transverse slices from the hippocampus of 100- to 150-g rats prepared as previously described (Callaway and Ross 1995). For these experiments, the composition of the Krebs solution was (in mM) 124 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 26 NaHCO3, 1.24 NaH2PO4, and 10 glucose. Temperature was regulated at 30-32°C. Intracellular recordings were made with sharp microelectrodes (60-120 MOmega resistance) pulled from thick-wall, 1.5-mm-diam glass. Sharp electrodes were used to avoid the potential problem of washout of intracellular buffers and second messengers (see Lancaster and Zucker 1994). For optical recordings on vagal motoneurons, the tips of the electrodes were filled with either 0.7-4.0 mM fura-2, 0.2-0.5 mM Calcium Green-1, or 2.0-8.3 mM furaptra (all from Molecular Probes, Eugene, OR) dissolved in 200 mM KAc. The shanks were filled with 4 M KAc. For measurements on pyramidal neurons, the tips were filled with 0.1-0.2 mM Calcium Green-1 in 200 mM KAc. Cells were filled with indicator by diffusion from the tip and iontophoresis (0.2-0.5 nA hyperpolarizing current for several minutes). The final concentrations inside the cells were unknown, but we tried to inject the lowest amount of each indicator that would give a clear activity-dependent fluorescence signal on our apparatus. In general, higher concentrations of each indicator were needed to detect signals from the dendrites. Fura-2 and furaptra fluorescence were excited and detected using a filter cube containing a 380 ± 5 nm excitation filter, a 410-nm dichroic mirror, and a 495-nm, long-pass emission filter. For measurements of Calcium Green-1 fluorescence the filters were: excitation, 485 ± 10 nm; dichroic, 505 nm; and emission, 515 nm. Far red light was blocked with an additional filter (Kopf C9788). High-speed optical recordings were made with a cooled slow-scan CCD camera (Photometrics, Tucson, AZ) operated in the frame transfer mode (Lasser-Ross et al. 1991). Typical frame intervals were 25-30 ms. The preparation was viewed and fluorescence detected using either a ×25 water-immersion lens (Leitz, FRG) or a ×40 water-immersion lens (Zeiss, FRG).

[Ca2+]i changes are expressed as Delta F/F (in percent) where F is the fluorescence intensity at resting membrane potential (corrected for tissue autofluorescence) and Delta F is the time-dependent change in fluorescence (corrected for bleaching). The bleaching correction was made by subtracting from the time-dependent change the signal from a trial when the cell was not stimulated. This correction was always <2% for a 6-s sweep when Calcium Green-1 was used and <1% when fura-2 was used. Intrinsic changes from uninjected neurons were generally undetectable and were ignored. No attempt was made to calibrate the fluorescence changes in concentration units. To correspond with physiological expectations for the underlying [Ca2+]i changes, Delta F/F is plottedupwards in figures where Calcium Green-1 was used and -Delta F/Fis plotted upwards when fura-2 was used.

The recovery time courses of the fluorescence transients were fit to single exponentials using three parameters---time constant (time to reach 1/e of the peak value), peak value, and final value. In most cases, the best-fit final value coincided with the fluorescence level before stimulation. If there was a significant difference between these values, the data from that cell were ignored. No attempt was made to correct for nonlinearities due to possible saturation of the high-affinity indicators. Saturation would make the recovery time slower than measured with a nonsaturating indicator. Therefore, the calculated optical time constants are possibly overestimates of the true values. The recovery time course of the sAHP also was fit to a single exponential. The starting point was selected arbitrarily at some point after the peak. This choice reduced complications due to the rising phase of the AHP, fast AHP components like IC and the M current, and possible nonlinearities due to the closeness of the peak potential to the reversal potential. For both the spatially averaged [Ca2+]i change and the sAHP there was no theoretical reason to expect that the true recovery time course should be exponential. However, the fits were reasonable and allowed a simple way to compare the time courses quantitatively.

More than 60 vagal motoneurons were measured using fura-2, 36 cells using Calcium Green-1, and 5 cells using furaptra. Confirming experiments on rat pyramidal neurons were made on 10 cells, all with Calcium Green-1.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

In most healthy motoneurons, the soma and dendrites were >50 µm below the surface of the slice. Consequently, light scattering and absorbance in the tissue made it difficult to resolve the thin dendrites of the vagal motoneurons. Some cells, however, had processes that remained close to the surface. Figure 1A2 shows spike-evoked fluorescence changes in the soma and a dendrite of one of these cells that was filled with Calcium Green-1 (regions and cell shown in Fig. 1A1). At the time resolution of our apparatus (25 ms), the fluorescence increases peaked at the time of the action potential. The recovery time course was faster in the dendrites than in the soma. The lower part of the figure shows a different cell filled with fura-2 (Fig. 1B1). The gray scale image (Fig. 1B2) shows the spatial distribution of the change in Delta F/F in this cell, measured at the time of the peak response. There were increases in the soma and all dendritic locations. There were no locations without increases at the resolution of this experiment (each pixel covered an area of 2.86 µm2). Ca2+ spikes, evoked in saline containing 1 µM tetrodotoxin and 10 mM tetraethylammonium, also caused large fluorescence transients all over the cell (data not shown). These two results (rapid time to peak and widespread distribution) were found in all cells examined. Because there was little time for Ca2+ to diffuse from the site of entry, these results imply that there must be voltage-sensitive Ca2+ channels distributed all over the membrane of this cell. This result also was found for Purkinje neurons in the cerebellum (Ross and Werman 1987) and pyramidal neurons in the hippocampus (Jaffe et al. 1992; Regehr and Tank 1992) and cortex (Schiller et al. 1995; Yuste et al. 1994). The higher Delta F/F values in the dendrites compared with the soma probably reflect the fact that the surface-to-volume ratio is higher in the thinner dendritic compartments. It does not imply that there was a greater Ca2+ current density into the processes during the action potentials.


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FIG. 1. A1: fluorescence image of a vagal motor neuron filled with Calcium Green-1. Boxes, regions from where time-dependent traces were taken. Inset: action potential that evoked fluorescence transients. Scales: 20 mV, 20 ms. A2: spike-evokedf l u o r e s c e n c e   t r a n s i e n t s   d e t e c t e d   f r o m   s o m a(shaded) and dendrite (). Sharp rise coincides with action potential. Time constant for recovery in soma is 1.8 s and 0.6 s in dendrites. Spikes were evoked by a brief, 200-ms depolarizing pulse through recording electrode. B1: fluorescence image of another cell filled with fura-2. Inset: train of action potentials that evoked fluorescence signals. Scales: 20 mV, 200 ms. B2: spatial distribution of Delta F/F measured from a time preceding train to time of last spike. There are clear increases at all locations over soma and dendrites. Each pixel covers an area of 2.86 µm2.

To detect signals in the sparse dendrites, we loaded the cells from electrodes containing a relatively high concentration of fura-2 (the actual concentration in the cells is unknown because we used sharp microelectrodes). Although useful for improving the signal-to-noise ratio of the measurements, the high concentration of indicator buffered the activity-dependent transients, slowing the recovery phase and reducing the peak amplitude of the fluorescence transients (Baylor and Hollingworth 1982; Helmchen et al. 1996; Regehr and Tank 1992). To try to reduce the effects of indicator buffering, we measured spike-evoked calcium transients using low concentrations of fura-2 and averaged many trials, low concentrations of the more sensitive indicator Calcium Green-1, and the low-affinity indicator furaptra. Figure 2 shows typical responses in the soma using each indicator.


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FIG. 2. Time course of spike-evoked fluorescence transients in soma detected with furaptra, Calcium Green-1, and fura-2. A1: response to a train of 5 action potentials evoked at 100-ms intervals with 20-ms depolarizing pulses. Cell loaded from a microelectrode containing 8.3 mM furaptra. Optical data average of 20 sweeps. Frame interval 25 ms. Each spike caused approximately an equal increment in fluorescence. B1: response to a train and a single spike in a cell filled from a microelectrode containing 2 mM Calcium Green-1. Spike interval 120 ms. Frame interval 25 ms. Both responses from a single trial. Fluorescence increment from first spike is clearly larger than increment from later spikes. C1: response to a train of spikes evoked at 40 ms intervals in a cell filled from an electrode containing 2 mM fura-2. Optical data average of 5 sweeps. Although individual steps are not visible, response appears to be linear. A2, B2, and C2: time course of fluorescence recovery in cell body of typical neurons. Time constants measured using each indicator were: furaptra (1,370 ms), Calcium Green-1 (1,700 ms), fura-2 (2,570 ms). Simultaneously measured slow afterhyperpolarization (sAHP) in each cell also is shown.

When furaptra was the indicator, the increments after each spike were about the same (Fig. 2A1). The Kd for Ca2+ binding to furaptra is >20 µM (Konishi et al. 1991; Raju et al. 1989), far above the peak spike-evoked [Ca2+]i increase detected in most neurons (e.g., Helmchen et al. 1996). Therefore, the response of this indicator is expected to be linear in [Ca2+]i, and the equal increments in fluorescence for each action potential imply equal amounts of Ca2+ entry. In contrast, when Calcium Green-1 was the indicator, the step for the first action potential was clearly larger than the steps for the later spikes (Fig. 2B1). The Kd for Calcium Green-1 is ~0.24 µM (Eberhard and Erne 1991), in the range of the expected peak values. This suggests that the fluorescence changes using Calcium Green-1 were nonlinear (saturating) when many action potentials were activated close together in time (see also Regehr and Alturi 1995). The fluorescence response measured using fura-2 was linear in the number of action potentials (Fig. 2C1), although the Kd of this indicator (0.23 µM) (Baylor and Hollingworth 1988) is similar to the Kd of Calcium Green-1. The explanation for the difference between the responses using these two high-affinity indicators is probably the higher concentration of fura-2 in the cell, which buffered the transients, preventing the [Ca2+]i changes from reaching the nonlinear range of fura-2 (e.g., Neher and Augustine 1992). Less buffering was found with Calcium Green-1 because we were able to use lower concentration of this more sensitive indicator. This conclusion is consistent with the longer recovery times measured using fura-2 compared with the other two indicators (see below).

Only a few cells were filled with furaptra. The low affinity of this indicator made it difficult to measure responses in the vagal motoneurons with a good signal-to-noise ratio, even with averaging. The recovery time constant measured with this indicator in the experiment in Fig. 2A2 was 1.4 s. This time constant probably reflects some buffering by the indicator because we could not reduce the cytoplasmic furaptra concentration and still measure a signal.

Calcium Green-1 is more sensitive than fura-2 or furaptra. Therefore, we were able to measure fluorescence responses to a single action potential, using a significantly lower concentration of indicator in the pipette (typically, 0.2 mM compared with 0.7 mM for fura-2). The recovery times were slightly faster after a single spike than after five spikes (see also Regehr et al. 1994). The fastest time constant measured using Calcium Green-1 was 0.47 s, and the fastest recovery time constant using fura-2 was ~1.7 s. Figure 4 shows the distribution of time constants for those cells where the recovery time of the sAHP also was measured. The fura-2 time constants were generally slower than the responses measured using Calcium Green-1. Therefore, even at the lowest fura-2 concentration we used, there was buffering of the [Ca2+]i transients. Indeed, Helmchen et al. (1996) found buffering in pyramidal cell dendrites by fura-2 at concentrations <50 µM. We do not know the cytoplasmic concentrations of fura-2 in our experiments. But they were probably higher than this level because the microelectrodes typically contained >= 700 µM indicator.


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FIG. 4. Distribution of recovery time constants for sAHP and fluorescence transient after electrical stimulation. Points for cells containing fura-2 (black-triangle) are scattered along 45° line, suggesting that 2 times are related. Points for cells containing Calcium Green-1 (triangle ) are all significantly to left of line, showing that fluorescence recovery time was always much faster than sAHP with this indicator. However, there is some correlation between 2 rate constants because slower sAHP recovery times are associated with slower optical time constants.

The highest change in [Ca2+]i as a result of Ca2+ current through voltage-sensitive channels is just under the membrane. After the channels close, diffusion will equalize the concentration in different parts of the cell (Eilers et al. 1995; Hernandez-Cruz et al. 1990; Thompson 1994). Until that time, the recovery time course will be faster just under the membrane than at more interior locations. This conclusion also is supported by computational models of Ca2+ transients (e.g., Sala and Hernandez-Cruz 1990). With our nonconfocal apparatus, we could not accurately measure the dynamics of the fluorescence changes at different locations within the soma. The time courses shown in the figures are spatial averages over most of the cell body. Therefore, all the fluorescence records shown in this and other figures are likely to represent an overestimate for the recovery time and an underestimate for the peak amplitude just under the membrane.

Figure 3A1 shows simultaneous measurements of the somatic fluorescence transient and the AHP in experiments using fura-2. Figure 3A2 shows the same data with the AHP inverted to facilitate comparison with the Delta F/F change, and Fig. 3A3 shows the falling phase of both of these traces on a logarithmic scale. For most of the recovery time course, the data lie along straight lines. Later, some deviation appears that could reflect a second component or may just be noise in the baseline. The slope of both lines is about the same, indicating time constants of 3.1 s. The bottom panels show similar data for a cell filled with Calcium Green-1. The fluorescence recovery rate measured with Calcium Green-1 was more than twice as fast as in the cell containing fura-2 and more than twice as fast as the recovery time constant of the sAHP. Figure 4 shows the optical and electrical time constants for all the cells where both constants could be fit to single exponentials. The data for the fura-2-containing neurons are clustered along the 45° line representing equal time constants. The data from Calcium Green-1 filled neurons all show much faster optical than electrical recovery times. There was no significant difference in the recovery times of the sAHP in the cells filled with different indicators. The difference in optical recovery rates probably reflects the different amount of buffering by these two indicators and not a difference in their rate constants. In fact, in other experiments, Helmchen et al. (1996) measured recovery time constants with fura-2 as fast as 50 ms in the dendrites of pyramidal neurons.


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FIG. 3. Comparison between time course of fluorescence change and associated sAHP. A1: responses to 5 action potentials in a cell filled with fura-2. Optical data average of 3 sweeps. Traces displayed in their normal configuration. A2: same traces with sAHP inverted and scaled. A3: log plot of falling phase of both traces. Lines are almost parallel with a time constant for each of 3.1 s. B1-B3: corresponding plots from a cell filled with Calcium Green-1. Only 1 spike was evoked. Optical data average of 5 sweeps. Ordinate for Delta F in arbitrary units; ordinate for AHP in millivolts. Slopes are clearly not parallel. Time constant for fluorescence recovery was 1.5 s, and time constant for the sAHP was 4.3 s. Arrows, times of peak fluorescence change and maximum sAHP.

Measurements of the AHP voltage do not directly show the time course of the underlying conductance. In most experiments, the magnitude of the sAHP was small compared with the difference between the resting potential and the reversal potential. Therefore, the difference between the two time courses is expected to be small. Nevertheless, to estimate the true relationship between the conductance time course and that of the [Ca2+]i change, we repeated the experiments in cells where we varied the resting potential by injecting a constant current. Figure 5A shows a family of spike-evoked AHPs in a cell injected with Calcium Green-1. The AHP has a clear reversal potential (Yarom et al. 1985). The time course of the conductance change was estimated using the procedure developed by Hocherman et al. (1992). Briefly, the current-voltage curves at different times along the AHP were calculated, and the slope of each of these curves was used to estimate the conductance change. This time course is displayed in Fig. 5B, which also shows the time course of the simultaneously measured fluorescence transient for the trial with no holding current. The time course at the other holding potentials was about the same (not shown). The recovery time constant of these fluorescence changes was about twice as fast as the time constant of the conductance change.


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FIG. 5. Comparison between underlying conductance change of sAHP and corresponding fluorescence change measured with Calcium Green-1. A: AHPs after single action potentials when a vagal motoneuron was held at different holding potentials. Estimated holding potential for each trial is indicated next to each trace. There was a clear reversal when cell was hyperpolarized. B: comparison between computed conductance change and fluorescence change evoked with no holding current. Recovery phases were fit to single exponentials(). Time constant for fluorescence recovery was 1.05 ± 0.05 s, and time constant for gAHP was 2.03 ± 0.15 s. Both traces are shown normalized to same peak amplitude. Peak conductance change was 25 nS, and peak fluorescence change was 20%.

Although the time courses of the fluorescence transient and the AHP were not closely matched, there was some connection between the two. As shown in Figs. 4 and 7, the measured time constant of the sAHP appears to be linearly related to the measured time constant of the fluorescence transient, although the slope is different for Calcium Green-1 and fura-2. Thus longer AHPs always were associated with prolonged changes in fluorescence. High concentrations of exogenous buffers like ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) eliminate both the AHP and the [Ca2+]i change (see INTRODUCTION). High concentrations of buffers like fura-2 reduce their amplitudes and slow their time courses as shown in Fig. 6, A and B. The effect of exogenous buffers on the time course of the sAHP has been noted previously in CA1 pyramidal neurons (Lancaster and Zucker 1994; Zhang et al. 1995) and in neurons from the sensorimotor cortex (Schwindt et al. 1992b). These experiments did not measure the time course of the corresponding [Ca2+]i change.


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FIG. 7. Comparison between somatic fluorescence change measured with Calcium Green-1 and sAHP in hippocampal pyramidal neurons. A: responses to 1, 2, and 3 depolarizing stimuli (20 ms duration, each evoking 2 spikes) are shown. Three optical time constants are similar (372, 415, and 449 ms). Two recovery time constants for larger sAHPs are also similar (1,410 and 1,673 ms). Smallest was too noisy to measure. Similarity of electrical time constants and proportional amplitudes of sAHP suggests that peak of sAHP potential was not close to reversal potential. B: scatterplot of optical and electrical time constants in 10 cells where both fluorescence transient and sAHP were measured. Fluorescence recovery was always significantly faster than recovery time of sAHP, in most cases by more than a factor of 3.


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FIG. 6. Fluorescence transient and sAHP are affected by increasing concentration of fura-2. A: spike-induced fluorescence and electrical changes in a vagal motoneuron at beginning of experiment (1) and 80 min later (2). Electrode contained 0.75 mM fura-2. At end, with >5 times indicator in cell (as assayed by resting fluorescence in soma), change in Delta F/F was smaller and much slower, and sAHP was almost eliminated. B: fractional change in these parameters in a different cell as indicator concentration (represented as mean somatic fura-2 fluorescence level at resting potential) increased. Rate constant for fluorescence recovery, Delta F/F amplitude, and AHP amplitude are shown normalized to values at beginning of experiment. Values of all of these parameters were reduced as fura-2 concentration increased.

Measurements on CA1 pyramidal neurons

Many studies examining the pharmacology and kinetics of the sAHP have been made on hippocampal neurons. To determine whether our results were generally valid, we repeated some of these experiments on pyramidal neurons from the CA1 region. In the hippocampal slice preparation, we could find healthy cells close to the surface. Therefore, we were able to use even lower concentrations of Calcium Green-1 and still detect measurable responses after intracellular stimulation. Figure 7A shows three trials from a pyramidal neuron that was filled from an electrode containing ~100 µM Calcium Green-1. A single 20-ms depolarizing stimulus evoked two action potentials followed by a small AHP. The simultaneously recorded fluorescence transient recovered with a time constant <400 ms. Two and three stimulating pulses, producing more spikes, evoked larger AHPs and fluorescence transients. The incremental amplitudes of the second and third transients were smaller than the first, suggesting that the indicator was beginning to saturate---a result similar to that found in vagal motoneurons. The recovery time courses of the fluorescence transients were about the same. With this concentration of indicator, we could not reliably detect signals from the dendrites. However, Callaway and Ross (1995), using electrodes containing 500 µM Calcium Green-1, found that the recovery time at all dendritic locations was <200 ms. Similarly, Helmchen et al. (1996), using minimal concentrations of fura-2, measured a recovery time of <100 ms in the proximal apical dendrite. Therefore, as found in the vagal motoneurons, the recovery time in the soma is slower than in the dendrites.

The recovery time constant of the AHP was much slower than the fluorescence transient, ~1.5 s. A similar time constant was measured for both the two- and three-pulse trials, suggesting that the peak amplitude of the AHP was not close to the reversal potential. Consequently, the underlying conductance change is likely to have a similar time course. Indeed, direct measurements of IsAHP in voltage-clamp measurements (e.g., Madison et al. 1987; Zhang et al. 1995) show similar recovery times.

Figure 7B summarizes measurements on 10 neurons. In all cells, the recovery time of the fluorescence transient was significantly faster than the recovery time of the sAHP, the same result found in vagal motoneurons.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

There are three results in this paper that are particularly relevant to an analysis of the relationship of the sAHP to the change in [Ca2+]i. First, action potentials caused an increase in [Ca2+]i in all locations in the cell, to at least as far as we could detect fluorescence in the dendrites (typically 100-200 µm from the soma). The dendrites in vagal motoneurons are sparse and thin and not all in one plane. Therefore, we could not follow the fluorescence further, although it is known that the dendrites can extend >400 µm (Yarom et al. 1985). In other cell types, it is known that spike-evoked [Ca2+]i changes can be detected out to the tips of the dendrites (Jaffe et al. 1992; Ross and Werman 1987; Schiller et al. 1995). In particular, single Na+ action potentials evoked a clear [Ca2+]i increase at the tips of the apical dendrites of hippocampal CA1 pyramidal neurons (Callaway and Ross 1995; Spruston et al. 1995).

The relatively uniform spatial distribution of fluorescence increases in Fig. 1B2 suggests that Ca2+ channels are distributed uniformly. However, several factors limit the accuracy with which this statement can be made. The spatial dimension of the pixel elements used in these experiments was finite. In the experiment in Fig. 1D, each pixel covered an area of 2.86 µm2. Also, there was light scattering in the tissue because the cell was not on the surface of the slice. Indeed, Fig. 1D shows that the distribution of the fluorescence increases covered an area slightly larger than the boundaries of the soma and dendrites (Fig. 1B1). In addition, Ca2+ can diffuse several microns during the interval after Ca2+ entry at the beginning of stimulation to the time when the image was taken. Therefore, these uniform fluorescence patterns are still in agreement with some heterogeneity in the distribution of Ca2+ channels, on a scale of 3-5 µm. Consistent with this possibility, Thompson (1994), Gola et al. (1990), Hirano and Hagiwara (1989), and O'Dell and Alger (1991) have presented evidence for clustered distribution of Ca2+ channels.

Second, the amount of Ca2+ entry in the soma appeared to be equal for each action potential in a train and the peak Delta F/F at all locations was reached at the end of the train. Equal fluorescence steps were detected using the low-affinity indicator furaptra and high concentrations of fura-2. The nonlinearity in the Calcium Green-1 response is reasonably attributed to saturation of the indicator (see RESULTS). Equal steps for each action potential also were detected using the low-affinity absorbance indicator arsenazo III (W. N. Ross and R. Werman, unpublished observations). The equal increments of [Ca2+]i for each action potential argues against a significant contribution of Ca2+-induced Ca2+ release (CICR), because this process would be expected to cause larger step increments once the basal [Ca2+]i level was raised (e.g., Llano et al. 1994). This kind of mechanism also would lead to a delayed time to peak of the fluorescence transient if the internal stores were at any distance from the site of Ca2+ entry. In addition, in CA1 pyramidal neurons caffeine, ryanodine, dantrolene, or thapsigargin, all of which affect Ca2+-release from intracellular stores, had no affect on the AHP (Zhang et al. 1995). Nevertheless, it is still possible that CICR could contribute to the [Ca2+]i changes evoked in these experiments. For example, the release could be concentrated in a few localized sites. However, the important point is that the time to peak of the fluorescence change at all locations occurred at the time of the spikes and not at the peak of the sAHP.

Third, the recovery time course of the fluorescence change did not match the time course of the AHP or the underlying conductance change. This conclusion follows from experiments using low concentrations of indicator and measurements of Delta F/F in both the soma and dendrites. Similar results were found in both vagal motoneurons and hippocampal pyramidal neurons. Previously (Knopfel and Gahwiler 1992; Knopfel et al. 1990), a close correspondence between the recovery of the fluorescence transient in the soma and the time course of the sAHP was observed in CA3 pyramidal neurons in slice cultures. However, inspection of the records in those papers indicates that the measured recovery times of the fluorescence changes in the soma were >1 s, suggesting that they were probably slowed by the buffering by fura-2. This slowing would make their time courses appear closer to that of the sAHP (see Figs. 3A and 4).

The time course of the fluorescence transients (Delta F/F) measured in our experiments are expected to differ from the true [Ca2+]i transients just under the membrane in several ways. First, they are spatial averages over the soma or dendrites. Until diffusional equilibrium is reached, the submembrane [Ca2+]i transients are expected to have a faster rise time and a faster recovery time than the measured fluorescence changes (see RESULTS). Second, the measured fluorescence transients are slowed by the response time of the apparatus and the kinetics of the Ca2+:indicator reaction. These effects are expected to be insignificant in these experiments. There is little delay in the apparatus (Lasser-Ross et al. 1991) and the reaction time introduces a delay of <10 ms (Baylor and Hollingworth 1988), much smaller than the recovery times of the transients. Third, the addition of indicator at concentrations comparable with the effective concentration of endogenous buffer is expected to slow the [Ca2+]i time course and the fluorescence transient (e.g., Helmchen et al. 1996). This effect will be smaller for low-affinity indicators and for highly fluorescent indicators that can be used at low concentrations. Fourth, the magnitude of the fluorescence change is a nonlinear function of the [Ca2+]i change. The nonlinearity is more significant for high-affinity indicators like Calcium Green-1. Fifth, the indicator is a mobile buffer, shuttling Ca2+ away from the membrane faster than the normal diffusion by mostly immobile buffers in the cytoplasm. All except the shuttling effect are expected to make the true recovery time of the [Ca2+]i transient faster than the measured Delta F/F time course. The effect of shuttling is expected to be small (Sala and Hernandez-Cruz 1990) and is less significant at low indicator concentrations. More importantly, it should affect the sAHP in a parallel manner if the underlying conductance change depends directly on [Ca2+]i. Therefore, comparisons between the time course of the fluorescence transient and the sAHP should be valid.

Implications for the mechanism controlling the sAHP

Lancaster and Zucker (1994) found that raising [Ca2+]i uniformly throughout a CA1 pyramidal neuron by releasing Ca2+ from a photolabile chelator caused an immediate increase in the AHP conductance. This suggests that the Ca2+-dependent rate constants are fast and rules out a kinetics-based explanation for the slow time to peak (Hocherman et al. 1992). To explain the delayed rise of the AHP after action potentials, they suggested that Ca2+ must diffuse from the site of entry to the site of the K+ channels underlying the AHP. One possibility is that the two kinds of channels might be in different parts of the cell. In support of this idea, they performed experiments that suggested that spike-evoked Ca2+ entry might be restricted to parts of the dendritic tree. However, recent experiments by Callaway and Ross (1995) and Spruston et al. (1995) indicate that spike-evoked entry occurs throughout the dendrites of pyramidal cells, and the experiments in this paper suggest that the same conclusion applies to vagal motoneurons. In another variation of this model, Zhang et al. (1995) proposed that Ca2+ entered throughout the neuron, and that the sAHP was dominated by K+ channels in the soma. On the basis of experiments where exogenous chelators were introduced into the cytoplasm, they predicted that Ca2+ diffusing from the dendritic compartment would cause the [Ca2+]i in the soma to peak at some time after the end of the stimulus, leading to a delayed AHP peak conductance. Our data rule out this kind of model because a delayed time to peak in the soma or any other part of the cell was not observed in our experiments in either motoneurons or pyramidal cells.

It is possible that Ca2+ and K+ channels are distributed throughout the neuron, but are not very close to each other. Lancaster and Zucker (1994) calculated that a mean separation of 3 µm could account for the delayed rise time of the sAHP if the diffusion constant of Ca2+ is 0.12 × 10-6 cm2 s-1 (Allbritton et al. 1992). Gola et al. (1990) came to a similar conclusion concerning the separation of Ca2+ and K+ channels in Helix neurons. As mentioned above, our fluorescence data are consistent with a punctate distribution of Ca2+. Therefore, microdiffusion could account for the delayed rise in the sAHP.

However, there are some arguments against a model of simple Ca2+ diffusion as an explanation for the slow rise time of the sAHP (see also Sah 1996). One problem is the large Q10 of the sAHP rise time (Sah and McLachlan 1991; B. Etinger and Y. Yarom, unpublished observations). Simple Ca2+ diffusion is expected to be much less sensitive to temperature. Lancaster and Zucker (1994) suggested that the temperature sensitivity could reside in the binding of Ca2+ to cytoplasmic buffers or to the K+ channels themselves, but there is no experimental evidence for this sensitivity. A second problem is that the introduction of exogenous mobile Ca2+ buffers (like EGTA, BAPTA, or fura-2) should increase the effective diffusion constant for Ca2+ (Irving et al. 1990) and, therefore, should shorten the time to peak of the sAHP. Such shortening has not been observed. Indeed, the general experience is that added buffers prolong the sAHP (Sah 1993; Zhang et al. 1995; our experiments).

If short-range diffusion is not the explanation for the rise time of the sAHP, then another interpretation is needed for the photorelease experiments of Lancaster and Zucker (1994). One possibility is that the caged Ca2+ in their experiments activated more than one class of Ca2+-activated K+ channel, including some with a fast response time to Ca2+ entry through voltage-sensitive Ca2+ channels. Arguing against this possibility, Lancaster and Zucker (1994) showed that isoprenaline blocked the flash-induced AHP. Because isoprenaline also blocked the sAHP, they suggested that caged Ca2+ only activated the sAHP channels. This would require that the level of [Ca2+]i reached in their experiments was not high enough to activate the other K+ channels. This is possible (Lancaster et al. 1991), but the actual [Ca2+]i levels reached in their experiments is not known. More work is needed to clarify this issue.

The biggest difference between our data and a model where the conductance of the sAHP depends directly on [Ca2+]i is the different recovery time constants of the fluorescence transients and the sAHP. When using low concentrations of indicator, the measured recovery time of the fluorescence change was faster than the sAHP at all locations in the cell. In fact, in many cells, the fluorescence appeared to return to baseline levels at a time when the sAHP was still active (e.g., Figs. 3B and 7). We attempted to measure the time course of the fluorescence transient under conditions where this time course accurately reflected the underlying [Ca2+]i change. However, if the indicator response was nonlinear or if the indicator was buffering the [Ca2+]i change, then the difference between the true [Ca2+]i recovery time course and the sAHP would be even greater (see above).

It is hard to reconcile these data with Lancaster and Zucker's experiments (1994), which showed that the sAHP conductance can be eliminated at the moment [Ca2+]i is reduced by activating a caged Ca2+ chelator. This experiment suggests that the sAHP depends directly on elevated [Ca2+]i. However, some of their records show that the conductance was not completely blocked by the chelating flash (see Fig. 5 in their paper). Therefore, their experiments might be consistent with a mechanism that requires a second synergistic factor, activated by Ca2+, to control the conductance. This factor could be important in determining the time course of the sAHP (see also Schwindt et al. 1992a). Another possibility, consistent with this idea, is that resting [Ca2+]i levels are high enough to activate sAHP channels in the presence of the cofactor, but activation of the caged chelator buffers the [Ca2+]i level so low that the cofactor is ineffective. Simultaneous measurements of [Ca2+]i levels in experiments where [Ca2+]i is photolytically raised or lowered might clarify this issue.

Interestingly, the experiments using fura-2 as an indicator showed a closer correspondence between the fluorescence dynamics and the sAHP, in agreement with previous measurements on hippocampal CA3 pyramidal neurons (Knopfel and Gahwiler 1992; Knopfel et al. 1990). The recovery time course in these experiments was considerably slower than in the experiments using Calcium Green-1 because of the greater buffering by fura-2. This suggests the possibility that when the time course of the [Ca2+]i change is slow, it becomes the dominant factor controlling the time course of the sAHP.

In summary, neither the rising phase nor the falling phase of the sAHP matches the time course of the measured [Ca2+]i transient in vagal motoneurons. The discrepancy between the sAHP and the expected time course of [Ca2+]i just under the membrane is expected to be even greater. Some additional controlling factor appears to be at work.

    ACKNOWLEDGEMENTS

  We thank H. Meiri for excellent technical assistance.

  This work was supported in part by a grant from the Office of Naval Research and National Institute of Neurological Disorders and Stroke Grant NS-16295. W. N. Ross was a Fogarty Senior International Fellow (F06 TW01993).

    FOOTNOTES

  Address for reprint requests: W. Ross, Dept. of Physiology, New York Medical College, Valhalla, NY 10595 E-mail: ross{at}nymc.edu

  Received 16 December 1996; accepted in final form 29 April 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society