The Journal of Neurophysiology Vol. 79 No. 2 February 1998, pp. 688-694
Copyright ©1998 by the American Physiological Society

Ca²⁺-Induced Ca²⁺ Release Mediates a Slow Post-Spike Hyperpolarization in Rabbit Vagal Afferent Neurons

Kimberly A. Moore¹, Akiva S. Cohen¹, Joseph P. Y. Kao², and Daniel Weinreich¹

¹ Department of Pharmacology and Experimental Therapeutics; and ² Medical Biotechnology Center and Department of Physiology, University of Maryland, School of Medicine, Baltimore, Maryland 21201-1559

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Moore, Kimberly A., Akiva S. Cohen, Joseph P. Y. Kao, and Daniel Weinreich. Ca²⁺-induced Ca²⁺ release mediates a slow post-spike hyperpolarization in rabbit vagal afferent neurons. J. Neurophysiol. 79: 688-694, 1998. The relation between Ca²⁺-induced Ca²⁺ release (CICR) elicited by action potentials (APs) and a Ca²⁺-dependent slow post-spike hyperpolarization (AHP_slow) in acutely dissociated adult rabbit nodose neurons was studied using microfluorimetric calcium measurements in conjunction with standard intracellular current- and voltage-clamp recording techniques. The magnitude of the AP-induced transient increase in [Ca²⁺]_i (Ca_t) was used to monitor CICR. There was a close correlation between the magnitude of the Ca_t and the AHP_slow current over the range of 1-16 APs (r = 0.985). Functional CICR blockers, ryanodine (10 µM), thapsigargin (100 nM), 2,5-di(t-butyl)hydroquinone (10 µM) or cyclopiazonic acid (10 µM), selectively reduced the peak amplitude of the AHP_slow 91%. In five neurons, simultaneous recordings of the Ca_t and the AHP_slow revealed that both responses were blocked in parallel. These findings indicate that CICR is necessary for the generation of the AHP_slow in rabbit nodose neurons. The Ca_t rises and decays significantly faster than the AHP_slow. This temporal disparity suggests that activation of the AHP_slow by Ca²⁺ may require additional signal transduction steps.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Afferent information in the peripheral nervous system is encoded by modulation of action potential (AP) frequency. Activation of distinct classes of potassium channels can dramatically affect the frequency and the pattern of neuronal firing. In ~35% of the ~16,000 vagal somata (inferior or nodose ganglion neurons) of the rabbit, the pattern of AP firing can be effectively modified by a Ca²⁺-dependent K⁺ current. This current produces a slowly developing and persistent post-spike hyperpolarization (AHP_slow) that plays a significant role in the regulation of membrane excitability, and is responsible for spike frequency accommodation in these neurons (Fowler et al. 1985; Higashi et al. 1984; Weinreich and Wonderlin 1987). Following a single AP, theAHP_slow displays a slow rise time-to-peak (0.3-0.5 s) and a long duration (3-15 s). The critical importance of the AHP_slow in mediating spike frequency accommodation is exemplified by observations that its inhibition by various endogenous autacoids results in an increase in firing frequency from <0.1 to >10 Hz (Undem and Weinreich 1993; Weinreich and Wonderlin 1987). Consistent with a functional role of the AHP_slow, some of these autacoids also produce an augmentation in impulse activity recorded in vagal afferents in vivo (Coleridge and Coleridge 1984). The AHP_slow is not restricted to vagal afferent neurons; analogous slow afterpotentials have been characterized in sympathetic, myenteric, and CNS neurons (reviewed by Sah 1996).

Observations of the AHP_slow recorded in rabbit nodose neurons, and analogous data from other nerve cells, suggest that Ca²⁺ discharged from a Ca²⁺-induced Ca²⁺ release (CICR) pool may contribute to the generation of theAHP_slow. Intracellular calcium chelation with ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) (Higashi et al. 1984) or bis-(o-aminophenoxy)-N,N,N,N-tetraacetic acid (BAPTA) (Cohen et al. 1994) abolishes the AHP_slow. Manipulations that increase [Ca²⁺]_i, such as intracellular injection of Ca²⁺, or applications of ouabain or low concentrations of caffeine, augment and prolong the AHP_slow (Higashi et al. 1987). CICR channel modulators (e.g., ryanodine) or sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitors [e.g., thapsigargin or 2,5-di(t-butyl)hydroquinone (DBHQ)] block an analogous AHP_slow in sympathetic (Jobling et al. 1993; Kawai and Watanabe 1989, 1991), parasympathetic (Yoshizaki et al. 1995), and vagal dorsal motor nucleus neurons (Sah and McLachlan 1991).

In rabbit nodose neurons, Ca²⁺ influx produced by 1-7 APs is amplified 5- to 10-fold by CICR (Cohen et al. 1997). The time courses of the CICR-dependent transient increases of intracellular free Ca²⁺ and the AHP_slow appear qualitatively similar (Cohen et al. 1994), suggesting that mobilization of Ca²⁺ from a CICR pool may underlie the AHP_slow. In the current work we describe experiments using physiological stimuli, in conjunction with manipulations of CICR, to demonstrate that CICR is essential for the development of the AHP_slow.

	METHODS

Abstract Introduction Methods Results Discussion References

Cell dissociation

New Zealand white rabbits of either sex weighing 1-2 kg were obtained from Robinson Services (Winston-Salem, NC) and killed by pentobarbital sodium overdose (100 mg/kg), as approved by the Institutional Animal Care and Use Committee. Dissociated nodose neurons were prepared as described previously (Leal-Cardoso et al. 1993). After a 3- to 12-h incubation at 37°C, neurons were maintained at room temperature (22-24°C) to minimize neurite outgrowth and were suitable for experimental use for at least 3-4 days (Magee and Schofield 1991; and our own observations). There were no observable differences in visual appearance or basic electrophysiological properties of the cells based on the sex of the animals or length of time neurons were kept in culture.

Electrode fabrication, recording chamber, and drug delivery

Intracellular recording microelectrodes were fabricated on a Flaming/Brown model P-97 micropipette puller (Sutter Instrument, San Francisco, CA). The aluminosilicate micropipettes (Sutter) had resistances ranging from 30 to 70 M when filled with 4 M potassium acetate or 3 M potassium chloride.

A custom recording chamber provided superfusion (3-5 ml/min) of a 25-mm coverslip with physiological salt solution via a gravity-flow system. It was mounted on the stage of an inverted microscope (Zeiss IM35) equipped with a ×40 phase-contrast oil-immersion objective (Fluor, NA 1.3, Nikon) to allow direct visualization of neurons for intracellular recording and fluorescence measurements. In some experiments where only electrical measurements were made, we used a compound microscope equipped with Hoffman optics (×400).

Electrophysiological recording

Standard intracellular stimulating and recording techniques were used to monitor electrical activity with "sharp" micropipettes (see Christian et al. 1989 for details). Current-clamp recordings were made with an Axoclamp-2A amplifier (Axon Instruments, Foster City, CA) either in bridge (filtering at 10 kHz) or in the discontinuous mode (sample rate 5 kHz, filtering at 3 kHz). AP-induced AHP_slow currents (I_AHP) were recorded using a hybrid voltage-clamp technique. Varying numbers of APs were evoked by transmembrane depolarizing current pulses (3 nA, 3 ms, 10 Hz). One hundred milliseconds after the final depolarizing current pulse, the amplifier was electronically switched from current-clamp to voltage-clamp mode to record the I_AHP. Current and voltage signals were digitized with a Neurocorder (Neurodata Instruments, Delaware Water Gap, NJ) for storage on videocassette tapes for off-line analysis. The membrane input resistance (R_in) of the cell was monitored by measuring the magnitude of the electrotonic voltage transient produced by hyperpolarizing current pulses (100 pA, 150 ms). Analysis of electrophysiological data were performed using pClamp 6.2 software (Axon Instruments).

During experiments the cells were superfused with 22-24°C Locke solution that had the following composition (in mM): 136 NaCl, 5.6 KCl, 1.2 NaH₂PO₄, 14.3 NaHCO₃, 1.2 MgCl₂, 2.2 CaCl₂, and 10 dextrose (continuously equilibrated with 95% O₂-5% CO₂; pH 7.2-7.4).

Reagents

Reagents were procured from the following vendors: thapsigargin from LC-laboratories (Woburn, MA), ryanodine and cyclopiazonic acid from Calbiochem (La Jolla, CA), and fura-2/acetoxymethyl ester (AM), BAPTA/AM, and BAPTA sodium salt from Molecular Probes (Eugene, OR). Inorganic salts were obtained from VWR (Piscataway, NJ).

Unless otherwise noted, drug solutions were prepared daily from concentrated (>10 mM) stock solutions that were stored frozen. Drugs were delivered via the superfusate by switching a three-way valve to a reservoir containing a known concentration of the drug in oxygenated Locke solution.

Ca²⁺ measurements

To measure Ca²⁺, cells on coverslips were incubated for 45-60 min at room temperature (22-24°C) in a solution containing 1 µM fura-2/AM as previously described (Cohen et al. 1994). After incubation the coverslip was placed in the recording chamber and superfused with Locke solution. Fura-2 fluorescence measurements were performed with a DeltaScan Illumination System [Photon Technology International (PTI), South Brunswick, NJ] coupled to the microscope through a fiberoptic cable. Each neuron under study was alternately illuminated with 340-nm and 380-nm light and the fluorescence emission, after passing through a 510-nm band-pass filter, was sampled by a photomultiplier tube, the output from which was digitized and stored for subsequent analysis. Instrument control, data acquisition, and analysis were performed using FELIX 1.1 software (PTI) running on a dedicated microcomputer.

[Ca²⁺]_i calibration

All fura-2 fluorescence records were corrected for background fluorescence by subtracting the light intensity measured after cell lysis with digitonin. Values of intracellular [Ca²⁺] ([Ca²⁺]_i) were calculated using the equation of Grynkiewicz et al. (1985) where R is the ratio F₃₄₀/F₃₈₀, R_min and R_max are the minimum and maximum values of the ratio, attained at zero and saturating Ca²⁺ concentrations, respectively. F₃₄₀ is the fluorescence emitted by the dye when excited at 340 nm, and F₃₈₀ is the fluorescence emitted by the dye when excited at 380 nm. S_f2/S_b2 is the ratio of fluorescence intensities for Ca²⁺-free and Ca²⁺-bound indicator measured with 380-nm excitation. R_min, R_max, and S_f2/S_b2 were determined from six acutely dissociated neurons used specifically for calibration purposes.

Data are expressed as means ± SE. Analysis of variance(ANOVA) and Student's t-test were used to assess significant differences between calculated means; P < 0.05 was considered significant. Unless specified, results were replicated in at least three different neurons.

	RESULTS

Abstract Introduction Methods Results Discussion References

Effect of BAPTA on the AHP_slow

AP-induced elevations of [Ca²⁺]_i (Ca_t) recorded in rabbit nodose neurons are strictly dependent on an intact CICR process (Cohen et al. 1997). Additionally, superfusion of acutely isolated rabbit nodose neurons with the AM ester form of the Ca²⁺ chelator BAPTA (25 µM) concomitantly abolishes both the AHP_slow and Ca_t (Cohen et al. 1994). In the current work, we have extended these results and tested whether BAPTA treatment affected Ca²⁺ influx. In 11 neurons incubated with 10-30 µM BAPTA/AM, the AHP_slow was completely blocked within 2-7 min (Fig. 1A). By contrast, incubating neurons with the membrane-impermeant sodium salt of BAPTA (20-30 µM) for 8 min produced no measurable effect on the AHP_slow (n = 3). Although the reduction of the AHP_slow by intracellular BAPTA is most likely due to Ca²⁺ buffering, BAPTA could conceivably alter Ca²⁺ influx through voltage dependent calcium channels (VDCCs). However, because the fast Ca²⁺-dependent afterhyperpolarization (AHP_fast) following each AP was unaffected by intracellular BAPTA loading, it appears that Ca²⁺ influx through VDCCs is not compromised (cf. Fig. 1, Ba and Bb; n = 10). When the superfusate was switched to one containing 100 µM CdCl₂, a blocker of Ca²⁺ influx, the AHP_fast was reduced in amplitude (Fig. 1Bc). The small remaining component of the AHP_fast is presumably a reflection of the delayed rectifier. In this neuron, abolition of the AHP_slow was accompanied by a marked increase in the excitability of the neuron as evidenced by the lowering of the threshold for AP firing, and an increase in the average frequency of firing, measured during a depolarizing ramp, from 1 to 5.5 Hz (Fig. 1C).

View larger version (27K): [in this window] [in a new window]   FIG. 1. Effect of bis-(o-aminophenoxy)-N,N,N,N-tetraacetic acid/acetoxymethyl ester (BAPTA/AM) on slow post-spike hyperpolarization (AHPslow) and on excitability in an isolated nodose neuron. A: AHPslow elicited by 2 action potentials (APs) in control Locke solution and in Locke solution containing 10 µM BAPTA/AM. APs were evoked by transmembrane depolarizing current pulses (4 nA, 1.5 ms, 10 Hz) and are truncated. Dashed line represents the resting membrane potential (60 mV). Resting membrane input resistance was 70 M. Bath application of BAPTA/AM blocks the AHPslow within 5 min without changing the resting membrane potential or membrane input resistance. B: responses digitized at a higher rate. The AHPfast, which precedes theAHPslow, is unaffected by BAPTA/AM (compare Ba with Bb). The Ca2+ dependence of the AHPfast is illustrated in Bc, where the neuron is superfused with 100 µM CdCl2 for 30 s, which blocks most of the AHPfast, and all of the AHPslow. The residual component of the AHPfast recorded in CdCl2 is presumably mediated by the delayed rectifier current. C: depression of the AHPslow markedly increases neuronal excitability. The average number of APs induced by a current ramp protocol (1 nA, 2 s) increased from 1 to 5.5 Hz when the AHPslow was blocked. Scale bar represents 3 mV, 2 s in A; 15 mV, 0.25 s in B; 15 mV, 0.5 s in C.

Relation between the number of APs and the magnitude of the AHP_slow current

We tested the dependence of the AHP_slow on CICR by examining whether the magnitude of the AHP_slow current (I_AHP) saturates with increasing numbers of APs in a manner parallel to that observed for the Ca_t (see Fig. 1 in Cohen et al. 1997) (and see Fig. 2). Neurons were current clamped at their resting membrane potential (approximately 60 mV) while varying numbers of APs were evoked by suprathreshold transmembrane depolarizing current pulses (2 ms, 10 Hz). One hundred milliseconds after the last AP, the neuron was voltage clamped to approximately 60 mV to measure the magnitude of the resulting I_AHP. Over the range of 1-8 APs, the amplitude of the I_AHP increased steeply. Beyond eight APs, the I_AHP amplitude began to level off, almost reaching a plateau by the 40th AP. Over the range of 1-40 APs, the relation between I_AHP amplitude and number of APs was well fit by a rectangular hyperbola (² = 2.35, r = 0.975, n = 10; Fig. 2B). These data are remarkably similar to those relating the amplitude of the Ca_t to the number of APs (dashed curve in Fig. 2B, redrawn from Cohen et al. 1997). When the I_AHP elicited by a given number of APs is plotted against the amplitude of the Ca_t evoked by the same number of APs, there was a close correlation (r = 0.985) between the magnitudes of Ca_t and I_AHP (Fig. 2C), suggesting a potential mechanistic linkage between the two.

View larger version (21K): [in this window] [in a new window]   FIG. 2. Effect of varying numbers of APs on the amplitude of the IAHP recorded in acutely isolated adult rabbit nodose neurons. A: IAHPs were evoked by APs using a hybrid voltage-clamp technique (see METHODS). APs were produced by transmembrane depolarizing current pulses (3 nA, 3 ms, 10 Hz) from a holding potential of 60 mV. One hundred ms following the last AP, the amplifier is switched to voltage-clamp mode (60 mV holding potential) to record the IAHP. The number of APs is indicated to the left of each trace. B: filled circles represent the mean IAHP amplitude (±SE) elicited by varying numbers of APs. The number of observations is indicated near each data point. IAHPs are normalized to the maximal response recorded in a given neuron. The normalized amplitudes of the IAHPs depicted in A are also shown (open circles). The continuous curve is a rectangular hyperbola fit to the mean data (2 = 2.35, r = 0.975). Dashed curve is the least-squares rectangular hyperbola relating the normalized Cat amplitude to the number of APs (originally presented in Cohen et al. 1997). C: IAHP amplitude elicited by a given number of APs (1, 2, 4, 8, and 15) is plotted against the amplitude of the Ca2+ transient elicited by the same number of APs (Ca2+ transient data from Cohen et al. 1997). Values are mean ± SE. There is a linear relation between the amplitude of the Ca2+ transient and the IAHP over the range of 1-15 APs (r = 0.985).

Effects of modulators of CICR on the magnitude of the AHP_slow

We have previously reported that ryanodine and SERCA inhibitors, such as thapsigargin, DBHQ, and cyclopiazonic acid (CPA) (reviewed by Inesi and Sagara 1994) can abolish caffeine- and AP-induced Ca_ts in rabbit nodose neurons (Cohen et al. 1997), where a robust process of CICR underlies AP-induced Ca_ts. In the current experiments, we investigate the effects of these reagents on the AHP_slow.

When neurons were superfused with Locke solution containing ryanodine (10 µM), thapsigargin (100 nM), DBHQ (10 µM), or CPA (10 µM), the amplitude of the AHP_slow was reduced >90% (Table 1 and Fig. 3). Figure 3, A and B, illustrates experiments where neurons were superfused with either ryanodine or thapsigargin. Although these compounds disable CICR by entirely different mechanisms, in these experiments they both abolished the AHP_slow completely. The time required to abolish the AHP_slow by these CICR antagonists (20 and 18 min, respectively) was similar to the time required to block the Ca_t (Cohen et al. 1997). At the concentrations used, ryanodine and thapsigargin do not alter either the AP waveform or the amount of Ca²⁺ influx per AP (Cohen et al. 1997).

View larger version (16K): [in this window] [in a new window]   View larger version (19K): [in this window] [in a new window]   FIG. 3. Effects of Ca2+-induced Ca2+ release (CICR) inhibitors on the AHPslow and the AP-induced Ca2+ transient. A: superimposed traces of an AHPslow elicited by 4 APs, recorded in control Locke solution and 20 min after switching to Locke solution containing ryanodine (10 µM). Ryanodine completely abolished the AHPslow. B: analogous experiment with thapsigargin (100 nM) recorded in another nodose neuron. Eighteen min after switching to Locke solution containing thapsigargin, the AHPslow was completely blocked. C: effect of 2,5-di(t-butyl)hydroquinone (DBHQ) on the AP-induced Ca2+ transient and on the AHPslow. Top traces: superimposed Ca2+ transients evoked by a train of 4 APs (10 Hz) recorded in control Locke solution and 5 min after switching to Locke solution containing 10 µM DBHQ. Bottom traces: AHPslows recorded simultaneously with the Ca2+ transients. DBHQ treatment completely blocked both the Ca2+ transient and the AHPslow. Resting [Ca2+]i was 91 nM. Fluorescence data were acquired at 10 Hz. D: time course of block of the AHPslow and the Ca2+ transient for the neuron shown in C. Resting membrane potentials in A-C were 56, 58, and 67 mV, respectively. AP amplitudes are truncated.

In five neurons, we simultaneously recorded the Ca_t and the AHP_slow in normal Locke solution and in Locke solutions containing DBHQ (n = 2), ryanodine (n = 2), or thapsigargin (n = 1). The average values for control Ca_t andAHP_slow, evoked by four APs, were 28 ± 4.3 nM and 7 ± 2.0 mV, respectively. Drug treatment completely abolished both the Ca_t and the AHP_slow in all five neurons. The data in Fig. 3C illustrate the effects of DBHQ on the Ca_t and the AHP_slow recorded in the same neuron. Five minutes after switching to a superfusate containing DBHQ, the Ca_t and the AHP_slow were concomitantly blocked. Parallel inhibition of the Ca_t and the AHP_slow in this neuron is illustrated in Fig. 3D. Collectively, these data support the conclusion that CICR is an important determinant of the AHP_slow in rabbit nodose neurons.

Comparison between the time course of the AHP_slow and the Ca_t

There are two mechanisms by which Ca²⁺ from AP-induced CICR could regulate the AHP_slow. First, Ca²⁺ may initiate signaling cascades that ultimately control the K⁺ channels underlying the AHP_slow. Alternatively, Ca²⁺ itself could activate the K⁺ channels directly, and thus exert moment-by-moment control of the AHP_slow. If the AHP_slow is directly dependent on Ca²⁺ released from the CICR pool, the AHP_slow and the rise in [Ca²⁺]_i elicited by an AP might display similar kinetics. Quantitative kinetic comparisons between these two variables are, unfortunately, subject to some uncertainty, because the time course of the Ca_t reflects global changes in [Ca²⁺]_i, whereas the kinetics of the AHP_slow are determined by events at the plasma membrane. Nonetheless, we determined the time-to-peak and 10 to 90% decay time for both the AHP_slow and the Ca_t elicited by 1-8 APs (Table 2). The time-to-peak for Ca_ts elicited by 1-8 APs was 1.0 ± 0.06 s (n = 32). The AHP_slow evoked by 1-8 APs peaked in 1.9 ± 0.13 s (n = 25), and was significantly slower in reaching peak than the Ca_t (P < 0.0001). The Ca_t also decayed much more rapidly than the AHP_slow (2.8 ± 0.42 s, n = 24, versus 6.7 ± 0.38 s, n = 26; P < 0.0001). These quantitative differences are graphically illustrated by Fig. 4, in which corresponding Ca_ts and AHP_slows are scaled and overlaid. Although our preliminary observations (Cohen et al. 1994) suggested a similar time course for the AHP_slow and the Ca_t, quantitative analysis of a much larger data set now reveals significant temporal differences between these parameters.

View larger version (12K): [in this window] [in a new window]   FIG. 4. Comparison of the temporal profiles of AP-evoked Ca2+ transients and the corresponding AHPslows. A: superposition of Ca2+ transient (thick line) and AHPslow (thin line) evoked by 4 APs. The AHPslow has been inverted and scaled for ease of visual comparison with the Ca2+ transient. Arrows mark the peaks of the Ca2+ transient and AHPslow. B and C: superpositions analogous to that shown in A, but for 8 and 14 APs, respectively. Resting membrane potential and [Ca2+]i were 55 mV and 70 nM, respectively. All traces were recorded in the same neuron. Population data for these experiments are presented in Table 2.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Our principal finding is that Ca²⁺ release from the CICR pool is necessary for the development of the AHP_slow in nodose neurons. In virtually all rabbit nodose neurons, there exists a CICR pool that can be activated by Ca²⁺ influx resulting from a single AP (Cohen et al. 1997). By contrast, neurons with AHP_slows are randomly distributed and occur in only ~35% of the population (Fowler et al. 1985). Thus expression of the AHP_slow is dependent on a neuronal property independent of the CICR pool. The most parsimonious explanation is that the expression of the AHP_slow K⁺ channel determines whether a nodose neuron exhibits a AHP_slow. The recent cloning of a small conductance, Ca²⁺-activated K⁺ channel (Köhler et al. 1996) with characteristics similar to the AHP_slow current may soon allow direct testing of this hypothesis.

In rabbit nodose neurons, APs induce transient increases in [Ca²⁺]_i (Ca_ts) that are dependent on an intact CICR pool (Cohen et al. 1997). If the Ca²⁺ trigger is increased by progressively increasing the number of APs from 1 to 64, Ca²⁺ influx per AP remains constant, but Ca²⁺ release from the CICR pool (i.e., the magnitude of the Ca_t) first increases sharply with the number of APs but then plateaus. This is reflected by the hyperbolic relation between the magnitude of the AP-evoked Ca_t and number of APs (dashed curve in Fig. 2B, redrawn from Cohen et al. 1997). In the current work, a similar hyperbolic relation was observed when the magnitude of I_AHP was plotted against a varying number of APs (Fig. 2B). Over the range of 1-16 APs, there was a close correlation (r = 0.985) between the magnitudes of the Ca_t and the I_AHP (Fig. 2C). This strong correlation suggests that CICR could underlie the AHP_slow.

There are several interpretations of the hyperbolic relation between the I_AHP and the number of APs. First, the amount of Ca²⁺ influx per AP might decrease progressively during a train of APs. However, we have demonstrated previously that over the range of 1-32 APs, the Ca²⁺ influx per AP does not change (Cohen et al. 1997). Thus the plateauing of the I_AHP-AP relation with increasing numbers of APs must reflect another process. A second possibility is that the number of AHP_slow potassium channels in nodose neurons is limiting. This too is unlikely because when the concentration of extracellular Ca²⁺ is doubled, the plateau of the I_AHP-AP relation is elevated (unpublished observations). Finally, saturation of the I_AHP amplitude could reflect exhaustion of the releasable content of the CICR pool. In principle, application of caffeine, the standard CICR agonist, before and during the plateau phase could test whether the CICR pool was exhausted. Unfortunately, caffeine activates not only CICR but also a novel calcium influx pathway in these neurons (Hoesch et al. 1997) thereby complicating interpretation of such experiments.

Pharmacological evidence that supports the involvement of CICR in the generation of the AHP_slow was obtained by using two functionally distinct classes of reagents to abolish CICR. Ryanodine disrupts CICR by interfering with intracellular Ca²⁺ release channels (the ryanodine receptors) (Fill and Coronado 1988). On the other hand, SERCA inhibitors (thapsigargin, DBHQ, and CPA) inhibit the ATPases that transport Ca²⁺ into stores, including the CICR pool (Inesi and Sagara 1994), thus allowing dissipation of the stores through leakage. When neurons were treated with SERCA inhibitors or with ryanodine, at concentrations and incubation times previously shown to block caffeine- and AP-induced Ca_ts (Table 2) (Fig. 5 in Cohen et al. 1997; also unpublished observations), the AHP_slow was consistently abolished (Fig. 3 and Table 1). Because these reagents do not affect Ca²⁺ influx (Cohen et al. 1997), these pharmacological data support the contention that, under physiological conditions, Ca²⁺ release from the CICR pool is necessary for development of the AHP_slow. What remains unresolved, however, is whether Ca²⁺ released from the CICR pool is sufficient, by itself, to activate and sustain this persistent afterpotential. The time course of elevated [Ca²⁺]_i near the plasma membrane can be approximated by the duration of the Ca²⁺-dependent fast post-spike afterpotential (AHP_fast, 150 ms) that precedes the AHP_slow (Fowler et al. 1985). Assuming that Ca²⁺ influx gates the AHP_slow potassium channels directly (Köhler et al. 1996; Sah 1996), it seems unlikely that AP-induced Ca²⁺ influx alone could sustain this protracted afterpotential.

The significant temporal disparity between the AHP_slow¹ and the underlying Ca_t (Fig. 4 and Table 2) also argues against any model where Ca²⁺ ions alone are sufficient to activate the AHP_slow directly. Rather, the incommensurate time courses of the AHP_slow and the Ca_t favor a model in which activation of the AHP_slow by Ca²⁺ is mediated by additional signal transduction steps. In this regard, it is notable that Lasser-Ross et al. (1997) have inferred a similar mechanism for Ca²⁺ activation of the AHP_slow in vagal motoneurons, where they suggest that additional second-messenger systems may be involved. Our results and those of Lasser-Ross et al. (1997) concerning the temporal characteristics of the AHP_slow and the Ca_t are concordant in all but one important respect. In vagal motoneurons, the peak of the Ca_t coincides with the last AP. In contrast, in vagal afferent (nodose) neurons, there is a delay between the last AP and the peak of the Ca_t. This delay probably reflects the contribution of CICR to the calcium transient.

Several processes could contribute to the slow time-to-peak and long duration of the AHP_slow. These include slow diffusion of Ca²⁺ ions, CICR, an enzymatic signal transduction cascade, or a combination of these factors. In CA1 pyramidal neurons of the hippocampus, there exists a well-characterized AHP_slow with slow kinetics and physiological and pharmacological properties similar to the AHP_slow recorded in nodose neurons (reviewed by Sah 1996). When intracellular photolabile Ca²⁺ chelators ("caged" calciums) were used to rapidly increase [Ca²⁺]_i in these neurons, the latency between Ca²⁺ photorelease and the Ca²⁺-induced membrane hyperpolarization was no longer than the membrane time constant (Lancaster and Zucker 1994). These results suggest that in CA1 pyramidal neurons Ca²⁺ directly gates AHP_slow K⁺ channels and that the slow onset kinetics could arise from Ca²⁺ channels residing on the proximal dendrites, distal to the AHP_slow potassium channels that are uniformly distributed in the somata. Acutely isolated nodose neurons are essentially spherical structures lacking dendritic processes. Thus, in these neurons, the delayed onset of the AHP_slow is unlikely to be the result of slow diffusion of Ca²⁺ from distal sources. The high temperature coefficient (Q₁₀  3.5) for the rising phase of the AHP_slow in rabbit nodose neurons (Fowler et al. 1985) also argues against simple Ca²⁺ diffusion (Q₁₀  1.3) as an explanation for the slow kinetics (see also Sah and McLachlan 1991). One possibility is that the CICR system imposes a time dependence on the AHP_slow; another may be that opening of the AHP_slow potassium channels requires an enzymatic signal transduction cascade. In this regard, it will be useful to learn whether rapid photorelease of Ca²⁺ in nodose neurons produces an instantaneous or delayed membrane hyperpolarization.

In summary, a CICR pool in nodose neurons releases Ca²⁺ in response to Ca²⁺ influx evoked by single APs. One physiological role for CICR in vagal afferents is to activate and control the AHP_slow that is responsible for spike frequency adaptation.

	ACKNOWLEDGEMENTS

  The authors thank Dr. Brad Undem for constructive suggestions on an earlier draft of this manuscript. The first two authors contributed equally to this work.

  This work was supported by National Institutes of Health Grants NS-22069 to D. Weinreich, GM-46956 to J.P.Y. Kao, and training grant NS-07375 to K. A. Moore.

	FOOTNOTES

¹   Fowler et al. (1985) demonstrated that the AHP_slow and its conductance follow an identical time course.

  Address for reprint requests: D. Weinreich, Dept. of Pharmacology and Experimental Therapeutics, Rm. 522 Health Science Facility, 685 West Baltimore St., University of Maryland, School of Medicine, Baltimore, MD 21201-1559.

  Received 11 August 1997; accepted in final form 10 October 1997.

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Abstract Introduction Methods Results Discussion References

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