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Ca(2+)-induced Ca2+ release phenomena in mammalian sympathetic neurons are critically dependent on the rate of rise of trigger Ca2+.

The role of ryanodine-sensitive intracellular Ca2+ stores present in nonmuscular cells is not yet completely understood. Here we examine the physiological parameters determining the dynamics of caffeine-induced Ca2+ release in individual fura-2–loaded sympathetic neurons. Two ryanodine-sensitive release components were distinguished: an early, transient release (TR) and a delayed, persistent release (PR). The TR component shows refractoriness, depends on the filling status of the store, and requires caffeine concentrations ≥10 mM. Furthermore, it is selectively suppressed by tetracaine and intracellular BAPTA, which interfere with Ca2+-mediated feedback loops, suggesting that it constitutes a Ca2+-induced Ca2+-release phenomenon. The dynamics of release is markedly affected when Sr2+ substitutes for Ca2+, indicating that Sr2+ release may operate with lower feedback gain than Ca2+ release. Our data indicate that when the initial release occurs at an adequately fast rate, Ca2+ triggers further release, producing a regenerative response, which is interrupted by depletion of releasable Ca2+ and Ca2+-dependent inactivation. A compartmentalized linear diffusion model can reproduce caffeine responses: When the Ca2+ reservoir is full, the rapid initial Ca2+ rise determines a faster occupation of the ryanodine receptor Ca2+ activation site giving rise to a regenerative release. With the store only partially loaded, the slower initial Ca2+ rise allows the inactivating site of the release channel to become occupied nearly as quickly as the activating site, thereby suppressing the initial fast release. The PR component is less dependent on the store's Ca2+ content. This study suggests that transmembrane Ca2+ influx in rat sympathetic neurons does not evoke widespread amplification by CICR because of its inability to raise [Ca2+] near the Ca2+ release channels sufficiently fast to overcome their Ca2+-dependent inactivation. Conversely, caffeine-induced Ca2+ release can undergo considerable amplification especially when Ca2+ stores are full. We propose that the primary function of ryanodine-sensitive stores in neurons and perhaps in other nonmuscular cells, is to emphasize subcellular Ca2+ gradients resulting from agonist-induced intracellular release. The amplification gain is dependent both on the agonist concentration and on the filling status of intracellular Ca2+ stores.


i n t r o d u c t i o n
Ca 2 ϩ -induced Ca 2 ϩ release (CICR) 1 is crucial for excitation-contraction coupling in mammalian cardiac muscle. According to a widely accepted scheme, the initial depolarization of the sarcolemmal membrane triggers Ca 2 ϩ influx by opening voltage-gated Ca 2 ϩ channels. This Ca 2 ϩ influx activates ryanodine receptor/Ca 2 ϩ release channels (RyR) on the sarcoplasmic reticulum, from which Ca 2 ϩ is then released into the cytosol, producing the large Ca 2 ϩ transients required to initiate contraction (Wier, 1990). Experiments in rat cardiac myocytes have shown that voltage-gated Ca 2 ϩ influx is the most efficient stimulus to trigger Ca 2 ϩ release (Sham et al., 1995). A recent model proposes that single L-type Ca 2 ϩ channels in the membrane of t-tubules are functionally coupled and in close proximity to small clusters or RyRs in the sarcoplasmic reticulum, which together constitute a Ca 2 ϩ release unit (Stern, 1992;Cannell et al., 1995).
The molecular elements responsible for CICR (voltage-gated Ca 2 ϩ channels and ryanodine receptor channels) are also present in vertebrate neurons (reviewed by Kostyuk and Verkhratsky, 1994;Kuba, 1994;Verkhratsky and Shmigol, 1996). This leads to the notion that Ca 2 ϩ mobilization from intracellular stores mediated by RyR channels may constitute an important pathway for Ca 2 ϩ signaling (McPherson and Campbell, 1993). Nonetheless, several studies indicate that under physiological conditions, neuronal calcium transients originated by Ca 2 ϩ influx are not amplified to a large extent by CICR. In the best cases, only a modest augmentation and prolongation of the calcium transient over that which would be produced by the calcium influx alone has been observed (Thayer et al., 1988;Friel and Tsien, 1992;Nohmi et al., 1992;Thayer and Miller, 1990;Ivanenko et al., 1993;Usachev et al., 1993;Kostyuk and Verkhratsky, 1994;Kuba, 1994;Shmigol et al., 1995). Moreover, prolonged depolarizations are often needed to raise [Ca 2 ϩ ] enough to initiate CICR (Llano et al., 1994;Verkhratsky and Shmigol, 1996). This behavior is characteristic of low feedback loop gain models of CICR (Stern, 1992).
If nerve cells are not optimized for widespread amplification of Ca 2 ϩ transients resulting from voltage-gated Ca 2 ϩ influx, what is the role of their ryanodine-sensitive Ca 2 ϩ stores? The answer to this question requires a better understanding of the functional organization of ryanodine receptors and neuronal caffeine-sensitive stores. For instance, we need to establish if released Ca 2 ϩ , by interacting with regulatory sites on the RyR channel, can participate in feedback loops that may promote or prevent further release. A related question is whether or not neighboring Ca 2 ϩ release channels and calcium stores are diffusionally coupled. Finally, we need to discern to what extent calcium depletion of intracellular reservoirs control the release process.
The goal of this study was to analyze the mechanisms that determine the dynamics of Ca 2 ϩ release from ryanodine-sensitive intracellular Ca 2 ϩ stores in rat sympathetic neurons after its activation by a rapid caffeine application. Since it is likely that endogenous agonists act similarly to caffeine , the conclusions of our study should be applicable to Ca 2 ϩ release initiated by physiological phenomena as well (Pozzan et al., 1994). This paper presents unequivocal evidence of CICR phenomena when trigger Ca 2 ϩ is released at an adequately fast rate from intracellular stores. Our data also suggest that the regenerative release is interrupted by a combination of rapid depletion of releasable Ca 2 ϩ and Ca 2 ϩ -dependent inactivation of release. The most salient features of caffeine-induced Ca 2 ϩ mobilization described in this paper could be accounted for with a compartmentalized linear diffusion model which contains a caffeine-sensitive intracellular Ca 2 ϩ store capable of releasing Ca 2 ϩ in response to changes in cytosolic [Ca 2 ϩ ], separated from the plasma membrane by a diffusional space containing fixed Ca 2 ϩ buffers. m a t e r i a l s a n d m e t h o d s

Tissue Culture of Rat Sympathetic Neurons
Cultures of superior cervical ganglion neurons were prepared as follows: 10-d-old rats of either sex were used. Ganglia were re-moved under ether anesthesia and aseptic conditions. They were cleaned and chopped, and then incubated in Ca 2 ϩ and Mg 2 ϩ -free Hank's medium (Sigma Chemical Co., St. Louis, MO) with 1 mg/ml trypsin (Worthington Biochemical Corp., Freehold, NJ) and 2 mg/ml DNAse I (Sigma Chemical Co.) for 30 min at 37 Њ C. After digestion, trypsin was inactivated by dilution in DMEM medium (Gibco BRL, Gaithersburg, MD) containing 10% FCS and 1 mg/ml trypsin inhibitor (Sigma Chemical Co.). The tissue was then incubated in Hank's medium with 2 mg/ml collagenase (Worthington Biochemical Corp.) and 2 mg/ml DNAse I for 30 min at 37 Њ C. Tissue fragments were then triturated with a fire-polished siliconized Pasteur pipette. Cell suspension was centrifuged at 800 rpm for 10 min, washed twice in Hank's medium (Gibco BRL) and resuspended in fresh DMEM (Gibco BRL) supplemented with 10% FCS (Gibco BRL). Cells were seeded on polylysine-treated No. 1 glass coverslips (1 ϫ 10 5 cells per well) and maintained for up to 3 wk supplemented with 20 ng/ml of 7S nerve growth factor (Sigma) at 37 Њ C in a humidified atmosphere of 95% air and 5% CO 2 . Medium was changed three times per week. Most experiments were carried out with cells that had been in culture from 1 to 5 d.

Measurements of Intracellular Ca 2 ϩ and Sr 2 ϩ Concentrations
Methods are described in detail elsewhere (Hernández-Cruz et al., 1995). Briefly, a coverslip containing sympathetic neurons was transferred to a recording chamber (Mod. RC-25; Warner Instruments, Hamden, CT) on an inverted microscope (Nikon Diaphot TMD; Nikon Corp., Tokyo, Japan). Cells were loaded with fura-2 by incubation with the acetoxymethyl (AM) ester form of the dye (fura-2/AM; Molecular Probes, Eugene, OR), at a final concentration of 1 M, with no dispersing agents added. Cells were allowed to load for 30-45 min at room temperature and then rinsed continuously for another 15 min before the beginning of the experiments.
Ca 2 ϩ or Sr 2 ϩ levels were determined by recording pairs of images using alternating illumination with 340-and 380-nm excitation. Dual wavelength excitation was provided by two nitrogen pulsed lasers (3-ns pulse duration), one emitting at its natural wavelength of 337 nm and the second one dye-tuned at 380 nm. These lasers were triggered alternatively at frequencies ranging from 2 to 15 Hz under computer control. (Biolase Imaging System, Newton MA). Background images at 340-and 380-nm illumination were obtained from an area of the coverslip free of cells. These images were stored separately and used for on-line background subtraction. We found that a correction for cell autofluorescence was not essential for calibration purposes. The key elements of the fluorescence Ca 2 ϩ imaging system were a high numerical aperture UV objective (Nikon UV-F 100X, 1.3 N.A.), an intensified charge coupled device camera (c2400-87; Hamamatsu, Bridgewater, NJ), and the Biolase Imaging System running under their FL-2 software. The system allows real-time simultaneous acquisition of fluorescence measurements from multiple areas of interest placed on individual cells or within a single cell. All Ca 2 ϩ determinations in this study were obtained from entire cells. Ca 2ϩ or Sr 2ϩ concentrations were calculated from fluorescence measurements at 340-and 380-nm excitation wavelengths using the formula: where the calculated dissociation constant (K D ) for fura-2 is 300 nM for Ca 2ϩ and 7.6 M for Sr 2ϩ (Kwan and Putney, 1990), F f /F b is the ratio of fluorescence values for X 2ϩ -free/X 2ϩ -bound indicator at 380-nm excitation, R is the ratio of fluorescence at 340/380 nm for the unknown [X 2ϩ ], and R min , R max are the ratio of fura-2 fluorescences at 340/380 nm of X 2ϩ -free and X 2ϩ -bound fura-2.
The values of F f /F b , R min , and R max for Ca 2ϩ were determined by measuring under identical conditions the fluorescence of a glass capillary 100-m in diameter containing calibration solutions with 50-100 M fura-2 (pentapotassium salt; Molecular Probes) and known Ca 2ϩ concentrations in the range from 10 nM to 40 M. Sr 2ϩ values were determined similarly, using solutions containing 100 M fura-2 and either no added Sr 2ϩ or 1 mM Sr 2ϩ . Although in situ calibrations were attempted in initial experiments, we found it very difficult to manipulate [Ca 2ϩ ] over the required range. Our Ca 2ϩ and Sr 2ϩ measurements, based exclusively on the in vitro calibrations could be underestimated to some extent because of effects of viscosity and dye binding to cytoplasmic constituents (Konishi et al., 1988). In previous experiments, we estimated that fura-2 reached intracellular concentrations between 30 and 50 M. This was done by comparing fluorescence levels attained in similar cells 10 min after breaking-in with patch pipettes filled with known concentrations of fura-2 pentapotassium (range 25-100 M). By comparing responses from cells loaded with fura-2 for 30-45 min with those from cells loaded with much less fura-2 (10-min incubation), we concluded that the additional Ca 2ϩ buffer activity introduced by the dye did not affect significantly the magnitude or the time course of Ca 2ϩ transients (unpublished data).

Solutions
Cells were continuously superfused with a recording solution containing (in mM) NaCl 130, KCl 3, CaCl 2 2, MgCl 2 2, NaHCO 3 1, NaH 2 PO 4 0.5, HEPES-Na 5, and glucose 5, pH 7.4. In some experiments CaCl 2 was replaced equimolarly by SrCl 2 . Test solutions were pressure-applied (10 psi) via independent puffer pipettes located within 100 m from the cell under examination. In some experiments, drugs (ryanodine, tetracaine, and the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-NNN ЈN Ј-tetraacetic acid [BAPTA-AM]) were applied via a third puffer pipette. Application of test solutions and drugs were separately controlled by solenoid valves of two Picospritzer II devices (General Valve, Fairfield, NJ) governed by an 486/AT computer running pClamp 5.1 (Axon Instruments, Foster City, CA). Control experiments showed that with this procedure, the external medium surrounding the cell is replaced within Ͻ100 ms. Most experiments were conducted in cells that had been kept in culture from 1 to 4 d. Experiments were carried out at 22-23ЊC. Test solutions used were: caffeine 10 or 20 mM, or tetracaine 500 M, dissolved in normal saline, and a depolarizing solution containing 140 mM KCl, 10 mM HEPES-Na and either 10 mM CaCl 2 or 10 mM SrCl 2 .

Sr 2ϩ Replacement Procedure
To substitute Sr 2ϩ for Ca 2ϩ both externally and internally, a coverslip containing sympathetic neurons was extensively superfused (45 min or longer) with normal external solution containing no Ca 2ϩ (no Ca 2ϩ added plus 500 M EGTA) and 2 mM SrCl 2 . During this period, the dish was exposed three times (1-min duration each) to a Sr 2ϩ -containing depolarizing solution (140 mM KCl, 2 mM SrCl 2 , 10 mM HEPES-Na, pH 7.35). In addition, to promote substitution of Ca 2ϩ with Sr 2ϩ in the intracellular stores, cells were bathed three times with saline containing 2 mM Sr 2ϩ and 10 mM caffeine (1-min duration each at 10-min intervals). The Sr 2ϩ replacement procedure had been completed before the beginning of fluorescence measurements.

Mathematical Modeling of Ca 2ϩ Mobilization from Intracellular Stores in Rat Sympathetic Neurons
The model consists of a linear arrangement representing the extracellular space, as well as the cytosol and the endoplasmic retic-ulum (ER), with boundary conditions located both at the plasma and at the ER membranes. The ER and the plasma membrane are coupled via n diffusional compartments. In the model, Ca 2ϩ diffuses freely between compartments and binds to fixed Ca 2ϩ buffers. Ca 2ϩ fluxes due to diffusion exchange with neighboring compartments and binding-unbinding to fixed buffers were computed by integrating Fick's law difference equations and mass action law differential equations, respectively. The boundary condition representing the plasma membrane allows Ca 2ϩ influx through voltage-gated Ca 2ϩ channels and Ca 2ϩ extrusion via Ca 2ϩ pumps. Similar Ca 2ϩ pumps are located at the boundary condition representing the membrane of the ER compartment, which also contains RyR channels which allow Ca 2ϩ release into the cytosol. These channels are regulated by two different cytosolic Ca 2ϩ binding sites operating with a first order kinetic scheme, which control activation and inactivation of the channel, respectively, and a lumenal regulatory binding site which controls channel conductance. The degree of activation or inactivation of this pathway is proportional to the fraction of total sites that are occupied with Ca 2ϩ . Caffeine-induced stimulation of RyRs was simulated by an abrupt change in the on-rate for Ca 2ϩ binding to the activation sites, with all remaining rate constants unaltered. A more detailed description of the model is included as an appendix. r e s u l t s

Rationale of Caffeine-induced Ca 2ϩ Release Experiments
Our basic experimental design allows the controlled activation of calcium influx through voltage-gated Ca 2ϩ channels and Ca 2ϩ release from intracellular stores in intact fura-2-loaded neurons (see Hernández-Cruz et al., 1995). Caffeine application provides two main advantages to analyze CICR phenomena in nerve cells with respect to voltage-gated Ca 2ϩ influx. First, since Ca 2ϩ is directly released from intracellular stores, [Ca 2ϩ ] in the microenvironment surrounding RyRs increases at a faster rate than when Ca 2ϩ diffuses from distant sources (i.e., the plasma membrane). Secondly, by increasing Ca 2ϩ affinity for the RyRs activation sites, caffeine promotes CICR (Sitsapesan and Williams, 1990). At the concentration used in these experiments (10 mM), caffeine produces half-maximal activation of release (Akaike and Sadoshima, 1989;Uneyama et al., 1993). This allows both up-and down-regulation of release by [Ca 2ϩ ] and other modulators (Hernández-Cruz et al., 1995). Previous work has shown that externally applied caffeine enters readily into cells, reaching equilibrium with extracellular concentration in Ͻ0.2 s (Hernández-Cruz et al., 1990;O'Neill et al., 1990;Kuba et al., 1990). Thus, the amount of Ca 2ϩ released will depend on three governing factors: (a) the prevalent cytosolic Ca 2ϩ concentration, (b) the number of RyRs opened per time unit by the drug, and (c) the calcium content of the store (Friel and Tsien, 1992;Hernández-Cruz et al., 1995;Shmigol et al, 1996). Changes of cytoplasmic [Ca 2ϩ ] resulting from Ca 2ϩ fluxes through the ER membrane can directly enhance (or inhibit) further release by interacting with regulatory Ca 2ϩ -binding sites on the RyR channels (Bezprozvanny et al., 1991;Györke and Patade, 1994;Meissner, 1994). Fig. 1 A depicts Ca 2ϩ transients recorded from a fura-2loaded sympathetic neuron in response to three brief applications of a high-K ϩ solution (arrows; see materials and methods), followed by several caffeine applications (10 mM, 40 s). Ca 2ϩ transients elicited by high-K ϩ and caffeine are termed here K ϩ -and C-Ca 2ϩ transients, respectively. Conditioning high-K ϩ pulses were applied to replete Ca 2ϩ stores before the first use of caffeine. Notice that the first Ca 2ϩ release comprises an early, fast release that decays before the end of the pulse, (transient release component, TR), followed by a slower and more sustained release (persisting release component, PR). When caffeine was applied again 140 s later, the TR component was obliterated, and only the PR component remained. Subsequent caffeine applications only produced smaller PRs with slower onsets. Fig.  1 B, obtained from a similar experiment, shows superimposed responses to the first and second caffeine applications. Both responses initiated with similar latencies, but the second one had a slower rate of rise and did not bring about a TR component. The first derivatives of these [Ca 2ϩ ] records are shown in the inset ( Fig. 1 C). Notice that during the second caffeine response the initial rate of rise was about half of that observed during the first response. The mean amplitudes of TR and PR components of release, measured shortly after the last conditioning high K ϩ pulse in 38 cells was TR ϭ 1,684.6 Ϯ 140 nM; PR ϭ 326.8 Ϯ 9.6 nM (mean Ϯ SE).

Two Kinetic Components of Caffeine-induced Ca 2ϩ Release
When exposed to caffeine for the first time, many cells show TR and PR components, even in the absence of conditioning high K ϩ applications (see Fig. 5). The TR component recuperates if caffeine is applied again 10-15 min later (data not shown). This slow recovery is greatly speeded up if one or more conditioning K ϩ -Ca 2ϩ transients are elicited during the interval between caffeine applications. This behavior is consistent with a use-dependent depletion of a slowly replenished caffeine-sensitive Ca 2ϩ store (Thayer et al., 1988;Friel and Tsien, 1992;Usachev et al., 1993).

Both Kinetic Components of Caffeine-induced Ca 2ϩ Mobilization Result from Intracellular Ca 2ϩ Release
It has been reported that caffeine can directly inhibit K ϩ currents in sympathetic neurons (Akaike and Sadoshima, 1989;Dryer et al., 1995). Therefore C-Ca 2ϩ transients could result in part from membrane depolarization as well as intracellular Ca 2ϩ release. It also has been shown that caffeine can directly activate Ca 2ϩ permeable channels in smooth muscle cells (Guerrero et al., 1994). The most rigorous test to rule out these po-tential problems would be to prevent completely and selectively Ca 2ϩ release from caffeine-sensitive stores. The plant alkaloid ryanodine is the most specific Ca 2ϩ release blocker known. Nevertheless, when ryanodine locks the release channel in a subconducting state (Russeau et al., 1987), it can significantly deplete Ca 2ϩ stores, hence increasing intracellular Ca 2ϩ levels by reducing the capacity of the cell to remove calcium (Kuba, 1994). This effect is most apparent when ryanodine (10-20 M) is applied continuously (see Fig. 8).
In preliminary experiments we noticed that 20 M of ryanodine can inhibit completely and irreversibly intracellular release after applications as brief as 5 s, without seriously affecting calcium homeostasis. Fig. 2 A shows the use-dependent inhibition of caffeine responses 1 and 4 min after a ryanodine application 5 s in duration. Fig. 2, B and C, depict the first derivatives of selected segments of these [Ca 2ϩ ] records. Ryanodine treatment did not change significantly basal Ca 2ϩ levels ( Fig. 2 A), or the magnitude or kinetics of the K ϩ -Ca 2ϩ transients. The time constant of an exponential function fitted to the decay phase of the K ϩ -Ca 2ϩ transients remained between 2.1 and 2.3 s. Also, the peak rate of rise (7.8 M s Ϫ1 in the control response), diminished only slightly after 1 and 4 min of exposure to the drug (7.1 and 6.4 M s Ϫ1 , respectively). Nevertheless, the amplitude and rate of rise of the C-Ca 2ϩ transients dropped from 2.8 M s Ϫ1 to 0.74 and 0.08 M s Ϫ1 after 1 and 4 min of ryanodine exposure (Fig. 2 C). Note that at 1 min post ryanodine, caffeine elicits a delayed and partially inhibited TR component, whereas only a reduced PR persists after 4 min. 10 min after ryanodine application, caffeine responses were completely abolished, both in this cell and in five other cells similarly examined (data not shown). Three conclusions can be drawn from these re-sults: (a) brief ryanodine applications can block Ca 2ϩ release without affecting resting Ca 2ϩ levels or the kinetics of K ϩ -Ca 2ϩ transients, (b) both kinetic components of the caffeine induced-Ca 2ϩ mobilization result entirely from intracellular Ca 2ϩ release, and (c) the TR component appears more susceptible to use-dependent inhibition of release by ryanodine than the PR component. 2

The Effects of Caffeine Pretreatment on the Kinetic Behavior of Subsequent Responses Are Not Due to Caffeine Desensitization
A conceivable explanation for the effects of caffeine pretreatment on the kinetic behavior of subsequent responses is that it produces a desensitization of Ca 2ϩ stores that persist for several minutes after the agonist is removed. To explore this possibility, the effect of pretreating the cell with a brief conditioning caffeine application on a test response was examined. To distinguish between the effects of caffeine per se from those resulting from depletion of the Ca 2ϩ store, we took advantage of the Ca 2ϩ -induced inhibition of Ca 2ϩ release characteristic of these cells (Hernández-Cruz et al., 1995). Conditioning caffeine applications were initiated at different intervals from a standard K ϩ -Ca 2ϩ transient. Caffeine, applied at intervals shorter than 10 s (when [Ca 2ϩ ] is still relatively high), is ineffective at releasing Ca 2ϩ . When this interval is increased, caffeine becomes progressively more effective. Fig. 3 A shows that when the conditioning caffeine application does not release any Ca 2ϩ , a test caffeine response 90 s later displays both TR and PR components (continuous trace). The same is true when only a small Ca 2ϩ release occurs during the conditioning caffeine application (dashed trace). However, when the conditioning caffeine application brings about a substantial Ca 2ϩ release, the TR component of the succeeding test response is completely abolished (dotted trace). This experiment rules out desensitization of the release, and clearly establishes that the diminution of the TR component during the second caffeine application only occurs if the first application produces substantial opening of Ca 2ϩ -release channels and/or Ca 2ϩ efflux from the intracellular stores. This inference is further supported by experiments shown in Fig. 3, B and C. First, two identical applications of 10 mM caffeine were given at an interval of 2 min (continuous traces, Fig. 3 B and C). The second response shown in Fig. 3 C displays the usual inhibition of the TR component. The experiment was then re- peated, but now the second response was elicited by delivering 20-mM caffeine with a third pipette, instead of 10 mM (dotted trace). This experiment was designed to try to activate more efficiently desensitized Ca 2ϩ stores with a higher dose of caffeine. Although the second response elicited with 20 mM caffeine was slightly larger (Fig. 3 C, arrow) and had a faster rate of rise ( Fig. 3 C, inset), it was still unable to trigger a complete TR response. Similar results were obtained in experiments where even higher caffeine concentrations (i.e., 40 mM) were used to elicit the second response (data not shown).

The Kinetic Behavior of Caffeine-induced Ca 2ϩ Release Depends upon the Filling Status of Intracellular Stores
As mentioned above, the obliteration of the TR component during subsequent applications of caffeine is a consequence of the opening of Ca 2ϩ release channels and/or Ca 2ϩ liberation from internal stores during the first application. Experiments were conducted to try to decide between these possibilities. We compared the kinetics of release under conditions where presumably an equal number of release channels were opened, but with the Ca 2ϩ store filled up to different degrees. Fig. 4 A shows the gradual recovery of the TR component after the application of one to four small "filler" conditioning K ϩ -Ca 2ϩ transients. Fig. 4 B depicts the first derivative of Ca 2ϩ concentration records shown in Fig. 4 A. The plot in Fig. 4 C illustrates the relationship between the conditioning K ϩ -Ca 2ϩ transients versus the resulting Ca 2ϩ release (open circles) and the magnitude of the peak rate of rise of the C-Ca 2ϩ transients (filled squares). In this figure, as well as in Fig. 5, the areas under the K ϩ -and C-Ca 2ϩ transients are plotted rather than peak Ca 2ϩ concentrations to better represent the magnitude of the Ca 2ϩ increase. Notice that a TR component was not produced in this cell when caffeine was applied without a conditioning K ϩ -Ca 2ϩ transient. It is clear from Fig. 4 C that both the magnitude of the C-Ca 2ϩ transients and their peak rate of rise reach saturation after three to four conditioning K ϩ -Ca 2ϩ transients. These results are expected if the generation of the TR response requires sufficient loading of intracellular Ca 2ϩ reservoirs with limited capacity for Ca 2ϩ storage. Since the supply of additional Ca 2ϩ by transmembrane Ca 2ϩ influx readily reestablishes the transient component of release, a long-lasting "fatigue" of Ca 2ϩ release channels cannot explain the TR abatement phenomena.
A somewhat different behavior often observed in the recovery of the TR component is shown in Fig. 5. Unlike the cell in Fig. 4, this cell displayed a TR response when first challenged with caffeine ( Fig. 5 A, trace a). Cells exhibiting this behavior are common during the first few days in culture. Here, the replenishment of the Ca 2ϩ store was carried out by preceding each caffeine application with a single conditioning K ϩ -Ca 2ϩ transient, whose magnitude was regulated by varying the duration of the high K ϩ application. As shown in the plot shown in Fig. 5 B, the area under the C-Ca 2ϩ transient increases gradually as the area under the filler Ca 2ϩ transient increases. This suggests a close correlation between the filling status of the store and the magnitude of the subsequent Ca 2ϩ release. Interestingly, an abrupt increase in the magnitude of the C-Ca 2ϩ tran- sient occurs when the area under the filler Ca 2ϩ transient reaches a critical value. This discontinuity coincides with the generation of the TR component ( Fig. 5  A, trace k). These results support the notion that an allor-none regenerative mechanism underlies the TR component. Further increasing the area under the K ϩ -Ca 2ϩ transient did not increase the size of the TR component nor the area under the C-Ca 2ϩ transient (Fig. 5 A, trace l), as if the release were self-limited or the stores had a finite capability to accumulate Ca 2ϩ . Previous studies have shown that Ca 2ϩ stores of intact cardiac myocytes reach a limiting Ca 2ϩ content that cannot be exceeded regardless of the loading stimulation protocol used (Bassani et al., 1995). Feedback inhibition of Ca 2ϩ uptake by Ca 2ϩ load of intracellular stores (Favre et al., 1996) may explain the apparent limiting Ca 2ϩ content. Notice that the response elicited by the initial caffeine application (trace a) is very similar to that produced when the reservoir is full (see traces k and l). This suggests that in resting sympathetic neurons a mechanism exists that ensures that caffeine-sensitive Ca 2ϩ stores remain filled nearly to the limit of their capacity, thereby allowing an expeditious Ca 2ϩ efflux whenever RyRs open, either in response to Ca 2ϩ or other endogenous agonist. 3 We also tested whether or not lower concentrations of caffeine were capable of eliciting a TR response provided that Ca 2ϩ stores were replete. Under conditions where 10 mM caffeine invariably gave a TR component, 3 or 5 mM of caffeine was unable to produce it (data not shown). These results indicate that the development of the TR component requires both a high Ca 2ϩ content of intracellular stores and the activation of a sufficient number of Ca 2ϩ release channels. Either of these conditions, occurring separately, do not support a TR response.

Tetracaine and Intracellular BAPTA Suppress the TR Component
Local anesthetics tetracaine and procaine affect excitation-contraction coupling by reducing Ca 2ϩ -induced Ca 2ϩ release (Endo, 1985). Thus, if the TR component was the result of regenerative activation by CICR, it should be reduced or even abolished by tetracaine. For these experiments, tetracaine was applied from a third puffer pipette for 30 s (only during the interval between the last conditioning K ϩ -Ca 2ϩ transient and the test caffeine application). This maneuver allowed us to explore the effects of tetracaine on the kinetics of the release mechanism without affecting voltage-gated Ca 2ϩ influx or Ca 2ϩ loading of intracellular stores. Fig. 6 A shows control responses from a sympathetic neuron to the filler high K ϩ applications and then to 10 mM caffeine. A TR component is clearly observed in response to caffeine. As shown in Fig. 6 B, the use of 500 M tet- (C, open circles and left y-axis) Relationship between the total area under the conditioning K ϩ -Ca 2ϩ transients and the area under the resulting Ca 2ϩ release. (Filled squares and right y-axis) Peak rate of rise obtained from the derivatives shown in B. A transient component was absent from this cell when caffeine was applied without a conditioning K ϩ -Ca 2ϩ transient. Notice that both the magnitude and peak rate of rise of caffeine responses reached saturation after 3-4 conditioning high K ϩ applications. Horizontal arrow indicates resting [Ca 2ϩ ]. racaine before the caffeine application completely suppressed the TR component, with less effect on the PR component. The TR component recovered after 5 min of tetracaine wash-out (see Fig. 6 C). Similar results were obtained in 5 other experiments. In 13 additional experiments, tetracaine reduced the amplitude of both TR and PR release components to 1,177.8 Ϯ 187 and 269 Ϯ 24.1 nM (mean Ϯ SE), respectively. These values are 30 and 20% smaller, respectively, than the control data (see above). In many of these cases, the upstroke of the TR component was delayed with respect to their control response after exposure to tetracaine (see Fig.  6 D). This delay (measured as the time interval between the peaks of the derivatives of the Ca 2ϩ transients, see Fig. 6 E), ranged form 1.5 to 7.48 s (4.5 Ϯ 0.63 s; mean Ϯ SE; n ϭ 13), and probably results from tetracaine not being present long enough to abolish the positive feedback mechanism that underlies the TR component.
The tetracaine sensitivity of the TR component supports the notion that it may constitute a regenerative response fueled by CICR. This presumption was corroborated by experiments where the kinetics of caf-  feine-induced release were examined before and after the cells had been incubated with the acetoxymethyl ester form of the fast calcium buffer BAPTA (10 M). A combination of BAPTA-AM concentration and incubation time was found such that the time constants of decay of K-Ca 2ϩ transients increased slightly, but their amplitude did not show a drastic reduction (see Fig. 7 C). This condition indicated that intracellular BAPTA had reached sufficient concentration to began competing with Ca 2ϩ extrusion mechanisms, but not enough to significantly compete with fura-2 for Ca 2ϩ binding. As shown in Fig. 7, A and B, after 10 min of incubation, BAPTA had two main effects: (a) it slowed the decay of the filler K ϩ -Ca 2ϩ transients (a monoexponential fit to the decay phase of the third K ϩ -Ca 2ϩ transient gave a time constant of 6.4 s before and 10.6 s after BAPTA), and (b) it eliminated the TR component. Similar results were obtained in 5 additional experiments. In 9 other cases, where the TR component was spared (see an example in Fig. 7 C), the combined amplitude of TR and PR components diminished after incubation with BAPTA from 923.6 Ϯ 91.4 nM to 739.5 Ϯ 91.4 nM (mean Ϯ SE). In addition, TR and PR components after BAPTA treatment were only 500.3 Ϯ 59.6 and 257.7 Ϯ 33 nM, respectively. It is noteworthy that after incubation with BAPTA, the separation between the TR and PR components became much less discernible (Fig. 7 C), as if BAPTA was also impeding Ca 2ϩ binding to inhibitory regulatory sites on the RyRs which may be partially responsible for the decay of the TR component (see below).
All experiments depicted so far support the hypothesis that CICR underlies the fast release component initiated by caffeine in nerve cells: This component results entirely from intracellular Ca 2ϩ release and is inhibited by tetracaine and intracellular BAPTA, which interfere with Ca 2ϩ -mediated positive feedback loops by two completely different mechanisms. The suppression by intracellular BAPTA or tetracaine of a fast kinetic component of calcium release in skeletal muscle is one of the strongest evidences supporting the assertion that it constitutes a release component controlled by calcium (Jacquemond et al., 1991;Pizarro et al., 1992). An important property of the TR component is that its appearance requires the simultaneous activation of a significant fraction of Ca 2ϩ -release channels together with an adequate loading of intracellular Ca 2ϩ stores. This property suggests a dependence on the rate of rise of trigger calcium.

Mechanisms Involved in the Termination of the TR Component: Sr 2ϩ Substitution
The kinetics of Ca 2ϩ release depicted in this study are consistent with a Ca 2ϩ -dependent dual feedback con-trol mechanism proposed for cardiac cells (Fabiato, 1985). According to this model, the abrupt termination of the TR component could be explained by Ca 2ϩ -dependent inactivation of release. However, other factors, such as adaptation of RyR/Ca 2ϩ release channels (Györke and Fill, 1993;Stern, 1996) or rapid depletion of releasable Ca 2ϩ from intracellular stores cannot be ruled out. Ca 2ϩ substitution with permeant divalent cations like Sr 2ϩ or Ba 2ϩ has provided valuable insights on Ca 2ϩ dependent phenomena, such as Ca 2ϩ -mediated inactivation of voltage-gated Ca 2ϩ channels (Tillotson, 1979). For the most part, these ionic replacement experiments have been useful because Ba 2ϩ and Sr 2ϩ do not interact as efficiently as Ca 2ϩ with regulatory binding sites (Oberhauser et al., 1988). Since Sr 2ϩ binds to fura-2 with relatively high affinity, and the spectral properties of the complex are similar to those of fura-2 bound with Ca 2ϩ ( Kwan and Putney, 1990), we decided to examine the effects of substituting Sr 2ϩ for Ca 2ϩ on the kinetics of TR and PR components. We were hoping that this maneuver would provide clues to better understand the mechanisms that underlie onset and termination of the release process. Fig. 8 shows fura-2 fluorescence changes obtained from a rat sympathetic neuron after undergoing the Sr 2ϩ replacement procedure described in materials and methods. As shown in the first half of Fig. 8, both depolarization and caffeine-induced fluorescence changes continue in Sr 2ϩ -containing saline. This demonstrates that Sr 2ϩ substitutes well for Ca 2ϩ both in voltage-gated influx and agonist-mediated intracellular release. Control experiments showed that responses to both stimuli were eliminated after manipulating the cells in a similar fashion but with solutions lacking both Ca 2ϩ and Sr 2ϩ (data not shown). Sr 2ϩ transients reach much higher concentrations than Ca 2ϩ transients do. This could be due in part to the fact that Ca 2ϩ binding proteins and Ca 2ϩ extrusion mechanisms bind Sr 2ϩ less efficiently than Ca 2ϩ . Nonlinearity of the dye signal should not be a problem here, since the maximum concentrations reached (about 10 M) are similar to the k d of fura-2 for Sr 2ϩ (7.6 M; Kwan and Putney, 1990). The kinetics of Sr 2ϩ transients shown in Fig. 8 is quite different from that recorded in the presence of Ca 2ϩ . Most noticeably, Sr 2ϩ excursions decay more slowly, reflecting a less efficient Sr 2ϩ uptake and extrusion by metabolic pumps (Rasgado-Flores et al., 1987;Berman and King, 1990). Further diminution of buffering ca-pacity is observed when the cell is stimulated repeatedly. The time constants of monoexponential functions fitted to the decay phases ( d ) of depolarization-induced (small arrows) and caffeine-induced (large arrows) Sr 2ϩ signals, increased progressively from their initial values 7.3 and 6.8 s, respectively, (first pair of responses in Fig.  8) to 10.2 and 11.84 s (sixth pair of responses). Near the middle of the recording, 20 M ryanodine was added to the bath solution and alternate stimulation with high-K ϩ and caffeine was restated 2 min later. Shortly after its application, ryanodine produced a step increase in resting [Sr 2ϩ ] and reduced the rate of decay of the next caffeine response ( d ϭ 19.7 s). Once the use-dependent suppression of caffeine responses was established (asterisks), the rate of decay of depolarization-induced Sr 2ϩ transients increased significantly, from a d ϭ 10.0 s (first deflection after stimulation reinitiated) to 17.0, 17.1, 18.7, and 19.9 s, in the following four responses. The effects of continuous ryanodine exposure are similar to those observed in the presence of Ca 2ϩ (data not shown) and are consistent with a locking of the release channels in an open state (Pessah and Zimanyi, 1991). Fig. 9 compares fluorescence changes recorded from another cell, first, when it was bathed in Ca 2ϩ -containing saline (Fig. 9 A) and then in Sr 2ϩ -containing saline ( Fig. 9 B), immediately after undergoing the Sr 2ϩ replacement procedure (see materials and methods). The stimulation protocol consisted of four conditioning 0.5-s pulses of high K ϩ with 10 mM of either Ca 2ϩ or Sr 2ϩ to preserve the filling status of intracellular stores, Figure 8. Recording of fura-2 fluorescence changes in a sympathetic neuron after undergoing the Sr 2ϩ replacement procedure (see materials and methods). High K ϩ (1 s; short arrows) and 10 mM caffeine (20 s; long arrows) were applied alternatively with independent puffer pipettes. Near the middle of the recording, 20 M ryanodine was added to the bath, and 2 min later, alternate stimulation with high-K ϩ and caffeine restated. Caffeine responses are eliminated in a use-dependent manner after exposure to ryanodine (asterisks). Horizontal arrow indicates resting [Sr 2ϩ ]. followed by two pulses of 10 mM caffeine in saline containing either Ca 2ϩ or Sr 2ϩ , to examine the kinetics of release. Besides the differences mentioned before, the most notorious dissimilarities were in the dynamics of release from intracellular stores. In the presence of Ca 2ϩ , the first caffeine application elicited TR and PR components, with distinct rates of decay ( dTR ϭ 4.2 s, dPR ϭ 43.9 s). In contrast, only a single component of release with a monoexponential decay ( dPR ϭ 11.5 s) was apparent after the Sr 2ϩ replacement procedure. Fundamental differences also existed in the response to the second caffeine application: While in Ca 2ϩ the TR component was abolished with only the PR component remaining ( dPR ϭ 59 s), a significant initial fast release persisted in Sr 2ϩ . Moreover, the rate of rise of the second caffeine response in Sr 2ϩ was almost as large as the first one (compare Fig. 9, C and D). The decay of the second caffeine-induced Sr 2ϩ release was also fitted with a single exponential function ( d ϭ 11.1 s). Fig. 9 C shows the first derivatives of the signals resulting from transmembrane Ca 2ϩ influx and intracellular Ca 2ϩ release. If we focus our attention in the last K-Ca 2ϩ transient and in the ensuing C-Ca 2ϩ transient, when the Ca 2ϩ content of the store is similar (Fig, 9 C, asterisks), it is clear that the latter rises faster, possibly because of the positive feedback amplification that underlies the generation of the TR response, as opposed to the modest amplification exerted on Ca 2ϩ signals originated from Ca 2ϩ influx. The same comparison in Fig. 9 D (asterisks) reveals that the differences are smaller in Sr 2ϩ . These results are consistent with the notion that Sr 2ϩinduced Sr 2ϩ release may operate with lower amplification gain than CICR in rat sympathetic neurons.
The kinetics of caffeine-induced Sr 2ϩ mobilization is consistent with Sr 2ϩ release operating with lower feedback loop gain (both positive and negative) than the release of Ca 2ϩ . Consequently, a distinct TR response was not produced in Sr 2ϩ , and the diminution of the initial fast release during the second caffeine challenge was less pronounced. When Ca 2ϩ was the divalent cation involved, substantial amplification by CICR determined a conspicuous TR response, whose abrupt termination may result of both rapid depletion of releasable Ca 2ϩ and Ca 2ϩ -dependent inactivation/adaptation of RyR channels (see discussion).  Caffeine application was simulated by increasing the association rate constant for Ca 2ϩ binding to the activation sites, which greatly increases the open probability of release channels. When intracellular reservoirs are filled with sufficient Ca 2ϩ (first caffeine response), a steep initial release of Ca 2ϩ is produced (Fig. 10 B, thin  line). Because of their faster association rate constant, the activation sites are occupied more quickly by the step-like increase in [Ca 2ϩ ] than the inactivation sites ( Fig. 10 C, left inset), promoting further release that in turn may act as trigger Ca 2ϩ to activate additional RyRs. This positive feedback loop is responsible for the regenerative response here termed TR component. The early phase of fast release is terminated by way of two independent processes: (a) slower but more prolonged occupation of the inactivation Ca 2ϩ binding sites of the RyRs, leading to a decrease in open probability (Fig. 10  C, thick line), and (b) depletion of [Ca 2ϩ ] in the ER compartment ( Fig. 10 B, thin line), which reduces the driving force for calcium efflux. A more sustained release at a lower rate is maintained throughout the caffeine application (PR component). When caffeine is reapplied after a relatively short delay, a smaller Ca 2ϩ ef-  . Simulation similar to that of Fig. 10, but with eleven depolarizing pulses given during the interval between the two caffeine applications to rapidly refill intracellular Ca 2ϩ stores. The initial parameters used were the same as those in the simulation of Fig. 10, with the addition of Ca 2ϩ flux pulses of Ca ChDur ϭ 0.5 s in duration, interstimulus interval Ca Chint ϭ 5 s and a rate Ca Chmax ϭ 40 s Ϫ1 m Ϫ2 . flux is produced because the stores have not refilled completely (Fig. 10 B, thick line). Consequently, Ca 2ϩ accumulates more slowly at the cytoplasmic side of the RyRs, with the change in [Ca 2ϩ ] resembling more a ramp than a step. In this case, the inactivating sites of the RyR are occupied with Ca 2ϩ almost as quickly as the activation sites (Fig. 10 C, right inset), thereby suppressing the regenerative TR response. The situation is comparable to that of voltage-gated sodium channels induced to open with a slowly rising voltage ramp. Inactivation overcomes activation, thus preventing the generation of an action potential. As shown in Fig. 11, our model also predicts the recuperation of the TR response if additional Ca 2ϩ is supplied by way of voltage-gated Ca 2ϩ influx before the second caffeine application. These episodes of additional Ca 2ϩ influx increase significantly the Ca 2ϩ content of intracellular stores (Fig. 11 B, thick  line), but since they produce relatively slow changes in [Ca 2ϩ ] at the RyR regulatory sites, inactivation dominates activation and no CICR is produced (Fig. 11 C; compare thick and thin lines). The production of a TR component during the second caffeine application results from the increased initial Ca 2ϩ efflux, which, by allowing a faster occupation of the activation sites with Ca 2ϩ than the inactivation sites (Fig. 11 C, inset), provide the means for triggering a regenerative release response.

The Main Kinetic Aspects of Caffeine-induced Release Can Be Reproduced with a Simple Mathematical Model
According to this very simplified model, the filling status of intracellular stores is one of the most relevant aspects governing the kinetics of Ca 2ϩ release, by determining the rate of change of cytosolic [Ca 2ϩ ] and the occupation rate of activation Ca 2ϩ binding sites of the RyRs. Undoubtedly, a multiplicity of other parameters, such as spatial organization of Ca 2ϩ stores and buffering mechanisms, as well as interaction between neighboring release channels and Ca 2ϩ stores, must come into play to determine the spatial distribution and release dynamics of Ca 2ϩ signals in real cells (Stern, 1992). Nevertheless, the conclusions from this work provide certain constraints as to which functional roles neuronal Ca 2ϩ stores can and cannot perform. In this regard, ryanodine-sensitive stores appear to be organized to emphasize subcellular Ca 2ϩ gradients initiated by agonist-induced intracellular release, rather than to produce widespread amplification of Ca 2ϩ signals originated from transmembrane Ca 2ϩ influx. Naturally, this does not exclude the possibility that ryanodine-sensitive stores located near the plasma membrane may produce substantial local amplification of Ca 2ϩ signals originated from transmembrane Ca 2ϩ influx.

d i s c u s s i o n
Voltage-gated Ca 2ϩ influx in nerve cells is not very effective to initiate widespread CICR under physiological conditions . A likely explanation is that Ca 2ϩ must diffuse from the plasmalemma to the endoplasmic reticulum over relatively long distances, and [Ca 2ϩ ] cannot rise sufficiently fast near the RyRs to effectively activate them (Hernández-Cruz, et al., 1995;Shmigol et al., 1995). Conversely, Ca 2ϩ released directly from intracellular reservoirs is more likely to efficiently change [Ca 2ϩ ] in the microenvironment surrounding the RyRs. This implies that both positive and negative feedback regulation of Ca 2ϩ release by Ca 2ϩ (Fabiato, 1985) should be more apparent after intracellular release than after transmembrane calcium entry. These assumptions are supported by the experiments described here. One possible exception to this scheme would be the RyRs located on specialized endoplasmic reticulum organelles, called subsurface cisternae (Watanabe and Burnstock, 1976). In fact, the most convincing examples of physiologically relevant CICR phenomena after transmembrane Ca 2ϩ influx in nerve cells involve the regulation of plasmalemmal channels, presumably by Ca 2ϩ released from submembrane domains (Kawai and Watanabe, 1989;Sah and McLachlan, 1991;Yoshizaki et al, 1995).
This study was aimed to identify the physiological parameters that determine the dynamics of Ca 2ϩ release from ryanodine-sensitive Ca 2ϩ stores in rat sympathetic neurons. For simplicity, we focused on Ca 2ϩ release phenomena triggered by caffeine, but the same conclusions should be applicable to Ca 2ϩ release initiated by natural agonists . Caffeine-induced release comprises two ryanodine-sensitive kinetic components, a fast initial release (TR component), and a delayed, persistent release (PR component). The early event shows refractoriness resulting from the depletion of releasable Ca 2ϩ , and requires both the adequate activation of Ca 2ϩ release channels and sufficient loading of intracellular stores. This behavior suggests a strong dependence on the rate of rise of trigger calcium. Tetracaine and BAPTA-AM, which interfere with Ca 2ϩmediated positive feedback loops by two different mechanisms, suppress the TR component, supporting the notion that it is a CICR phenomenon. Our data also suggest that the abrupt termination of the TR component results both from rapid depletion of releasable Ca 2ϩ and Ca 2ϩ -dependent inactivation or adaptation of RyRs.

Caffeine-induced Intracellular Ca 2ϩ Mobilization in Nerve Cells Comprises Two Kinetic Components, One of which Is a CICR-dependent Phenomenon
This work provides evidence for two separate components of caffeine-induced Ca 2ϩ release. The TR component is expected from the regenerative nature of CICR if released Ca 2ϩ can promote its own release by a positive feedback mechanism (see below). The PR compo-nent may result from a sustained nonregenerative activation of release balanced with Ca 2ϩ uptake (Kuba, 1994).
The TR component can only be observed when Ca 2ϩ stores are replete. A similar loading-dependent property has been described for InsP 3 -induced Ca 2ϩ release in smooth muscle cells (Iino and Endo, 1992) and Ca 2ϩ release from the sarcoplasmic reticulum of intact cardiac myocytes (Bassani et al., 1995). Several circumstances may combine to explain the effects of increasing the Ca 2ϩ content of the store: (a) an increased transmembrane Ca 2ϩ gradient, (b) an increased rate of Ca 2ϩ release (Donoso et al., 1995), (c) an increased open probability of release channels (Vélez and Suárez-Isla, 1992;Sitsapesan and Williams, 1994;Tripathy and Meissner, 1996), and (d) an increased caffeine sensitivity of overfilled neuronal Ca 2ϩ stores . Also, CICR is strongly dependent on the rate of rise of trigger calcium, a phenomenon that probably involves fast competition between Ca 2ϩdependent activation and inactivation of release channels (Fabiato, 1985(Fabiato, , 1992; see below). Thus, the filling status of the Ca 2ϩ store may affect the kinetics of release to the extent that it affects the initial rate of Ca 2ϩ release. Accordingly, a second TR response cannot be generated (even with a high caffeine concentration) when Ca 2ϩ stores are partially depleted, probably because [Ca 2ϩ ] does not rise sufficiently fast to initiate CICR. Experiments in cardiac myocytes have demonstrated that the fractional Ca 2ϩ release (the percentage of SR Ca 2ϩ content released by a twitch) increases steeply as the SR Ca 2ϩ load increases. Conversely, Ca 2ϩ release is almost abolished when SR content falls below 40% of its maximum (Bassani et al., 1995).
Our results are inconsistent with the "quantal Ca 2ϩ release" hypothesis according to which individual stores release their content in an all-or-none manner once their threshold for caffeine is reached (Cheek et al., 1994). If this scheme were true, application of caffeine concentrations Ͻ10 mM should elicit CICR phenomena involving low-threshold caffeine-sensitive Ca 2ϩ stores. Likewise, the recruitment of Ca 2ϩ stores with higher caffeine threshold should give rise to TR responses of their own, regardless of prior activation of low-threshold Ca 2ϩ stores. This type of behavior was not observed (see Fig. 3 C). Recent experiments in a skeletal muscle cell line (Györke and Györke, 1996), suggest that the incremental Ca 2ϩ release phenomenon cannot be accounted for by "quantal" Ca 2ϩ release but by adaptive control of release units (Györke and Fill, 1993).
The TR component has properties expected from a CICR phenomena: (a) it exhibits refractoriness, (b) it requires the activation of minimum fraction of Ca 2ϩrelease channels (threshold), (c) it requires sufficient loading of intracellular Ca 2ϩ reservoirs (i.e., it depends on the rate of rise of trigger Ca 2ϩ ), (d) it behaves as an all-or-none regenerative process once a critical amount of loading is achieved, and (e) it is inhibited by drugs that restrict or abolish Ca 2ϩ -mediated positive feedback loops. One important conclusion of these experiments is that CICR in nerve cells is possible whenever the conditions are met which allow the generation of adequate trigger Ca 2ϩ signals. Interestingly, freshly dissociated sympathetic neurons, or cells that have been in culture for 1-3 d, maintain their caffeine-sensitive Ca 2ϩ stores occupied nearly to the limit of their capacity, despite the absence of spontaneous electrical activity. This physiological status supports an effective Ca 2ϩ efflux whenever RyR open, either in response to Ca 2ϩ or any other endogenous agonist (Bassani et al., 1995). This differs from the situation in older cultures of sympathetic neurons or in most studies with central neurons, where in order to obtain full-sized caffeine responses, ER stores have to be charged with releasable Ca 2ϩ beforehand (Shmigol et al., 1994).

Tetracaine and BAPTA-AM Suppress the TR Component of the Release by way of Different Mechanisms
The local anesthetic tetracaine reduces Ca 2ϩ -induced Ca 2ϩ release in several preparations (Endo, 1985). Presumably, these effects result from direct inhibition of channel activity by binding to high-affinity, regulatory sites on the RyRs (Xu et al., 1993). In our experiments, tetracaine reached the cell only during the interval between the last conditioning K ϩ -Ca 2ϩ transient and the following caffeine application. This was necessary to prevent tetracaine effects on voltage-gated Ca 2ϩ influx and Ca 2ϩ loading of intracellular stores that would have resulted from inhibition of voltage-gated Ca 2ϩ channels with the tetracaine concentrations used here (Sugiyama and Muteki, 1994). In preliminary experiments, when tetracaine was applied continuously, rather than for a short period of time, we observed that the TR responses were always blocked. The brief exposure to tetracaine in the experiments reported here underestimates its effects on caffeine-induced Ca 2ϩ release. In fact, they clearly show that CICR is more sensitive to tetracaine than voltage-gated Ca 2ϩ influx, since it was able to delay, and in some cases suppress the TR component, sparing the PR component. These results are consistent with the reported inhibition by tetracaine of Ca 2ϩ -gated channel activity by an allosteric mechanism rather than by blockade of the RyR channel (Xu et al., 1993).
In contrast to tetracaine, BAPTA does not interact directly with the RyRs. We used this high-affinity fast diffusible Ca 2ϩ buffer to try to prevent the interaction between released Ca 2ϩ and RyRs regulatory Ca 2ϩ binding sites. A measure of the ability of exogenous Ca 2ϩ buff-ers to compete with other Ca 2ϩ binding sites is their association rate constant (k on ). Since BAPTA's k on is almost diffusion limited ‫2.6ف(‬ ϫ 10 8 M Ϫ1 s Ϫ1 ; see Kao and Tsien, 1988) and free BAPTA is likely to equilibrate with cytosolic Ca 2ϩ in Ͻ3 s (Adler et al., 1991), it is expected to compete effectively for Ca 2ϩ with the RyR activating sites, thus limiting the feedback loop gain that underlies CICR phenomena. In some experiments, BAPTA was able to suppress the TR component without affecting Ca 2ϩ transients induced by Ca 2ϩ influx (see Fig. 7 B). In other cases, the TR component was spared, but the separation between TR and PR components became less discernible (see Fig. 7 C, dotted trace). These effects of BAPTA on the kinetics of decay of the caffeine-induced release are reminiscent of the effects of Sr 2ϩ substitution (see Fig. 9 B). In both cases, the decay of the TR component becomes slower, conceivably as a result of a decreased interaction of the divalent cation with inhibitory regulatory binding sites on the RyRs (see below). Cells massively loaded with fura-2, (1-h incubation or more) often display alterations of Ca 2ϩ dynamics similar to those produced by incubation with BAPTA AM.

Substitution of the Permeant Divalent Cation Provides Kinetic Information on the Onset and Termination of the TR Component of the Caffeine Response
A model of RyR dynamics has been postulated for cardiac cells, whereby the cytoplasmic side of the channel possess two regulatory Ca 2ϩ binding sites, one that is fast and positively regulatory and another that is slow and negatively regulatory (Fabiato, 1985). A feedback control by Ca 2ϩ on IP 3 -mediated Ca 2ϩ release has been demonstrated in flash-photolysis experiments of caged IP 3 and caged Ca 2ϩ in permeabilized smooth muscle fibers (Iino and Endo, 1992). Caffeine-induced release in mammalian sympathetic neurons exhibits a bellshaped dependence on cytosolic Ca 2ϩ concentration, which could give rise to positive feedback in the earlier phase of release followed by negative feedback due to Ca 2ϩ -dependent inactivation (Hernández-Cruz et al., 1995). Although the kinetics of Ca 2ϩ release depicted in the present study was seemingly consistent with this dual feedback control mechanism, it was uncertain to what extent the termination of the TR component could depend on Ca 2ϩ -dependent inactivation since for the most part, the reported biphasic Ca 2ϩ dependence is more closely related to steady-state conditions. Furthermore, other factors related to the release process, such as adaptation of RyRs (Györke and Fill, 1993) and Ca 2ϩ depletion of intracellular stores, could also influence this process.
The Ca 2ϩ content of the caffeine-sensitive store diminishes after the generation of a TR response (see Figs. 1 and 3 A). We can speculate that if local depletion of releasable Ca 2ϩ occurs at a sufficiently fast rate, the feedback gain that sustains the activity of release channels would be significantly reduced, thus playing a role in the termination of the TR. Ca 2ϩ -dependent inactivation or adaptation, described for RyRs isolated from cardiac sarcoplasmic reticulum (Györke and Fill, 1993), could also participate in terminating the release. Direct evidence for the relative contribution of these mechanisms to the termination of release is difficult to obtain in intact cells, because manipulations that affect Ca 2ϩ binding to the inactivation site(s) are likely to affect binding to the activation site(s) as well. Also, cytoplasmic Ca 2ϩ removal mechanisms (buffering, sequestration, and extrusion of Ca 2ϩ ) can become affected in unpredictable ways.
Sr 2ϩ permeates through both plasmalemmal and RyR Ca 2ϩ channels (Hagiwara and Ohmori, 1982;Williams, 1992) and is taken up by neuronal intracellular stores (Rasgado-Flores et al., 1987). Moreover, Sr 2ϩ influx can induce Ca 2ϩ release from caffeine-sensitive stores in smooth muscle cells (Grégoire et al., 1993) and binds to fura-2 with high affinity, producing the same type of changes as Ca 2ϩ in spectral properties of the dye (Kwan and Putney, 1990). Nevertheless, in part because of its larger ionic radius (Hille, 1992), Sr 2ϩ is not expected to interact with Ca 2ϩ binding sites on the RyR channel as efficiently as Ca 2ϩ . An impaired interaction of Sr 2ϩ with regulatory binding sites has been well documented for Ca 2ϩ -activated K ϩ channels, where Sr 2ϩ is ‫061ف‬ times less effective than Ca 2ϩ as a channel agonist (Oberhauser et al., 1988). Given these properties, we reasoned that by examining the effects of substituting Ca 2ϩ for Sr 2ϩ we could gain clues to better understand the mechanisms that underlie the dynamics of caffeineinduced Ca 2ϩ release.
As shown in Fig. 8, Sr 2ϩ can substitute for Ca 2ϩ both in voltage-gated influx and caffeine-induced intracellular release in rat sympathetic neurons. Caffeine-induced signals were abolished by ryanodine, demonstrating that they indeed represent Sr 2ϩ release from intracellular stores. A gross contamination by Ca 2ϩ in these experiments was ruled out because (a) external Ca 2ϩ concentration was maintained below 100 nM throughout the experiment by excluding Ca 2ϩ and adding 0.5 mM EGTA to all external solutions, and (b) fura-2 fluorescence signals disappeared altogether when Sr 2ϩ was omitted from the external medium. A potentially significant problem could result if a small amount of intracellular Ca 2ϩ persists after the Sr 2ϩ replacement procedure. The presence of a residual resting Ca 2ϩ would seriously affect [Sr 2ϩ ] determined with fura-2, because of its higher affinity for Ca 2ϩ (k d ϭ 0.3 M). Nevertheless, this problem should not affect significantly the kinetics of intracellular Sr 2ϩ transients.
Fluorescence changes recorded in the presence of Sr 2ϩ were different from those obtained in Ca 2ϩ . For instance, they decayed more slowly, reflecting a less efficient handling of Sr 2ϩ by metabolic pumps (Rasgado-Flores et al., 1987). Most importantly, the kinetics of release from intracellular stores was markedly dissimilar. Rather than the TR and PR components elicited by the first caffeine application when the stores are replete with Ca 2ϩ , only a single component of release was apparent in Sr 2ϩ , with a decay time constant ( d ϭ 11.5 s) intermediate between those of TR and PR components in Ca 2ϩ -containing saline ( dTR ϭ 4.2 s, dPR ϭ 43.9 s). Two possible explanations exist for this result: (a) the decay of the TR component slows as a result of deficient buffering and (b) TR and PR components fused into one.
The simplest interpretation for a monoexponential decay of caffeine-induced Sr 2ϩ release is that Sr 2ϩ inactivates its own release much less efficiently than Ca 2ϩ and that the kinetics of decay are now dominated by the rate of depletion of intracellular stores. If Sr 2ϩ were as effective as Ca 2ϩ at activating RyRs, then caffeine should deplete more because of uninterrupted activation in the absence of counteracting inactivation. Nevertheless, as shown in Fig. 9 B, a substantial amount of fast Sr 2ϩ release persists during the second caffeine challenge. This could indicate that stores become less depleted of Sr 2ϩ at the end of the first caffeine application. This would be possible if feedback activation of RyR is also less effective when Sr 2ϩ is the flux carrier. Alternatively, intracellular stores could become equally or even more depleted in the presence of Sr 2ϩ , but as inactivation is less effective, Sr 2ϩ activation of the RyR channels could still evoke fast release in spite of the reduced intraluminal Sr 2ϩ concentration. Comparison of activation and inactivation kinetics of single neuronal RyRs incorporated into lipid bilayers, upon step applications of Ca 2ϩ and Sr 2ϩ , should help us decide among these possibilities.
When the first derivatives of signals associated to transmembrane Ca 2ϩ influx and intracellular Ca 2ϩ release are compared under similar conditions of Ca 2ϩ content of the intracellular pool ( Fig. 9 C, asterisks), it is apparent that the latter rises faster. This difference could result from the positive feedback amplification that underlies the generation of TR responses. However, when Sr 2ϩ substitutes for Ca 2ϩ , the differences in rate of rise between signals associated with influx and release are smaller (Fig. 9 D, asterisks). The tentative conclusion of these results is that Sr 2ϩ -induced Sr 2ϩ release operates with lower amplification gain than CICR in sympathetic neurons.
In summary, the kinetics of caffeine-induced Sr 2ϩ mobilization is consistent with the idea that the release of Sr 2ϩ operates with a lower feedback loop gain (both positive and negative) than the release of Ca 2ϩ . When Ca 2ϩ is the flux carrier, a regenerative TR response can be generated as a result of amplification by CICR. The abrupt termination of this fast release, in spite of the continuous presence of the agonist, appears to be the outcome of both rapid depletion of releasable Ca 2ϩ and Ca 2ϩ -dependent inactivation or adaptation of release. Single channel recordings of neuronal RyRs would be required to determine more precisely the contribution of Ca 2ϩ -dependent inactivation and adaptation to this process.

Mathematical Modeling of Caffeine-induced Ca 2ϩ Mobilization
Our mathematical model attempts to describe qualitatively how the macroscopic phenomena observed in real cells relate to the basic properties of CICR. The model belongs to the class of common pool models (see Stern, 1992), meaning that trigger calcium reaches the RyRs via the same cytosolic calcium pool into which the calcium is released. These models are intrinsically unstable. If they are conferred with low loop gain (i.e., calcium release relatively insensitive to cytosolic [Ca 2ϩ ]), they produce only a moderate amplification of transients produced by the trigger calcium. When the gain is set to a higher value, more amplification is produced, but all-or-none Ca 2ϩ transients develop that evolve autonomously when cytoplasmic calcium reaches a certain threshold. In our variant of common pool model, Ca 2ϩ entering through plasmalemmal Ca 2ϩ channels is insufficient to induce CICR because of the effects of diffusion over several cytosolic compartments. Nevertheless, Ca 2ϩ entering through this pathway is readily taken up by intracellular stores, greatly affecting their filling status.
Judging by the modest amplification of Ca 2ϩ signals resulting from transmembrane Ca 2ϩ influx, CICR in nerve cells constitute an example of low loop gain systems. However, as shown in this study, trigger Ca 2ϩ originating from intracellular stores can produce regenerative responses characteristic of high loop gain models. The solution to this apparent discrepancy resides in the rate of rise of trigger Ca 2ϩ , which determines the effective gain of CICR: If Ca 2ϩ rises sufficiently fast, activation can overcome inactivation and regenerative responses are generated. Conversely, for slowly developing changes in Ca 2ϩ concentration, CICR operates with low gain. The notion that the rate of rise of trigger Ca 2ϩ is the most important parameter determining the extent of release is supported by experimental and theoretical studies (Fabiato, 1985(Fabiato, , 1992Tang and Othmer, 1994;Stern 1996). The main conclusion of our results with the model is that the loading status of intracellular stores determines the effective gain of CICR, regardless of the source of trigger Ca 2ϩ . Since it is conceivable that this physiological parameter undergoes cellular control, both globally and locally, it may represent a potentially important mechanism for regulating CICR.
Interaction between neighboring RyRs and calcium stores is intrinsic to common pool models provided that they are sufficiently close to one another so that they respond to each other's released Ca 2ϩ . Our model does not specifically consider interactions between release channels. In heart cells, RyRs are organized in discrete release units coupled to single sarcolemmal dihydropyridine channels, but largely uncoupled from neighboring release units, an arrangement that provides high gain but prevents uncontrolled regenerative behavior (Stern, 1992).
In avian heart cells, where excitation-contraction coupling depends on Ca 2ϩ diffusion from one release unit to the next one (because of the lack of t-tubules; Sommer et al., 1991), the distance between individual release units and its nearest neighbors is about 135 nm. Presumably this distance allows effective propagation of a wave of Ca 2ϩ release between neighboring release units (Protasi and Franzini-Armstrong, 1996). The spatial organization of neuronal intracellular calcium pools and release units is largely unknown (Pozzan et al., 1994). Sympathetic neurons possess abundant ryanodine binding sites (Hernández-Cruz et al., 1995) but lack privileged communication pathways (i.e., t-tubules) between the plasmalemmal voltage-gated Ca 2ϩ channels and the majority of their Ca 2ϩ release channels. The necessity of using caffeine concentrations close to the half-maximal dose in order to elicit regenerative responses in nerve cells (this study), suggest that overall, the interaction between release channels is limited. It is possible that the distance between neighboring release units does not favor a significant interaction. Alternatively, Ca 2ϩ -dependent inactivation and local depletion of releasable Ca 2ϩ could extinguish channel activity shortly after it has been initiated by the stimulus. A detailed study on the spatial organization of release units in nerve cells is required to understand the reasons for their apparently limited interaction.
This study suggests that neuronal CICR is designed to favor amplification of Ca 2ϩ signals originated from intracellular sources like IP 3 or ryanodine-sensitive Ca 2ϩ stores themselves, rather than amplification of voltagegated Ca 2ϩ influx. The gain of this amplification mechanism may be regulated by the density of RyRs, the filling status of the Ca 2ϩ stores and the local concentration of physiological activators of release (Mészáros et al., 1993;Hua et al., 1994). We propose that the primary role of ryanodine-sensitive intracellular stores in nerve cells (and possibly of other nonmuscular cells) is to improve signal to noise ratio by intensifying subcellu-lar Ca 2ϩ gradients generated by agonist-induced intracellular Ca 2ϩ release. This local Ca 2ϩ signaling function of CICR may be particularly relevant for confined cellular compartments, like synaptic terminals, dendritic spines, or submembrane domains . a p p e n d i x

Mathematical Model
To compute Ca 2ϩ concentration changes in the different compartments of the nerve cell, the cytosolic space was divided into three regions: an outmost shell (compartment 0; closer to the plasma membrane), a region composed of n Ϫ 1 intermediate, nonborder compartments, and an inmost shell (compartment n; closer to the intracellular store membrane). The endoplasmic reticulum was represented by two diffusionally connected compartments, one that takes up Ca 2ϩ from the cytosol and another one that releases it into the cytosol.
The net change in Ca 2ϩ concentration in any compartment c at all times is given by the difference between Ca 2ϩ influx and efflux: , where [Ca 2ϩ ](t,c) is the Ca 2ϩ concentration of compartment c, and J in (t,c), J out (t,c) are the net Ca 2ϩ influx and efflux into/from compartment c. In general, for an arbitrary compartment c, Ca 2ϩ fluxes resulting from diffusion exchange with adjacent compartments, DF(c) and exchange with fixed buffers, BF(c) are given by: and (A3) where D Ca is the Ca 2ϩ diffusion coefficient and dx the thickness of compartment c, B Tot (c) and B_Ca(t,c) are total and Ca 2ϩ -bound fixed buffers in compartment c, k onB and k offB are association and dissociation rate constants of fixed buffers with Ca 2ϩ . The Ca 2ϩ pump activity responsible for Ca 2ϩ extrusion from compartment 0 through the plasma membrane (Pump pm ) and Ca 2ϩ removal from compartment n through the ER membrane (Pump erm ) is represented as follows: The magnitude of this Ca 2ϩ transfer function is given by the pump maximal transfer rate per unit of area, Rate p , multiplied by the fraction of the total number of Ca 2ϩ binding regulatory sites which are occupied with Ca 2ϩ : PUMP_Ca(t)/PUMP Tot . Here, PUMP Tot is the total number of Ca 2ϩ binding sites and PUMP_Ca(t) the number of sites bound with Ca 2ϩ . ds is the area section of plasma or endoplasmic reticulum membrane involved. Occupation with Ca 2ϩ of regulatory sites is governed by a first order mass action law: where k onP and k offP represent association and dissociation rate constants for Ca 2ϩ binding, respectively.
[Ca 2ϩ ] changes in the outmost shell (compartment 0). These changes are given by: In this expression, Ca Ch (t) describes the depolarization-induced opening of a voltage-gated Ca 2ϩ conductance, given by: where Ca Chmax is the maximum Ca 2ϩ permeability per membrane area unit, Ca ChInt is the time interval between l successive depolarizations, Ca ChDur is the duration of each stimulus, and ds is the plasma membrane section area of compartment 0. u(t Ϫ a) is a square pulse Heavyside function defined as follows: Ca 2ϩ efflux from the outmost shell (compartment 0) by the plasmalemmal Ca 2ϩ pump is given by: Since Ca 2ϩ diffusion through the plasma membrane is negligible, the diffusional fluxes of the outmost shell (compartment 0), can be written as follows:  [Ca 2ϩ ] changes in nonborder shells (compartments 1 to n Ϫ 1). In each of these compartments, Ca 2ϩ fluxes due to the opening of Ca 2ϩ channels and Ca 2ϩ pump activity are absent. Therefore, the changes in Ca 2ϩ , are simply given by: (A10) [Ca 2ϩ ] changes in the inmost shell (compartment n). The Ca 2ϩ concentration changes in compartment n can be computed as follows: (A11) The first term represents Ca 2ϩ influx due to Ca 2ϩ mobilization through RyR release channels. Here, the function RyR(Ca 2ϩ ,t) represents the macroscopic Ca 2ϩ permeability per unit membrane area of the endoplasmic reticulum compartment capable of releasing Ca 2ϩ to the cytosol (ERt). Release channels possess three regulatory Ca 2ϩ binding sites: two cytosolic sites, which regulate activation and inactivation of the channel, and a third luminal site, which regulates channel conductance. The macroscopic Ca 2ϩ permeability of these channels is given by: where RyR max is the peak macroscopic permeability per unit of area, aRyR_Ca(t, n)/ARyR Tot is the fraction of activating sites occupied with Ca 2ϩ . Likewise, iRyR_Ca(t, n)/iRyR Tot is the fraction of inactivating sites occupied with Ca 2ϩ , and lRyR_Ca(t, ER t )/lRyR Tot is the fraction of luminal sites occupied with Ca 2ϩ . N is the cooperativity of the lumenal regulatory site and ds erm is the area of the section of the ER membrane. These regulatory Ca 2ϩ binding sites are occupied with Ca 2ϩ according to the law of mass action: where k onaRyR(caff, t) , k offaRyR , k oniRyR , k offiRyR , k onlRyR , and k offRyR are the association and dissociation rate constants of cytosolic and lumenal regulatory Ca 2ϩ binding sites of the release channel. A caffeine dependence has been introduced to the association rate constant of the activating site. This parameter is used to simulate agonist-induced stimulation of the release channel as follows: where k on Ϫ Caff and k on ϩ Caff are the association rate constants before and after and during caffeine application, respectively. Caff Int is the interval between m caffeine stimuli and Caff Dur is the duration of each caffeine stimulus.
The Ca 2ϩ uptake from the inmost shell (compartment n) by the ER Ca 2ϩ pump is given by a transfer function similar to that of the plasmalemmal Ca 2ϩ pump.

(A17)
Since Ca 2ϩ diffusion through the ER membrane is negligible, the diffusional fluxes of the inmost cell (compartment n), can be written as follows:   Ca 2ϩ distribution within the ER compartment. In this model the ER comprises two diffusionally connected compartments. The first one (ERs), removes Ca 2ϩ from the cytosol by way of the ER Ca 2ϩ pump. The second one (ERt) releases Ca 2ϩ into the cytosol via Ca 2ϩ release channels. The change in Ca 2ϩ concentration in compartment ERs is given by: where ds ER is the thickness of the ER compartment and vol ER is the volume of each ER compartment, and the change in Ca 2ϩ concentration in the ER compartment that releases Ca 2ϩ into the cytosol is given by: (A20) All equations were numerically integrated using a finite difference approximation (Euler method) with the aid of the Scope 3.5 simulation package.