Functional IP₃- and ryanodine-sensitive calcium stores in presynaptic varicosities of NG108–15 (rodent neuroblastoma × glioma hybrid) cells

Abstract

1. Presynaptic varicosities of the model neuronal cell line NG108–15, a cholinergic neuroblastoma cell × glioma cell hybrid capable of innervating striated myotubes, were examined for the presence of inositol 1,4,5-trisphosphate (IP₃)-sensitive and Ca²⁺-activated (ryanodine-sensitive) Ca²⁺ stores using confocal microscopic imaging of Ca²⁺-sensitive fluorescent dye loaded into the cells.

2. Initial demonstration of the presence of IP₃ receptors and ryanodine receptors in the NG108–15 varicosities was obtained using immunocytochemistry.

3. Treatment of NG108–15 cells with bradykinin (0.1 μM), whose receptor is linked to IP₃ generation, and separately, caffeine (10 mM), an activator of endoplasmic reticulum ryanodine receptors, resulted in substantial increases in [Ca²⁺]_i in the varicosities.

4. K⁺-evoked changes in [Ca²⁺]_i in the varicosities were reduced (52 %) after emptying the ryanodine-sensitive Ca²⁺ store using caffeine (10 mM), but were not affected by prior depletion of the IP₃-sensitive Ca²⁺ store using thapsigargin (1 μM).

5. Bradykinin-induced changes in [Ca²⁺]_i were abolished following depletion of the IP₃-sensitive Ca²⁺ store using thapsigargin (1 μM) and were reduced (72 %) by prior emptying of the ryanodine-sensitive Ca²⁺ store with caffeine (10 mM).

6. The same results were obtained when the varicosities of the NG108–15 cells had formed synaptic junctions with co-cultured rat hindlimb myotubes.

7. Taken together, the results suggest that, in the varicosities, activation of the IP₃ pathway evoked the release of Ca²⁺ from the IP₃-sensitive store, which, in turn, secondarily induced the release of Ca²⁺ from the ryanodine-sensitive store via Ca²⁺-induced Ca²⁺ release, and that depolarization-induced Ca²⁺ entry evoked Ca²⁺-induced Ca²⁺ release only from the ryanodine-sensitive store. Thus, functional internal Ca²⁺ stores are inherent components of presynaptic varicosities in this neural cell line.

Calcium ions play key roles in a wide array of neuronal processes. Most cells, neuronal and non-neuronal, utilize two main sources of Ca²⁺ for generating signals: Ca²⁺ entry across the plasma membrane and Ca²⁺ release from internal stores. In neurons, release of Ca²⁺ from internal stores has been found to mediate and, often, amplify Ca²⁺ changes induced by neurotransmitter receptors, resulting in the activation of signalling pathways leading, for example, to altered gene expression (see Ghosh et al. 1994).

One major site for calcium stores in cells is the endoplasmic reticulum (ER) wherein two functionally distinct compartments can release Ca²⁺, in one case via the inositol 1,4,5-trisphosphate (IP₃) receptor and in the other via the ryanodine receptor (for review, see Berridge, 1998). IP₃ formation, which can be induced in neurons by a variety of neurotransmitter receptors including muscarinic, adrenergic, serotonergic and glutamatergic receptors (Fisher & Agranoff, 1987), is generated in response to the activation of phospholipase C and results in the release of Ca²⁺ from stores bearing IP₃-sensitive channels (Ferris & Snyder, 1992). For example, activation of metabotropic glutamatergic receptors has been found to increase the cytoplasmic level of IP₃, and hence [Ca²⁺]_i, in a variety of mammalian neurons, such as striatal, hippocampal and cerebellar neurons (Sladeczek et al. 1985; Nicoletti et al. 1986; Murphy & Miller, 1989). In the case of cerebellar Purkinje cells, release of Ca²⁺ via the IP₃-sensitive Ca²⁺ channels in stores located mainly in the dendritic tree plays an essential role in the induction of long-term depression of the parallel fibre-Purkinje cell synapse (Inoue et al. 1998). In contrast, Ca²⁺ release from caffeine/ryanodine-sensitive stores results from activation of Ca²⁺-sensitive Ca²⁺ channels in the ER upon elevation of cytoplasmic [Ca²⁺], the latter often via voltage-gated Ca²⁺ channels. Such Ca²⁺-induced Ca²⁺ release (CICR) has been demonstrated in sensory, sympathetic, hippocampal and cortical neurons (Thayer et al. 1988a, b; Tsai & Barish, 1995; Seymour-Laurent & Barish, 1995). In some of these excitable cells, CICR can lead to the regenerative release of Ca²⁺ from the stores. In addition to activating intracellular signalling pathways, CICR can secondarily regulate membrane ion channels. In the dorsal motor nucleus of the vagus, for instance, release of internal Ca²⁺ via the ryanodine-sensitive pathway activates a specific class of potassium channels, thereby modulating neuronal excitability (Sah & McLachlan, 1991).

Much less is known about calcium stores and their physiological roles in the presynaptic nerve terminal. Early studies produced conflicting results about the existence of a functional ER in isolated brain nerve endings (Blaustein et al. 1978; Nicholls & Åkerman, 1981; Rasgado-Flores & Blaustein, 1987). Calcium analysis and measurement of IP₃ formation yielded results indicating a potential role for the inositol phosphate pathway in synaptosomes (Audigier et al. 1988; Adamson et al. 1990; Brammer et al. 1991), though the major site of action of IP₃ may actually be the plasma membrane (Ueda et al. 1996), rather than an intrasynaptosomal store. The possibility of a caffeine/ryanodine-sensitive store in synaptosomes and in intact sympathetic nerve terminals has also been reported (Martinez-Serrano & Satrustegui, 1989; Peng, 1996; Smith & Cunnane, 1996). Even less is known about the nature and functional roles of the Ca²⁺ stores in presynaptic varicosities. To explore the possible involvement of calcium stores in signalling processes of presynaptic varicosities, we used differentiated NG108–15 neuroblastoma × glioma hybrid cells which elaborate large presynaptic-like varicosities along their neuritic arbors and demonstrated that, in this model system, the varicosities expressed both IP₃- and ryanodine-sensitive Ca²⁺ stores.

METHODS

Cell culture

NG108–15 cells

NG108–15 cells (kind gift of Dr M. Nirenberg, NIH) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % fetal bovine serum, 0.1 mM hypoxanthine, 1 μM aminopterin and 16 μM thymidine at 37°C in an incubator with a humidified atmosphere containing 8 % CO₂ (Nelson et al. 1976). To switch the cells to a differentiated state, NG108–15 cells were harvested, plated onto coverslips coated with Cell-Tak (Collaborative Biomedical Products) and cultured in DMEM containing 1 % fetal bovine serum and 1 mM dibutyryl-adenosine-3′:5′-cyclic monophosphate.

Co-culture of NG108–15 cells with rat hindlimb myotubes

Cultures of striated muscle cells were prepared according to Miles et al. (1987) by dissociation of muscle from the hindlimbs of 2- to 4-day-old rat pups that had been killed by rapid decapitation, using 0.5 % trypsin, pH 7.4, for 30 min at 37°C in Puck’s saline solution composed of: 150 mM NaCl, 6 mM KCl, 1 mM Na₂HPO₄, 0.5 mM K₂HPO₄ and 6 mM D-glucose; pH 7.4, for 30 min at 37°C. (The procedures using animals followed a protocol approved by the MCP Hahnemann University Animal Care Committee.) The cells were resuspended in DMEM plus 20 % fetal bovine serum and filtered through a 50 μm pore nylon filter (Tetko Inc). Cell suspensions were pre-plated for 1 h on Falcon plastic Petri dishes to remove fibroblasts. Cells that did not attach were then plated on sterile poly-L-lysine-rat tail collagen-coated coverslips at 4 × 10⁵ cells per coverslip. After 2 days, the medium was replaced with DMEM containing 2 % chick embryo extract and 10 % horse serum to promote fusion of the myoblasts into myotubes. After 3–4 days of incubation, 3 × 10⁴ NG108–15 cells were added to each muscle culture and the co-cultures were maintained in DMEM containing 2 % chick embryo extract, 10 % horse serum and 1 mM dibutyryl-adenosine-3′:5′-cyclic monophosphate to promote differentiation of the NG108–15 cells. Spontaneous fluctuations in [Ca²⁺]_i in the varicosities of the NG108–15 cells as well as corresponding contractions of the myotubes were evident after 2–3 days, with the contractions being completely inhibited on incubation with the neuromuscular junction blocker α-bungarotoxin, indicating the formation of functional cholinergic synapses (not shown).

[Ca²⁺]_i measurement

Cultures of NG108–15 cells or co-cultures of NG108–15 cells with hindlimb myotubes were washed with Hepes-buffered saline (HBS) composed of: 142 mM NaCl, 2.4 mM KCl, 1.2 mM K₂HPO₄, 20 mM D-glucose and 10 mM Hepes, pH 7.4; and then loaded with 5 or 10 μM of the fluorescent Ca²⁺ indicator Oregon-Green 488 BAPTA-1 (K_d= 225 nM; Molecular Probes) in HBS for 1 h at 37°C, using the acetoxymethyl ester derivative of the dye. (Similar results were obtained using either concentration of the dye, indicating that significant buffering by the dye did not occur under the conditions used.) Imaging was typically commenced within 30 min of dye loading. Coverslips were mounted in a microscope chamber for confocal imaging and images were acquired typically at 4 s intervals with a Bio-Rad model MRC-600 argon-krypton ion laser-scanning confocal imaging system attached to a Zeiss Axiovert 135M inverted microscope (×40 or ×63 oil-immersion epifluorescence objectives), detecting fluorescence emitted in response to 488 nm excitation. For rapid data collection, line-scan mode was used wherein a single laser line was repeatedly scanned across a structure every 10 ms, and the resultant fluorescence was collected as an image of successive lines. The samples were under constant perfusion at 3–6 ml min⁻¹ with HBS containing 1 mM Ca² plus, in most cases, 10 μM tetrodotoxin (TTX). Stimulatory agents were rapidly applied using a blunt patch pipette after recording the first five images (as baseline), as detailed previously (Nichols & Mollard, 1996). Flooding of the whole field in view occurred in < 1 s, with the change in local concentration surrounding any given structure occurring in < 50 ms as determined using indicator dye and imaging in line-scan mode.

All quantification of fluorescence intensities associated with individual varicosities, as recorded in raw grey scale digitized images, was performed using OPTIMAS image analysis software (Optimas Co., Seattle, WA, USA). Changes in fluorescence intensities over time are expressed as the ratio of the fluorescence intensity associated with a structure at any given point in time (F) to the fluorescent intensity measured at t₀ (F₀). As such, F/F₀ yields relative (uncalibrated) changes in [Ca²⁺]_i. Note, however, that as the fluorescence was collected from an ‘optical slice’ of ∼0.8 μm, owing to the shallow depth of field (Z-axis) of the confocal microscope, apparent differences in fluorescence intensity observed in different regions of the cells should largely not be a function of differences in volume. For averaged data, all varicosities within the microscopic field under study that responded on application of agonist were included in the quantification, n referring to the number of individual varicosities analysed in at least three independent experiments. Curves obtained on quantification were corrected for photobleaching, which was typically 1–2 %. For illustration, representative pseudocolourized images were processed using Adobe Photoshop (Adobe Systems Inc., Mountain View, CA, USA), without correction for photobleaching, and printed via a high resolution colour printer.

Immunocytochemistry and immunoblot analysis

Differentiated NG108–15 cells were fixed with 4 % paraformaldehyde in HBS and permeabilized with 0.1 % Triton X-100. After extensive washing with phosphate-buffered saline, immunostaining was performed as described in Nayak et al. (1999). The following primary antibodies were used: anti-synaptophysin monoclonal antibody (1:100 dilution; Calbiochem), anti-syntaxin 1 polyclonal antibody (1:100 dilution; Alomone Laboratories), anti-IP₃ receptor polyclonal antibody or anti-ryanodine receptor monoclonal antibody (1:100 dilution each; Chemicon). After extensive washing, appropriate fluorescent secondary antibodies were used in the presence of 10 % goat serum: fluorescein-conjugated goat anti-mouse antibody (1:1000 dilution; Gibco BRL), fluorescein-conjugated sheep anti-rabbit antibody (1:500 dilution; Sigma), Alexa 568 goat anti-rabbit antibody or Alexa 488 rabbit anti-mouse antibody (1:500 each; Molecular Probes). Immunostained structures were imaged using confocal microscopy as described (Nayak et al. 1999). No fluorescence was observed when the differentiated cells were stained with the fluorophore-conjugated secondary antibodies alone (not shown), a control used in each experiment to set the threshold level for imaging immunostained samples (see Nayak et al. 1999).

Sodium dodecyl sulfate-solubilized extracts of differentiated and undifferentiated NG108–15 cells were resolved by polyacrylamide gel electrophoresis (Laemmli, 1970) and blotted onto nitrocellulose. The nitrocellulose blots were immunostained with the anti-synaptophysin monoclonal antibody (1:100 dilution; Calbiochem) followed by horseradish peroxidase-conjugated goat anti-mouse second antibody (1:1000 dilution) and visualized using an ECL Plus staining kit (Amersham).

Electrophysiology

Varicosities in cultures of differentiated NG108–15 cells were identified morphologically (distinguished by characteristic elongated pod shape; see Fig. 1) and then isolated from cell bodies by rapidly severing the connecting neurites using bevelled microelectrodes (Hulsizer et al. 1991). After extensive perfusion with external recording solution (see below), ‘whole-varicosity’ configuration patch-clamp recording was performed using 4–6 MΩ fire-polished pipettes through an Axopatch-1B amplifier (Axon Instruments) controlled via AxoBASIC software. Recordings were filtered at 5 kHz (Bessell filter) and digitized at 10 kHz using a Digidata A/D converter. The compositions of the solutions used were as follows: external solution (mM): NaCl, 140; KCl, 2; MgCl₂, 1; CaCl₂, 2; Hepes, 10; mannitol, 10; glucose, 10; TTX, 0.01; pH 7.4; pipette (internal) solution (mM): caesium glutamate, 145; Cs-Hepes, 20; NaCl, 9.5; BAPTA-4Cs, 0.6; Mg-ATP, 2; pH 7.2. Osmolarity of each solution was checked prior to use and adjusted to 305 and 295 mosmol l⁻¹ for the external and internal solutions, respectively.

Figure 1. K⁺ depolarization induces changes in Ca²⁺ levels in individual presynaptic-like varicosities.

A, fluorescent immunostaining for the nerve terminal marker protein synaptophysin (Navone et al. 1986) and the t-SNARE protein syntaxin (Südhof et al. 1993) visualized by confocal microscopy. Left, transmitted confocal images. Inset in top sequence, an immunoblot of undifferentiated (U) and differentiated (D) NG108–15 cell extracts probed with the same anti-synaptophysin antibody revealing induction of synaptophysin (p38) on differentiation. Middle, fluorescent images. Lower sequence includes control using rabbit immunoglobulin. Right, luminance map. Arrows indicate varicosities. B, successive confocal images showing Ca²⁺ changes in an individual varicosity of a NG108–15 cell. Images were collected at 4 s intervals and the series shown corresponds to the time points indicated by the numbers on the graph. Quantification of relative (uncalibrated) changes in Ca²⁺ levels over time as ratios of fluorescence intensities (F) measured at varying times to the fluorescence measured at t₀ (F₀) in the presence or absence of 10 μM TTX. C, typical responses of differentiated NG108–15 cells to the application of 50 mM KCl shown in a sequence of two confocal microscopic images. Variability in basal fluorescence among the structures was probably due to variability in the loading of fluorescent dye. Colour bar indicates pseudocolour coding of fluorescence intensities over a grey scale of 256 from top (highest) to bottom (lowest).

RESULTS

Depolarization-induced changes in [Ca²⁺]_i in presynaptic-like varicosities

Upon differentiation NG108–15 cells extend long neurites, along which are found prominent presynaptic-like varicosities. These morphological changes are accompanied by the induction of neuronal proteins, especially those involved in presynaptic functions. For example, increased expression of neuron-specific voltage-gated calcium channels (Ichida et al. 1993; Lukyanetz et al. 1998) as well as proteins implicated in synaptic vesicle cycling (Han et al. 1991) have been noted. We have shown here that differentiation induced an increase in the expression of the synaptic vesicle marker synaptophysin (Fig. 1A, inset) which was highly concentrated in the varicosities of the NG108–15 cells as revealed by confocal imaging of immunostained differentiated cells (Fig. 1A). Significant immunostaining for the SNARE protein syntaxin was also observed in the varicosities; however, in contrast to synaptophysin, very strong immunostaining was apparent in the cell bodies as well (Fig. 1A). As with many primary nerve cells, differentiated NG108–15 cells exhibited spontaneous activity leading to oscillations of [Ca²⁺]_i in the varicosities, which could be completely blocked by pretreating the cells with TTX (Fig. 1B). (Consequently, all subsequent functional experiments were performed in the presence of TTX to insure that changes observed in varicosities would be the result of the local action of a given reagent rather than via the firing of the NG108–15 cell body.) On elevation of extracellular K⁺ in the presence of TTX, increased levels of relative [Ca²⁺]_i were observed in the varicosities and cell bodies of NG108–15 cells, measured via confocal imaging of cells previously loaded with the fluorescent Ca²⁺ indicator Oregon-Green 488 BAPTA (Fig. 1C). The responses were dependent on the presence of external Ca²⁺ (not shown) and could be blocked by prior incubation with 10 μM Cd²⁺, 10 μM Co²⁺ and 1 μM nitrendipine (Rondé & Nichols, 2000).

Functional Ca²⁺ stores in presynaptic-like varicosities

Whether presynaptic nerve terminals contain functional Ca²⁺ stores has been the subject of some controversy (Blaustein et al. 1978; Nicholls & Åkerman, 1981; Rasgado-Flores & Blaustein, 1987). As for presynaptic varicosities, in particular, direct demonstration of the presence of both ryanodine- and IP₃-sensitive stores and their involvement in regulating [Ca²⁺]_i has been lacking. We first examined whether these two major intracellular Ca²⁺ stores are present in the presynaptic-like varicosities of our model neuronal system. (As the NG108–15 cells fail to synapse on one another, the varicosities of NG108–15 cells cultured without appropriate target cells represent presynaptic-like structures prior to synapse formation.) Initial evidence for ryanodine and IP₃ receptors in varicosities was obtained using immunocytochemistry (Fig. 2), though the staining for the IP₃ receptor was somewhat patchy. In functional experiments, addition of caffeine, an activator of most forms of the ryanodine receptor (McPherson & Campbell, 1993), induced a rise in the relative [Ca²⁺]_i in the varicosities (Fig. 3A). The caffeine-induced rises in [Ca²⁺]_i in the varicosities were inhibited in a use-dependent manner by 10 μM ryanodine (Fig. 3A, top trace), which acts as an open-channel antagonist of the ryanodine receptor at low micromolar concentrations (Thayer et al. 1988a, b). In addition, activation of the bradykinin receptor, which couples to the phospholipase C pathway and thereby generates IP₃ formation (Yano et al. 1984), also evoked a rise in [Ca²⁺]_i in the varicosities (Fig. 3A), independent of the presence of external Ca²⁺ (not shown). These results suggest the existence of two separate calcium stores in varicosities.

Figure 2. Immunostaining for the ryanodine receptor and the IP₃ receptor.

Differentiated NG108–15 cells were immunostained using either an anti-ryanodine receptor monoclonal antibody (anti-RyR) or an anti-IP₃ receptor polyclonal antibody (anti-IP₃R), followed by appropriate fluorescent secondary antibodies (see Methods). Arrows indicate well-defined, prominent varicosities (left, transmitted images), each of which displayed positive staining (right, fluorescence images) over background (see control in Fig. 1A). (The patchy immunostaining for the IP₃ receptor was also observed using different antibodies and different fixation/permeabilization protocols.)

Figure 3. Involvement of Ca²⁺ stores in K⁺-, caffeine- and bradykinin-induced Ca²⁺ changes in individual varicosities.

A, quantification of caffeine-induced (left; n = 57) and bradykinin-induced (right; n = 20) increases in [Ca²⁺]_i in varicosities. Top trace, example of use-dependent block of successive caffeine-induced responses (10 mM caffeine) in an individual varicosity by low micromolar (10 μM) ryanodine, followed by a typical bradykinin-induced response (0.1 μM bradykinin). B, quantification of responses to KCl before and after an intervening 15 min treatment with 10 mM caffeine (left; n = 65) or to bradykinin before and after an intervening 15 min treatment with 1 μM thapsigargin (right; n = 20). Equivalent responses were obtained with successive stimulation with KCl or with bradykinin. C, quantification of changes in [Ca²⁺]_i induced by 0.1 μM bradykinin before and after a 15 min treatment with 10 mM caffeine (left; n = 42), by 50 mM KCl before and after a 15 min treatment with 1 μM thapsigargin (middle; n = 8), and by 10 mM caffeine before and after a 15 min treatment with 1 μM thapsigargin (right; n = 6). Peak (F_max/F₀) responses were averaged. Bars represent ±s.e.m.*P < 0.05 (Student’s paired two-tailed t test). D, recordings of ionic currents in acutely isolated varicosities (example shown at top) in ‘whole-varicosity’ (dialysed) configuration in response to voltage steps from a holding potential of −90 mV to −10 mV in the absence or presence of 10 mM caffeine. The large initial sharp peak (ranged from 1 to 5 nA with 2–10 ms durations) was sensitive to TTX, indicating a pronounced Na⁺ current, whereas the subsequent slowly tapering current (ranged from 100 to 200 pA with ≈300 ms durations) was sensitive to the presence of EGTA and is characteristic of slowly inactivating Ca²⁺ conductances in nerve terminals (Lemos & Nowycky, 1989). The tail current present after the end of the voltage step was most probably a Ca²⁺-activated Cl⁻ current, owing to its sensitivity to the level of the Ca²⁺ chelator BAPTA in the recording pipette, but it remains to be characterized.

To begin to investigate the roles of the calcium stores, each store was selectively depleted. Treatment of the NG108–15 cells with 10 mM caffeine for 15 min to specifically deplete the ryanodine-sensitive store reduced the subsequent Ca²⁺ response in the varicosities to local K⁺-induced depolarization by 52 ± 4.8 % (s.e.m.) when compared with control (Fig. 3B; average peak responses). Under the same conditions caffeine at concentrations < 1 mM was without effect (not shown). In addition, preincubation with caffeine had no effect on depolarization-induced Ca²⁺ currents, measured in acutely isolated varicosities using patch-clamp recording under ‘whole-varicosity’ (dialysed) configuration (Fig. 3D), eliminating a direct action of caffeine on Ca²⁺ channels. Finally, direct elevation of cyclic AMP levels by forskolin (10 μM) had no effect on K⁺-induced changes in [Ca²⁺]_i in the varicosities (not shown), excluding the possibility that caffeine was acting via inhibition of phosphodiesterase. These results suggest that upon K⁺-induced depolarization, Ca²⁺ enters through voltage-gated Ca²⁺ channels in the varicosities and then triggers the release of Ca²⁺ from ryanodine-sensitive stores, greatly amplifying the overall increase in [Ca²⁺]_i.

Next, we used thapsigargin, a highly potent inhibitor of the endoplasmic reticulum (ER) Ca²⁺-ATPase (Thastrup et al. 1990), to specifically deplete the IP₃-sensitive store. Incubation of NG108–15 cells with 1 μM thapsigargin completely blocked the bradykinin-induced increase in Ca²⁺ levels (Fig. 3B), indicating an essential role of the ER in mediating increases in [Ca²⁺]_i in the varicosities upon activation of the phosphatidyl inositide pathway. To determine whether the two Ca²⁺ stores are functionally independent, we examined the effect of caffeine and thapsigargin on the responses induced by application of bradykinin and KCl, respectively. Pretreatment with thapsigargin had no effect on K⁺-evoked changes in [Ca²⁺]_i, nor on caffeine-induced responses (Fig. 3C). In contrast, pretreatment with caffeine reduced the subsequent response to bradykinin by 71.9 ± 6 % (s.e.m.) (Fig. 3C; average peak responses). Whereas for some other systems there is evidence that thapsigargin can potentially release Ca²⁺ from either IP₃-sensitive or ryanodine-sensitive stores (see Treiman et al. 1998), our results would indicate that these two stores are functioning independently in these cells. Thus, it appears that Ca²⁺ release from IP₃-sensitive stores is not involved in depolarization-induced increases in [Ca²⁺]_i in the varicosities, based on the lack of effect of thapsigargin on K⁺-evoked changes in [Ca²⁺]_i. On the other hand, ryanodine-sensitive stores appear to be activated following release of Ca²⁺ from IP₃-sensitive stores by bradykinin, based on the inhibition by caffeine of the bradykinin-induced changes in [Ca²⁺]_i.

Though the stores may indirectly interact under certain circumstances, their physiological roles are probably distinct, which may be reflected in differences in the kinetics of the Ca²⁺ signals obtained on evoking IP₃ generation when compared with Ca²⁺ entry. To test for such differences, we analysed the time courses of the changes in [Ca²⁺]_i in the varicosities using the line-scan mode of the confocal microscope. For the comparison to be valid, we used KCl and bradykinin to evoke release of Ca²⁺ from each internal store, changes which are both initiated via signals first generated in the plasma membrane of the varicosities. On scanning individual varicosities every 10 ms, we observed that KCl induced a much sharper and faster increase in Ca²⁺ levels than did bradykinin (Fig. 4). No significant difference in the kinetics of the responses could be detected between the Ca²⁺ responses evoked upon addition of KCl when compared with responses evoked by caffeine, the latter directly activating the ryanodine receptors. These results indicate that Ca²⁺-induced Ca²⁺ release initiated on elevation of extracellular K⁺ has an onset in varicosities that is faster than 10 ms. In addition, no apparent difference was found in the kinetics of the changes in fluorescence at the edges of the varicosities when compared with their centres.

Figure 4. Comparison of the time course of K⁺-, bradykinin- and caffeine-induced Ca²⁺ changes in individual varicosities at high resolution.

Images of the time series of successive line scans obtained with the line-scan mode of the confocal microscope during stimulation of a varicosity with elevated K⁺ (A), bradykinin (B) or caffeine (C). Top, confocal image of a varicosity before stimulation. The white line indicated by the arrow shows the position of the laser line that was scanned across the varicosity every 10 ms. Each scan line was 512 pixels in length. Apparent initial drop in fluorescence was due to photobleaching. Graphs show quantification of the fluorescent line-scan signals in individual varicosities during stimulation. These curves represent the fluorescent intensities over time (each point = 1 line scan) corrected for photobleaching along a 20 pixel width line positioned in the middle of the line-scan sequence image (A and B) or lines positioned in the middle and edge (C). The X-axis of each line-scan sequence image (A and B) was aligned to the X-axis (time) of the corresponding graph.

Functional Ca²⁺ stores in presynaptic varicosities of NG108–15 cells innervating striated muscle cells

When NG108–15 cells are co-cultured with striated muscle cells, functional synapses form between the NG108–15 varicosities and the muscle cells in a manner similar to those of normal neuromuscular junctions (Nelson et al. 1976). To examine the effect of synaptic formation, or lack thereof, on the Ca²⁺ responses in the varicosities of the NG108–15 cells, rat hindlimb myotubes were co-cultured with differentiated NG108–15 cells (Fig. 5A), and the Ca²⁺ signals in the varicosities in contact with the myotubes were then analysed. To prevent firing of the NG108–15 cells and contractions of the myotubes, all experiments were done in the presence of TTX. Upon addition of caffeine, an increase in relative [Ca²⁺]_i was observed in both the varicosities and the myocytes (Fig. 5A). The presynaptic varicosities responded to the addition of caffeine and bradykinin with increases in relative [Ca²⁺]_i, reaching on average 1.5- and 2.0-fold basal values, respectively (Fig. 5B). Depleting the ryanodine-sensitive store or the IP₃-sensitive store by incubating the cultures with 10 mM caffeine or 1 μM thapsigargin, respectively, significantly reduced by 62.4 ± 6.9 % (s.e.m.) and 87.5 ± 8.3 % (s.e.m) the increases in [Ca²⁺]_i evoked on subsequent addition of KCl or bradykinin (Fig. 5C).

Figure 5. Involvement of Ca²⁺ stores in caffeine-evoked and bradykinin-induced Ca²⁺ changes in individual presynaptic varicosities innervating co-cultured hindlimb myotubes.

A, typical responses to the application of 10 mM caffeine in varicosities (arrows) making synaptic contacts with an underlying myotube. (Other underlying cells are mainly fibroblasts, with a few scattered NG108–15 cell bodies.) Confocal imaging detected fluorescence in the varicosities in a narrow plane of focus (≤ 1 μm), with little interference by the fluorescence associated with the underlying myotube. Note that whereas all of the indicated varicosities responded to agonist, the increase in fluorescence was not always manifested in a shift in colour (e.g. middle varicosity). B, quantification of caffeine-induced (left; n = 18) and bradykinin-induced (right; n = 11) increases in Ca²⁺ level in individual varicosities. C, responses to KCl before and after an intervening 15 min treatment with 10 mM caffeine (left; n = 10) or to bradykinin before and after an intervening 15 min treatment with 1 μM thapsigargin (right; n = 11). Bars represent ±s.e.m.

DISCUSSION

Analysis of stimulated changes in Ca²⁺ levels in readily identifiable varicosities of differentiated NG108–15 cells, as independent structures and as components of synaptic junctions on appropriate target cells, revealed that independent, functional Ca²⁺ stores are inherent constituents of these presynaptic elements. Both IP₃-sensitive and ryanodine-sensitive stores were found in this neuronal cell line to be present in the same varicosity, with release of Ca²⁺ from the IP₃-sensitive store secondarily inducing release of Ca²⁺ from the ryanodine-sensitive store via CICR, whereas Ca²⁺ entry via voltage-sensitive Ca²⁺ channels appeared to release Ca²⁺ only from the ryanodine-sensitive store (summarized schematically in Fig. 6). As CICR appears to have a very rapid onset in the varicosities (< 10 ms; Fig. 4), the slower kinetics of the bradykinin-induced increases in Ca²⁺ levels are probably limited by the kinetics of phosphatidyl inositol hydrolysis, which requires several seconds in these cells for significant elevation of IP₃ (peaking at 10–30 s; Yano et al. 1984), despite the significant involvement of CICR in these responses (Fig. 3).

Figure 6. Diagram of hypothetical model of the involvement of IP₃- and ryanodine-sensitive Ca²⁺ stores in the regulation of cytoplasmic [Ca²⁺] in an individual varicosity.

Ca²⁺ entry via voltage-sensitive Ca²⁺ channels following membrane depolarization (Ψ) leads to direct elevation of cytosolic Ca²⁺ level and to release (red arrow) of Ca²⁺ from the ryanodine-sensitive Ca²⁺ store (at left). Activation of bradykinin (BK) receptors leads to the formation of IP₃ via phospholipase C (PLC) and subsequent release (red arrow) of Ca²⁺ from the IP₃-sensitive Ca²⁺ store (at right). Ca²⁺ released (dashed black arrow) from the IP₃-sensitive Ca²⁺ store induces secondarily the release of Ca²⁺ from the ryanodine-sensitive Ca²⁺ store.

Presynaptic varicosities

The varicosities of NG108–15 cells display many features of typical presynaptic elements. They express the synaptic vesicle protein synaptophysin, among other nerve terminal proteins (e.g. Han et al. 1991), and, upon direct depolarization, display prominent changes in Ca²⁺ levels (Fig. 1). In acutely isolated preparations, they display voltage-dependent ionic currents (Fig. 3D) typical of mammalian nerve terminals (e.g. Lemos & Nowycky, 1989). In addition, they have previously been shown to contain abundant small clear vesicles, smooth ER-like cisternae, and mitochondria (Han et al. 1991). They also express high levels of choline acetyltransferase and release acetylcholine on stimulation (McGee et al. 1978). When co-cultured with striated muscle cells, the varicosities of the NG108–15 cells make cholinergic synaptic contacts, resembling normal neuromuscular junctions at an early stage of development (Nelson et al. 1976). Thus, as a model neuronal system, the NG108–15 cells permit examination of presynaptic elements before and after formation of synaptic junctions. In addition, direct patch-clamp recording allows direct assessment of different voltage-dependent ionic currents in the varicosities. From the results presented here, it appears that voltage-dependent ion channels and the two major Ca²⁺ stores probably function in nerve varicosities prior to synapse formation. As the level of internal Ca²⁺ plays an essential role in the motility of growing nerve endings (Kater et al. 1988) as well as in secretion, the question arises as to what roles these channels and stores have during the development of synaptic contacts between varicosities and their postsynaptic targets.

In comparison with typical mammalian brain nerve terminals or peripheral axon varicosities and terminals, the varicosities of the NG108–15 cells are quite large, averaging > 10 μm in diameter. As it remains uncertain as to whether functional Ca²⁺ stores exist in brain nerve terminals (see Introduction), which range in diameter from 0.5 to 2 μm (see Dunkley et al. 1986), the presence of both internal Ca²⁺ stores in varicosities could thus be a function of the size of the presynaptic structure. (Use of our approach of confocal imaging for analysis of individual isolated brain nerve terminals (see Nichols & Mollard, 1996) failed to measure any changes in basal or K⁺-evoked rises in [Ca²⁺]_i in individual synaptosomes on application of caffeine, ryanodine or thapsigargin (P. Rondé & R. A. Nichols, unpublished results); also, no pharmacological evidence for functional Ca²⁺ stores has been found for motor nerve terminals (David, 1999)). However, a significant proportion of the varicosities (10–20 %) have diameters around 2 μm in size, within the range noted above for brain nerve terminals and as found for varicosities when present on peripheral neuron axons (e.g. Brain & Bennett, 1997), and varicosities of such diameters clearly contained functional Ca²⁺ stores (data included in the results reported here), indicating that the presence of the stores is not a function of the size of the presynaptic structure in this neuronal cell line.

By definition, the varicosities reside along the length of axonal processes. ER is also usually found along the full course of each axon, where it appears to be composed of connecting tubular structures (Broadwell & Cataldo, 1984). Thus the presence of Ca²⁺ stores in the varicosities could result from their subcellular position in the neuron, as the ER in the axon probably runs through each varicosity. Whether the ER in the non-varicosity regions of the axon functions in Ca²⁺₁ regulation has yet to be determined. In the present results, evoked changes in [Ca²⁺]_i were observed in the neurites, but were largely confined to sites just adjacent to the varicosities, indicating that the Ca²⁺ may have arisen at these sites by diffusion out of the varicosities. These observations, together with a previous proposal that the ER subcompartments in the neuron, including the axon, are heterogeneous and anatomically discontinuous (Takei et al. 1992), raise an alternative possibility wherein the Ca²⁺ stores in the varicosities are entities separate from the ER found along the axon. At present, we cannot, however, rule out that the localization of the Ca²⁺ changes to the varicosities is largely a function of the concentration of receptors/channels at these sites.

Reticulum-like structures do also appear to extend from the axon into the terminal, often juxtaposed to mitochondria on the side of the terminals opposite to the synapse (McGraw et al. 1980); however, as noted above, the function of ER in the terminal remains to be demonstrated. Indeed, it has been suggested that control of brain nerve terminal [Ca²⁺]_i is largely based in the mitochondria (Nicholls & Åkerman, 1981). Using expression of organelle-targeted recombinant green fluorescent proteins having distinct spectral properties and, in separate experiments, low-affinity Ca²⁺ indicator trapped within the lumen of the ER, close functional interactions between the ER and mitochondria have been noted in non-neuronal cells (Rizzuto et al. 1998). The potential roles for the mitochondria in the varicosities in regulating [Ca²⁺]_i on release of Ca²⁺ from the internal stores and in contributing to the subsequent refilling of the stores will thus be of particular interest in future studies.

Internal Ca²⁺ stores in varicosities

Differences in the kinetics of the changes in [Ca²⁺]_i on evoked Ca²⁺ entry when compared with that obtained on generation of IP₃ suggest distinct physiological roles for the internal Ca²⁺ stores in the varicosities. The rapid response of the ryanodine-sensitive pathway (Fig. 4) during CICR and the apparent rapid equilibration of the Ca²⁺ across the terminal (see, for example, Peng & Zucker, 1993) would indicate that this store is involved in regulating [Ca²⁺]_i during typical nerve firing. Indeed, ryanodine and caffeine were previously found to substantially alter the rise in [Ca²⁺]_i in sympathetic terminals during electrical stimulation (Peng, 1996) as well as alter the resultant release of neurotransmitter (Smith & Cunnane, 1996). In contrast, the IP₃-regulated pathway may underlie a more modulatory physiological pathway, which, nonetheless, is capable of inducing the release of acetylcholine from the NG108–15 cells, independent of changes in membrane potential (Higashida & Ogura, 1991; note that those experiments were performed with NG108–15 cells innervating myotubes via short neurites without varicosities). The role of secondary CICR following activation of the IP₃-sensitive pathway in the regulation of neurotransmitter release remains an open question.

Conclusion

The present results demonstrate colocalization of functionally independent Ca²⁺ stores in presynaptic varicosities. How these stores contribute to the control of synaptic transmission between varicosities and target cells will be an essential issue to address in future studies.