©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Acetylcholine Differentially Affects Intracellular Calcium via Nicotinic and Muscarinic Receptors on the Same Population of Neurons (*)

Margaret M. Rathouz , Sukumar Vijayaraghavan , Darwin K. Berg (§)

From the (1)Department of Biology, University of California at San Diego, La Jolla, California 92093-0357

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Multiple receptor subtypes activated by the same ligand but coupled to different second messengers can produce divergent signaling in a cell, while receptors activated by different ligands but sharing the same second messenger can produce convergent signaling. We show here that chick ciliary ganglion neurons have three classes of receptors activated by the same neurotransmitter, acetylcholine, and that all three regulate the same second messenger, intracellular free calcium. Activation of muscarinic receptors on the neurons stimulates phosphatidylinositol turnover and induces calcium oscillations that are initiated and maintained by calcium release from caffeine/ryanodine-insensitive intracellular stores. Extracellular calcium is required to sustain the oscillations, while cadmium abolishes them. Activation of either of two classes of nicotinic receptors, distinguished both by location on the neurons and by subunit composition, induces a single, rapid elevation in intracellular calcium without inducing phosphatidylinositol turnover. The nicotinic responses are entirely dependent on extracellular calcium, show no dependence on release from internal stores, and do not display oscillations. Low concentrations of the native agonist, acetylcholine, induce repetitive calcium spikes in the neurons characteristic of muscarinic receptors, while higher concentrations induce nonoscillating increases in intracellular calcium that include contributions from nicotinic receptors. The three classes of receptors also differ in the acetylcholine concentration required to elicit a response. These differences, together with differences in receptor location and sources of calcium mobilized, may enable the receptor subtypes to target different sets of calcium-dependent processes for regulation.


INTRODUCTION

Intracellular calcium is widely used as a second messenger and regulates a vast array of cellular events. Examples include exocytosis, long-term synaptic modulation, cytoskeletal restructuring, gene expression, and cell death. Often cells have receptor types responding to different agonists but sharing an ability to elevate intracellular calcium ([Ca]) levels(1, 2) . The multiplicity of receptors enables different signaling pathways to converge on the same molecular targets. Less obvious is the virtue of a cell expressing several classes of receptors that elevate [Ca]in response to the same hormone orneurotransmitter.

Cholinergic receptors activated by the endogenous agonist acetylcholine (ACh)()produce elevations in [Ca] in many cell types. Muscarinic acetylcholine receptors, found on glands, smooth muscle, cardiac muscle, and neurons elevate [Ca] by stimulating release from intracellular stores (for review, see Ref. 3). Nicotinic acetylcholine receptors (nAChRs) found throughout the nervous system and in skeletal muscle (for review, see Ref. 4) are also effective at elevating [Ca]. Recently it has been shown that several types of neuronal nAChRs are highly permeable to calcium(5, 6, 7, 8) . Most prominent in this respect are nAChRs that bind Bgt and contain the 7 gene product(9, 10) .

Chick ciliary ganglion neurons provide a system for comparing the effects of nicotinic and muscarinic receptors on [Ca] in the same cells. The neurons have two major classes of nAChRs: mAb 35-AChRs that bind the monoclonal antibody mAb 35 and are primarily synaptic in location, and Bgt-AChRs that bind -bungarotoxin (Bgt) and are primarily nonsynaptic in location(11, 12) . mAb 35-AChRs collectively contain the 3, 4, and 5 gene products found in the neurons (13) and mediate synaptic transmission through the ganglion(14) . Bgt-AChRs contain the 7 gene product and function as ligand-gated ion channels(13, 15) , but they have yet to be assigned a physiological role in the ganglion. Both classes of receptors elevate [Ca] in the neurons(16, 17) . In addition, ciliary ganglion neurons have muscarinic receptors capable of increasing [Ca] and generating a slow inward current(18, 19) .

The experiments reported here use calcium imaging with the fluorescent indicator fluo-3 (20) to compare the abilities of cholinergic receptor subclasses on the neurons to increase [Ca]. The results demonstrate that both nicotinic and muscarinic receptors contribute to [Ca] elevations when activated by ACh but do so by relying on different sources of calcium and different mechanisms of activation. The two receptor classes also differ with respect to the temporal pattern of calcium changes they produce. All three of the receptor subtypes differ with respect to the ACh concentrations at which they demonstrably affect [Ca]. These differences may enable the receptors to achieve distinctive effects on calcium-dependent processes.


EXPERIMENTAL PROCEDURES

Cell Preparations

For inositol phosphate (IP) measurements, cultures were prepared with chick ciliary ganglion neurons from 8-day embryos and were maintained for 6 days before analysis as described previously(21) . For experiments measuring calcium-dependent fluorescence, cultures were prepared by dissecting ganglia from 14- to 15-day-old chick embryos, cutting them into halves, incubating them for 30 min at 37 °C in recording solution containing 1 mg/ml collagenase but no divalent cations, and then rinsing and triturating as described previously(22) . The cells were plated on plastic Costar dishes coated with poly-D-lysine and incubated in complete culture medium containing 3% (v/v) embryonic chick eye extract (21) for 1 h at 37 °C to allow attachment of cells to the substratum prior to analysis. No attempt was made to isolate or record selectively from ciliary or choroid neurons.

IP Release Assay

Ciliary ganglion cultures containing 8-10 dissociated ganglia per 35-mm dish were incubated overnight in inositol-free minimal essential medium containing 10% horse serum, 3% eye extract, and [H]inositol (1 µC/dish). The cells were then washed 5 times with rinse buffer (serum-free culture medium containing 10 mM LiCl) and incubated for an additional 15 min in the same buffer. Phosphatidylinositol turnover was initiated by removing the medium and adding 1 ml of the indicated agonist in rinse buffer. Unless otherwise indicated, all agonist incubations were for 20 min at 37 °C. Reactions were stopped by removing the media and scraping cells in 100 µl of NaPO buffer, pH 7.4, containing 10 mM LiCl and 0.1% Triton X-100. Lipids were extracted, and an aliquot of the chloroform phase was counted in a scintillation counter to determine incorporation of label into phosphatidylinositols. The aqueous phase was removed, diluted 3-fold with water, and incubated with 150 µl of the anion exchange resin AG-X8 (formate form) for 15 min at room temperature. The mixture was then centrifuged briefly, and the supernatant was removed. An aliquot of the supernatant was counted to determine the proportion of label existing as free inositol. The resin was then washed four times with 1 ml of 5 mM inositol. IPs were eluted with 500 µl of solution containing 1 M ammonium formate and 0.1 M formic acid and counted. Data were fit to curves using Inplot (GraphPAD, San Diego, CA).

Fluorescence Measurements

Cells were loaded with the calcium indicator fluo-3AM for 30 min at room temperature in the dark as described previously(16) . The cells were perfused with recording solution and examined for epifluorescence with a 16 neofluor water immersion objective and a 100-watt mercury vapor lamp fitted with a narrow band pass excitation filter (H485, Zeiss) on a Zeiss photomicroscope. Neutral density filters of 0.5-1.5 optical density were used to reduce photobleaching. Field images were scanned with a silicon-intensified target camera (Dage) and accumulated every 1.8 or 3.6 s. Intermittent illumination of the cells (0.4 or 0.8 s per image) was accomplished using a shutter and shutter-controlling device which also triggered the Quadra 680AV (or Macintosh II) computer to capture images (video frames). Squares of 10 10 pixels were selected at the centers of 10 neurons in a field. These were measured for fluorescence intensity throughout the recording period using Image (NIH) software. Background fluorescence (pixel intensity level) was subtracted from the fluorescence of the neurons throughout the recording period. Increments in intracellular calcium were expressed as a multiple (-fold increase) of basal level fluorescence for each neuron.

Solutions

Recording solution contained the following (mM): CaCl(2) , HEPES(10) , KCl (5.4), NaCl (116.4), MgSO (0.8), glucose (5.6), sodium succinate (0.4), and succinic acid (0.6). All agonists and antagonists were diluted in this solution unless otherwise indicated and were applied to neurons via a gravity-feed perfusion system. Some nicotine applications were made by addition of the agonist directly to the bath using a Pasteur pipette. The solution with elevated potassium concentration contained (mM): KCl (35.4), NaCl (86.4), CaCl(2) , HEPES(10) , MgSO (0.8), glucose (5.6), sodium succinate (0.4), and succinic acid (0.6). Calcium-free solution was recording solution in which the calcium was replaced with 0.5 mM EGTA.

Materials

White leghorn chick embryos were obtained locally and maintained at 37 °C in a humidified incubator. Bgt and neuronal bungarotoxin (nBgt) were purified from Bungarus multicinctus venom(23) . Fluo-3AM was purchased from Molecular Probes. Muscarine, atropine, 4-diphenylacetoxy-4-methylpiperidine methiodide (DAMP), nifedipine, verapamil (D600), -conotoxin, thapsigargin, and acetylcholine chloride were purchased from RBI. Ryanodine was purchased from Calbiochem; 2,5-di-(tert-butyl)-1,4-benzohydroquinone (DTBHQ) was a gift from Dr. N. C. Spitzer (University of California, San Diego). [H]Inositol was purchased from DuPont NEN. All other chemicals were purchased from Sigma unless otherwise indicated.


RESULTS

Repetitive [Ca]Elevations Induced by Muscarine-Bath application of 100 µM muscarine produced a rapid increase in [Ca] in ciliary ganglion neurons seen with fluo-3 microfluorimetry. In 45-65% of the neurons (range for >10 fields of 5-10 neurons each), repetitive oscillations in [Ca] were observed. The majority of these neurons (30-40% overall) showed persistent oscillations throughout the period of agonist application (Fig. 1A), while the remaining oscillators terminated their responses before the agonist was removed. The oscillations ranged in frequency from 1 to 5 per min. In 15-30% of the neurons tested, muscarine induced a single, brief elevation in calcium that quickly subsided to baseline despite the continued presence of agonist. In a small portion of the neurons (1-2%), calcium levels remained elevated throughout a 2.5-min application of agonist. Some neurons (10-30%) did not respond to muscarine. Both atropine and DAMP at 50 nM blocked the muscarine-induced elevations in calcium (Fig. 1, B and C). Although atropine is a general antagonist of muscarinic receptors, DAMP is more specific(3) . Of the muscarinic receptor subtypes capable of generating [Ca] oscillations, only M3 receptors are likely to be blocked by the concentrations of DAMP used here.


Figure 1: Blockade of muscarine-induced calcium oscillations by muscarinic antagonists. Neurons were loaded with the calcium indicator fluo-3AM and then challenged with 100 µM muscarine (black bar) after a challenge with 35 mM K in recording medium (open box). The resulting fluorescence (F) was expressed as a multiple of the basal fluorescence (F) at zero time. Neurons were bathed in control medium (A), 50 nM atropine (B), or 50 nM DAMP (C). Both the general muscarinic antagonist, atropine, and the M3 receptor subtype antagonist, DAMP, blocked the muscarine-induced responses.



IP Production by Muscarinic Agonists

Activation of muscarinic receptors in other systems has been shown to induce oscillatory changes in [Ca] similar to those observed here(1, 24, 25, 26, 27) . The induction of oscillations has been linked to production of inositol(1, 4, 5) -trisphosphate (IP) and subsequent release of calcium from intracellular calcium stores (28-30; for reviews see Refs. 31 and 32). To confirm that muscarine induces IP production in ciliary ganglion neurons and to investigate the receptor subtype responsible, neurons were incubated in [H]inositol overnight and then stimulated with cholinergic agonists. Muscarine at 50 µM induced the production of IPs within 1 min, and the accumulation continued for at least 30 min (Fig. 2A). Oxotremorine-M and carbachol were also effective at inducing IP production while nicotine and McN-A-343, an M1 subtype-specific agonist, were not (Fig. 2B). Both DAMP and atropine efficiently blocked muscarine-induced IP production while pirenzipine, an M1 antagonist, and methoctramine, an M2 antagonist, were much less effective (Fig. 2C). The rank order potencies for the several agonists and antagonists are those expected for M3 muscarinic receptors(3) .


Figure 2: Time course and pharmacology of muscarine-induced release of inositol phosphates from ciliary ganglion neurons. Ciliary ganglion neurons in culture were loaded with [H]inositol, rinsed, and challenged with 50 µM muscarine for the indicated times (A), with the indicated agonists for 20 min at the indicated concentrations (B), or with 50 µM muscarine for 20 min in the presence of the indicated antagonists which had been applied to the neurons at the indicated concentrations 2 min before (C). Values indicate the released radioactivity expressed as a percent of total radioactivity incorporated into the cells (A), of unstimulated basal release (B), or of the release induced by 50 µM muscarine in the absence of antagonists (mean ± S.E. from 4-6 cultures combined from 2-3 experiments for each panel) (C). Release as a function of time was induced by muscarine with a pharmacology characteristic of M3 receptors: activation by muscarine, carbachol, and oxotremorine-M but not by McN-A-343 or nicotine, and blockade by atropine and DAMP but only poorly by pirenzipine or methoctramine.



Taken together, the results suggest that muscarine elevates [Ca] in ciliary ganglion neurons by activating M3 receptors which stimulate the release of calcium from IP-dependent internal stores. [Ca] oscillations could then be accounted for by repetitive release from such stores(32, 33) . Alternatively, the IP-sensitive stores may only initiate the oscillations while a second set of stores maintains them via calcium-induced calcium release (CICR; 26, 30, 34).

Contributions from Microsomal Calcium Stores to Muscarinic Responses

A pharmacological approach was taken to determine if activation of muscarinic receptors releases calcium from intracellular stores and whether CICR plays a role. Thapsigargin and DTBHQ, compounds that inhibit endosomal calcium ATPases(35, 36) , were used to deplete endosomal stores by blocking reuptake of calcium. Caffeine and ryanodine were used to target a subset of stores for depletion that in other cells is necessary for CICR (37, 38; for review, see Ref. 31).

Thapsigargin had the most pronounced effect. At 1-5 µM, it increased the portion of neurons displaying an abbreviated response, i.e. 1-2 spikes, at the expense of neurons displaying a sustained or oscillatory response throughout the period of agonist application (Fig. 3B; ). The effect was more dramatic when a second test with muscarine was carried out on thapsigargin-treated cells. In this case, 50% of the neurons (7 out of 14 cells) showed no response to muscarine at all while only 14% showed oscillations. The majority of control neurons (no exposure to thapsigargin) from the same cultures responded vigorously to the second application of muscarine (74% oscillated; 23 cells), while only 9% did not respond. DTBHQ produced effects similar to those in neurons treated with thapsigargin. About half of the DTBHQ-treated neurons (47%; 19 cells) gave no response to a second application of muscarine while only a quarter (26%) showed oscillatory activity. The results indicate that thapsigargin- and DTBHQ-sensitive intracellular calcium stores contribute to muscarine-induced oscillations in [Ca].


Figure 3: Contributions of extracellular sources and intracellular stores to muscarine-induced oscillations in [Ca]. Neurons were loaded with fluo-3AM and tested for muscarine-induced increases in [Ca] as described in Fig. 1 while perfusing with vehicle (recording solution) (A), 1 µM thapsigargin plus indicated caffeine challenges (B), 10 µM ryanodine (C), 10 µM ryanodine plus indicated caffeine challenges (D), and calcium-free medium contemporaneously with the muscarine tests (E). Muscarine, black bars (hatched bars in E); 30 mM caffeine, stippled boxes; 35 mM K, open boxes. Little effect on muscarine-induced oscillations was caused by caffeine challenges alone, chronic ryanodine alone, or ryanodine combined with the indicated caffeine challenges to deplete caffeine-sensitive stores. Thapsigargin not only depleted the caffeine-sensitive stores but also truncated the muscarine-induced oscillations. Calcium-free medium eliminated the oscillations, leaving only a single initial spike. Extracellular calcium is necessary for oscillations (beyond the first spike), and thapsigargin-sensitive internal stores also contribute; caffeine-sensitive stores contribute neither to the initiation nor the maintenance of muscarine-induced oscillations in [Ca].



In contrast to thapsigargin and DTBHQ, caffeine and ryanodine had only minor effects on muscarinic responses. Together, caffeine and ryanodine produced no change in the percentage of neurons generating sustained oscillations to muscarine (), although the oscillations were somewhat more erratic than those observed in untreated neurons and usually did not return to baseline between spikes (Fig. 3D). A decrease was also seen in the percentage of neurons producing only one or two spikes (). These minor consequences of caffeine and ryanodine treatment on muscarine-induced oscillations can probably be attributed to effects of the compounds on buffering of cytosolic calcium or on the IP receptor, as suggested previously (39).

The observation that muscarine-induced oscillations in [Ca] can still be elicited in neurons after treatment with ryanodine and caffeine provides evidence that CICR is not required. Failure of caffeine to elicit a second response in such cells confirmed that caffeine-sensitive stores had been depleted by the caffeine/ryanodine treatment (Fig. 3D). Even in the absence of caffeine challenges, ryanodine alone should have been able to decrease the number of oscillations if CICR from caffeine-sensitive stores were involved because such stores would have emptied within the first few calcium spikes and could not have refilled(30, 34, 40) . This was not the case (Fig. 3C). The results indicate that ciliary ganglion neurons have caffeine-sensitive stores but they are not necessary for the muscarine-induced oscillations in [Ca]. The attenuation of muscarine-induced oscillations by thapsigargin demonstrates that ciliary ganglion neurons have additional stores that are caffeine-insensitive and contribute to the maintenance of the oscillations. These latter stores apparently have slower endogenous turnover rates than do caffeine-sensitive stores. Neither ryanodine nor thapsigargin alone produced a significant increase in intracellular free calcium (data not shown).

Contributions from Extracellular Calcium to Muscarinic Responses

Although intracellular calcium stores are required for muscarine-induced oscillations in [Ca], extracellular calcium may also be required to replenish the stores and sustain the oscillations as shown in other systems(40) . To examine the role of extracellular calcium in the muscarinic response, agonist was applied in a calcium-free solution containing 0.5 mM EGTA. Under these conditions, 100 µM muscarine produced only a single spike. The proportion of neurons responding to agonist did not change from control, but no cells produced either oscillatory or sustained responses throughout the period of agonist application. Similar results were obtained when the neurons were incubated in calcium-free medium with 0.5 mM EGTA for 1 min prior to challenging with muscarine (93% responding with a single spike, n = 29 cells), indicating that residual unbound calcium in the bath was unlikely to explain the single spikes observed. Most (90%) of the cells that did respond initially with a single spike in the absence of extracellular calcium failed to do so when challenged with muscarine a second time even though the cells were bathed with calcium between the muscarine tests (Fig. 3E).

Two conclusions emerge. First, extracellular calcium is not required for the initial calcium increase caused by muscarine because the receptors trigger release from internal stores, presumably in an IP-dependent manner. Second, extracellular calcium is required to sustain the muscarine-induced increases in calcium beyond the initial spike. Two pathways along which extracellular calcium could enter the cell to achieve this are voltage-dependent calcium channels (VDCCs), of which there are at least three types in ciliary ganglion neurons(41) , and calcium release-activated channels, whose current (I) has been described in other cell types (42, 43; for review, see Ref. 44).

Evidence that such channels are involved was obtained by showing that cadmium at 200 µM prevented muscarine-induced oscillations (40/40 neurons). In this case, muscarine induced only a single transient calcium elevation, similar to the response seen in calcium-free medium. In about one-third of the cadmium-treated neurons, the single spike was followed by a second calcium elevation that did not return to baseline even after removal of the agonist. No oscillations were observed. Cadmium at this concentration completely inhibits the current triggered by activation of VDCCs in ciliary ganglion neurons(41) ,()but may also act at I channels(45, 46) . Blockers of N- and L-type VDCCs, namely -conotoxin (at 1 µM) and the dihydropyridines nifedipine and verapamil (10 µM), block up to 90% of the voltage-dependent calcium current in these neurons(41, 47) but have no significant effect on the muscarine-induced oscillatory response (data not shown). Apparently the cadmium-sensitive channels required to sustain the oscillations do not include conventional L- and N-type VDCCs.

Absence of Contributions from Internal Stores to Nicotinic Responses

In addition to muscarinic receptors, ciliary ganglion neurons have two major classes of nicotinic receptors that can elevate [Ca], namely mAb 35-AChRs and Bgt-AChRs (16, 17). Because muscarinic and nicotinic receptors share the same endogenous ligand, it was important to compare the conditions under which they alter [Ca] and the mechanisms by which they do so. One issue was the calcium sources mobilized by the receptors. Previous studies demonstrated that calcium can enter the neuron directly through nAChR channels or indirectly through VDCCs activated by membrane depolarizations the receptors induce. Possible contributions to [Ca] from internal stores had not been examined for nicotine-induced responses.

Nicotine at 10 µM activates both Bgt-AChRs and mAb 35-AChRs(16, 17) . Repeated applications of 10 µM nicotine at 5-min intervals induced calcium-dependent fluorescence responses with little attenuation (Fig. 4A). To examine the role of internal calcium stores, either caffeine and ryanodine together or thapsigargin alone were applied to the neurons between applications of nicotine. Neurons bathed in 10 µM ryanodine were challenged with 30 mM caffeine to deplete caffeine-sensitive stores. No significant effect was observed on subsequent nicotine-induced responses (Fig. 4B); the mean peak response after treatment was 95 ± 4% (n = 22 cells) of the original responses from the same cells. Thapsigargin at 1 µM also had no effect (Fig. 4C). The mean peak response to nicotine after thapsigargin treatment was 102 ± 2% (n = 20 cells) of the response before treatment. A second application of nicotine after thapsigargin treatment induced a mean response that was 107 ± 3% of the response obtained before treatment. Controls in these cases received only vehicle (0.02% ethanol for 5 min) and yielded mean peak responses of 98 ± 3% and 103 ± 5% (first and second challenges with nicotine, respectively; n = 20 cells) of control responses from the same cells before exposure to vehicle (Fig. 4A). The experiments provided no evidence that nicotine-induced increases in [Ca] depend on intracellular calcium stores. In contrast, replacing the 2 mM calcium in the extracellular solution with 0.5 mM EGTA completely abolished the response to nicotine (Fig. 4D), confirming the dependence of nicotine-induced changes in [Ca] on extracellular calcium. Quick recovery of the nicotinic response (<5 min) was observed when calcium was added back to the extracellular solution.


Figure 4: Dependence of nicotine-induced increases in [Ca] on extracellular calcium but not on internal stores. Fluo-3AM-loaded neurons were stimulated with pulses of 10 µM nicotine (upward arrows) applied by pipette at the indicated times; the breaks indicate 5-min intervals during which the shutter was closed to minimize photodamage. After the first two nicotine tests, the neurons were perfused for 5 min (middle break, downward arrow) with vehicle (0.02% ethanol in recording medium) (A), 30 mM caffeine plus 10 µM ryanodine (B), or 1 µM thapsigargin (C), and then tested twice more with nicotine. D, some neurons were perfused with calcium-free medium (hatched bar) and tested with calcium-free nicotine (open upward arrow). Although the duration of the nicotinic responses varied among neurons, the amplitude of the responses was relatively constant for a given neuron. Only the calcium-free condition affected the response, eliminating it entirely and reversibly.



In the above experiments, nicotine was applied in single, brief applications to the bath as described previously(16) . For better temporal comparisons between nicotinic and muscarinic responses, neurons were also perfused with nicotine for 2.5 min as was done with muscarine. In these cases, nicotine induced a rapid rise in fluorescence followed by a partial decline to a level above baseline during the remainder of the 2.5-min period of agonist exposure. As in the case of brief applications, nicotine was unable to induce oscillations in [Ca].

The results show that nicotine-induced responses differ from muscarine-induced responses in two ways. Unlike the muscarinic response, nicotine-induced elevations in [Ca]do not appear to require release of calcium from internal stores, at least those sensitive to thapsigargin or caffeine plus ryanodine. Secondly, the responses do not oscillate in amplitude, even when nicotine is applied for the same length of time as muscarine. The nicotinic responses are similar to muscarinic responses, however, in showing a rapid rate of rise and in declining nearly to baseline in the continued presence of agonist (repeatedly so in the case of muscarine-induced oscillations). [Ca]Elevations Induced by ACh-The finding that at least three kinds of cholinergic receptors capable of elevating intracellular calcium levels can co-exist on ciliary ganglion neurons posed questions about the operating range of the receptor subtypes and their relative effectiveness. To examine this, dose-response measurements were made using the native agonist ACh. Contributions from individual receptor classes were distinguished pharmacologically.

ACh was bath-applied for 2.5 min to fluo-3-loaded neurons. At 1 µM, ACh induced oscillatory activity in about 40% of the neurons (Fig. 5A; ). Nicotinic receptors did not contribute significantly because no change was detected in the percentage of neurons that oscillated or in the nature of the oscillatory response when the neurons were incubated for 1 h with either 60 nM Bgt or 20 nM nBgt prior to testing (Fig. 5, B and C; ). The combination of 200 nM DAMP plus 20 nM nBgt completely eliminated any response in over 90% of the neurons (Fig. 5D; ). (Studies with DAMP alone were not considered informative since a recent report suggests it can partially inhibit nicotine-induced [Ca] increases in ciliary ganglion neurons(48) ; it did completely block the responses induced by 1 µM ACh.) The results are consistent with the response induced by 1 µM ACh being largely, if not exclusively, the product of M3 receptors.


Figure 5: Effects of selective receptor activation on [Ca] patterns induced by ACh. Neurons loaded with fluo-3AM were stimulated with 1 µM (stippled bars) and 10 µM (black bars) ACh and with elevated K (open boxes) while perfusing with recording solution (A), preincubating 1 h with either 60 nM Bgt (B) or 20 nM nBgt (C), or both preincubating with nBgt and perfusing with 200 nM DAMP (D). At 1 µM, ACh induces a muscarinic-like oscillatory response that is little affected by Bgt or nBgt (the truncation seen in C is within the range of variation normally seen for oscillations), but DAMP plus nBgt blocks the response. At 10 µM, ACh induces a sustained response that becomes oscillatory when nicotinic receptors are blocked with nBgt but not with Bgt, implying that mAb 35-AChRs are essential but not Bgt-AChRs. DAMP plus nBgt again completely blocks the response.



At 10 µM ACh, a large fraction of the neurons responded with sustained elevations in calcium that showed no oscillations; only a few neurons displayed the oscillatory pattern seen with the low concentration of ACh (Fig. 5A; ). Again, 60 nM Bgt produced no detectable alteration, indicating that Bgt-AChRs did not contribute significantly at this concentration of ACh (Fig. 5B; ). In contrast, 20 nM nBgt radically altered the response profile, shifting it to the oscillatory pattern seen at the lower ACh concentration (Fig. 5C; ). The combination of DAMP and nBgt again completely blocked the response (Fig. 5D; ). The results indicate that both M3 receptors and mAb 35-AChRs are activated by 10 µM ACh, and that the muscarinic response is either obscured or inhibited by contributions from mAb 35-AChRs under these conditions.

A somewhat different picture emerges at 50 µM ACh. The control response revealed little in the way of oscillatory behavior in most cases (Fig. 6A; ), but treatment with Bgt allowed oscillations to emerge late during the period of agonist application in a substantial fraction of the cells (Fig. 6B; ). Treatment with nBgt had an even more dramatic effect, producing more robust oscillations and increasing the proportion of cells displaying such responses (Fig. 6C; ). Combining DAMP and nBgt blocked the responses entirely (Fig. 6D; ; DAMP alone both at 10 and 50 µM ACh reduced but did not eliminate the response and did not change the proportion of cells responding). Clearly at high ACh concentrations Bgt-AChRs can contribute to the response and are joined here both by mAb 35-AChRs and M3 receptors on the neurons.


Figure 6: Contributions of receptor subtypes at high ACh concentrations. Neurons were loaded with fluo-3AM and stimulated with 50 µM ACh (black bars) and with elevated K (open boxes) while perfusing with recording solution (A), preincubating 1 h with either 60 nM Bgt (B) or 20 nM nBgt (C), or both preincubating 1 h with nBgt and perfusing with 200 nM DAMP while testing (D). Blockade of Bgt-AChRs with Bgt revealed oscillations late during the exposure to agonist while blockade of both Bgt-AChRs and mAb 35-AChRs with nBgt produced more pronounced oscillations, confirming that both classes of nAChRs contribute to responses induced by high concentrations of ACh. DAMP plus nBgt completely blocked the responses.




DISCUSSION

The findings reported here demonstrate for the first time that a single neuron can have as many as three kinds of receptors responding to the same neurotransmitter, driving the same second messenger, and displaying differences while doing so. In the present case where the transmitter is ACh and the second messenger is calcium, the differences can include the temporal pattern of calcium elevation produced, the source of calcium mobilized, and the agonist concentration required for the mobilization. These differences, together with previously reported differences in receptor localization, may enable individual receptor classes to exert unique regulatory effects, targeting different sets of calcium-dependent processes in the cells. Regulation of [Ca]by Muscarinic and Nicotinic Receptors-Results utilizing specific muscarinic agonists indicate that chick ciliary ganglion neurons have M3 type muscarinic receptors which trigger IP release and induce an oscillatory increase in [Ca]. Elevations in [Ca] caused by activation of muscarinic receptors have been detected previously in ciliary ganglion neurons, but oscillations were not described(18) . The lack of oscillations in that case may have resulted from the choice of agonist, oxotremorine-M. Since oxotremorine-M can activate both nicotinic and muscarinic receptors on ciliary ganglion neurons (49),()it might produce sustained elevations as seen with high concentrations of ACh.

Not all neurons from the ganglion responded to muscarine with oscillations. Variation in the pattern of calcium response has been seen in other systems (25) and has been attributed to differences in basal [Ca] levels(27) . Differences between choroid and ciliary neurons in their responsiveness to muscarinic agonists or in their abilities to manage [Ca]could also induce variation in the types of responses observed. Two recent findings supporting this possibility are that muscarinic receptor-mediated synaptic fatigue is seen in choroid neurons, but not ciliary neurons(50) , and that only half of ciliary ganglion neurons have a muscarinic receptor-induced slow inward current(19) . In contrast, all of the neurons have nicotinic responses.

Both intracellular and extracellular sources of calcium are mobilized to initiate and maintain muscarinic [Ca]oscillations, as demonstrated by the effects of store depletion and extracellular calcium removal on muscarine-induced oscillations. The ability of cadmium to block the muscarine-induced [Ca] oscillations is consistent with the involvement of either VDCCs or I channels in sustaining oscillations(41, 45, 46, 47) . Neither N- nor L-type calcium channels, however, are required since -conotoxin and dihydropyridines do not block the oscillations.

The patterns of [Ca] elevation and the sources of calcium mobilized clearly differ for nicotinic and muscarinic receptors on the neurons. Activation of either Bgt-AChRs or mAb 35-AChRs elevates [Ca] without producing oscillations. The responses depend completely on the presence of extracellular calcium which enters the neurons both through the receptors themselves because of their high permeability to calcium and through VDCCs activated by membrane depolarizations the receptors cause (16, 17). The results demonstrate that neither class of nAChRs induces PI turnover or relies on calcium release from internal stores to elevate [Ca].

The simplest explanation for the patterns of [Ca] changes reported here is that muscarine-induced oscillations are produced by cyclical emptying of IP-sensitive calcium stores into the cytosol, inhibition of the IP receptor by the elevated [Ca], and clearing of cytosol by membrane pumps to relieve inhibition and permit a new round of release when stores have refilled(26, 51, 52, 53, 54) . When nicotinic receptors are also activated, e.g. at high ACh concentrations, additional calcium influx raises the level of [Ca]sufficiently to maintain the inhibition of release from intracellular stores, and no oscillations are seen.

Both mAb 35- and Bgt-AChRs have the capacity to inhibit or override muscarinic oscillations but display different dependences on agonist. Only at the highest ACh concentration tested (50 µM) did Bgt-AChRs have a pronounced effect while mAb 35-AChRs contributed to the response both at 10 and 50 µM ACh. The results are consistent with previous calcium imaging studies showing that Bgt-AChRs are relatively insensitive to ACh, but seem at odds with electrophysiological studies indicating that the major component of the Bgt-AChR current response decays much more quickly than that of mAb 35-AChRs(15) . Bgt-AChRs also produce a slower, sustained component of the current response, however(15) , and it could be this second component that is most effective in masking oscillations induced by activation of muscarinic receptors. Additionally, Bgt-AChRs are thought to have the highest calcium permeability of any nicotinic AChR and may influence calcium-dependent block of oscillations with even a small current influx. Thus, each class of cholinergic receptor on these neurons appears to play a role in shaping the ACh-induced temporal pattern of [Ca]. Consequences of [Ca]Elevations Caused by Cholinergic Signaling-Increases in [Ca] are known to regulate a vast array of cellular events, often acting through specific kinases(34, 55, 56, 57) . The effects of nAChRs on calcium-dependent events in chick ciliary ganglion neurons are poorly understood, but recent experiments indicate that, in addition to stimulating neurotransmitter release, activation of the receptors induces neurite retraction (58) and release of the second messenger arachidonic acid from the cells (59) in a calcium-dependent manner. Even less is known about the impact of muscarinic receptors on calcium-dependent events in ciliary ganglion neurons, although one report indicates that muscarinic receptors in the ganglion can promote synaptic fatigue in developing choroid neurons when the cells are likely to be most vulnerable to overstimulation and excitotoxic cell death(50) .

Distinctive patterns of [Ca] elevation can be induced both in neurons and in non-neuronal cells depending on the type of receptor activated, the second messengers produced, and the source of calcium employed(2, 60, 61, 62) . The kinds of effects imposed on cellular processes can depend on the temporal pattern of [Ca] elevation as well as the source of calcium utilized(61, 63, 64, 65) . The fact that nicotinic and muscarinic receptors generate different patterns of [Ca]elevation in chick ciliary ganglion neurons and mobilize calcium from different sources suggests that the receptors may exert different effects on calcium-dependent events in the cells.

Two additional factors likely to be important in determining the role of individual receptor subtypes are receptor affinity for agonist and receptor location. Muscarinic receptors have the greatest impact on [Ca] levels at low concentrations of ACh, but their location on the neurons in vivo is unknown. At 10 µM, ACh recruits contributions to [Ca] from mAb 35-AChRs which are located primarily at synapses, and at 50 µM contributions are also apparent from Bgt-AChRs which are located primarily in nonsynaptic regions. (mAb 35-AChRs may obscure the contributions of Bgt-AChRs to [Ca] at lower ACh concentrations.) In vivo ACh is released from preganglionic terminals at specific sites juxtaposed to synaptic membrane on the ganglionic neurons(66) . No information is available on the concentration of ACh achieved in the synaptic cleft or on the area over which it diffuses before being hydrolyzed. It is also unknown whether ACh can be released either from preganglionic terminals at nonsynaptic sites or from ganglionic somata in vivo.

At the vertebrate neuromuscular junction, significant amounts of ACh diffuse only a relatively short distance from the site of release because of the high levels of acetylcholinesterase activity present (67). On the other hand, ACh released from the vagus nerve onto pacemaker neurons in the heart spreads up to 1 µm away within 0.2 ms while declining about 10-fold in concentration(68) . If synapses on ciliary ganglion neurons display a similar spread of transmitter, mAb 35-AChRs may elevate [Ca] during low frequency synaptic transmission, while extrasynaptic Bgt-AChRs may contribute more during high-frequency stimulation where buildup of ACh could allow a spatial spread beyond the immediate point of transmitter release (50). Speculation about possible synaptic activation of M3 receptors must await information on receptor location. It may be instructive to examine the contributions of individual receptor subtypes to [Ca] microdomains within the cells during ganglionic transmission.

  
Table: Effects on muscarine-induced responses of compounds that deplete internal calcium stores

Neurons were treated with vehicle (Control), 1 µM thapsigargin (Thapsigargin), 10 µM ryanodine plus 30 µM caffeine (Ryan/Caff), or 10 µM ryanodine alone (Ryanodine) and tested for muscarine-induced fluorescence responses as described in Fig. 3. Responses were scored as oscillatory/sustained (3 spikes or maintained fluorescent signal without spikes), single/double spikes (1 or 2 calcium transients), or no response at all (basal fluorescence) for the number of neurons indicated. The distribution of responding cells was significantly different from controls for thapsigargin treatment (p < 0.01; contingency test, analysis), but not for any of the other treatments.


  
Table: Effects of cholinergic antagonists on ACh-induced responses

Neurons loaded with fluo-3AM were tested for fluorescence responses induced by the indicated concentrations of ACh after treatment with the indicated blockers (Bgt at 60 nM, nBgt at 20 nM, and DAMP at 200 nM); the responses were scored as oscillatory or non-oscillatory (other responses). All conditions were significantly different from controls except for Bgt and nBgt at 1 µM ACh and Bgt at 10 µM ACh (contingency test, analysis except for small values where the Fisher Exact test was used).



FOOTNOTES

*
This work was supported by National Institutes of Health Grants R01 NS12601 and P01 NS25916. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Biology, 0357, University of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0357. Tel.: 619-534-4680; Fax: 619-534-0301; E-mail: dberg@ucsd.edu.

The abbreviations used are: ACh, acetylcholine; AChR, acetylcholine receptor; Bgt, -bungarotoxin; nBgt, neuronal bungarotoxin; mAb, monoclonal antibody; IP, inositol 1,4,5-trisphosphate; DAMP, 4-diphenylacetoxy-4-methylpiperidine methiodide; DTBHQ, 2,5-di-(tert-butyl)-1,4-benzohydroquinone; CICR, calcium-induced calcium release; VDCC, voltage-dependent calcium channel.

M. M. Rathouz and D. K. Berg, unpublished results.

Z. W. Zhang and S. Vijayaraghavan, unpublished observation.


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