Differential Modulation of Nicotinic Acetylcholine Receptor Subtypes and Synaptic Transmission in Chick Sympathetic Ganglia by PGE2

Chuang Du and Lorna W. Role

Department of Anatomy and Cell Biology in the Center for Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University, New York, New York 10032


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Du, Chuang and Lorna W. Role. Differential Modulation of Nicotinic Acetylcholine Receptor Subtypes and Synaptic Transmission in Chick Sympathetic Ganglia by PGE2. J. Neurophysiol. 85: 2498-2508, 2001. The diversity of neuronal nicotinic acetylcholine receptors (nAChRs) is likely an important factor in the modulation of synaptic transmission by acetylcholine and nicotine. We have tested whether postsynaptic nAChRs are modulated in a subtype-specific manner by prostaglandin E2 (PGE2), a regulator of neuronal excitability in both the central and peripheral nervous systems, and examined the effects of PGE2 on nicotinic transmission. Somatodendritic nAChRs in chick lumbar sympathetic ganglia include four nAChR subtypes distinguished on the basis of conductance and kinetic profile. Nanomolar PGE2 applied to the extrapatch membrane differentially regulates opening probability (Po), frequency and the opening duration of each nAChR channel subtype in cell-attached patches. PGE2 decreases the Po of the predominant nAChR subtype (36 pS) and significantly increases Po and open duration of the 23 pS subtype. The 23 pS subtype is gated by the alpha 7-selective agonist choline, and choline-gated currents are inhibited by alpha -bungarotoxin. To examine whether PGE2 modulates nAChRs at synaptic sites, we studied the effects of PGE2 on amplitude and decay of synaptic currents in visceral motoneuron-sympathetic neuron co-cultures. PGE2 significantly decreases the amplitude of miniature excitatory postsynaptic currents (mEPSCs), consistent with the predominant inhibition by PGE2 of all but the 23 pS subtype. The time constant of mEPSCs at PGE2-treated synapses is prolonged, which is also consistent with an increased contribution of the longer open duration of the 23 pS nAChR subtype with PGE2 treatment. To examine the presynaptic effect of PGE2, nanomolar nicotine was used. Nicotine induces facilitation of synaptic transmission by increasing mEPSC frequency, an action thought to involve presynaptic, alpha 7-containing nAChRs. In the presence of PGE2, nicotine-induced synaptic facilitation persists. Thus the net effect of PGE2 is to alter the profile of nAChRs contributing to synaptic transmission from larger conductance, briefer opening channels to smaller conductance, longer opening events. This subtype-specific modulation of nAChRs by PGE2 may provide a mechanism for selective activation and suppression of synaptic pathways mediated by different nAChR subtype(s) at both pre- and postsynaptic sites.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Neuronal nicotinic acetylcholine receptors (nAChRs) present a vast array of receptor subtypes through different subunit combinations. A variety of nAChR subtypes have been detected in vivo and in vitro (see Albuquerque et al. 1997; Changeux et al. 1998; McGehee 1999). nAChR subtypes can be intermingled or segregated in a subtype-specific manner through targeting to different subcellular locations including the somatodendritic (postsynaptic) versus the axonal (preterminal and presynaptic) domains (e.g. Moss and Role 1993; for reviews, see Changeux et al. 1998; MacDermott et al. 1999; Role and Berg 1996). Different nAChR subtypes may be subject to distinct second-messenger modulations or divalent cation interactions that modify channel activity (Amador and Dani 1995; Fenster et al. 1999; Pardi and Margiotta 1999; also see Albuquerque et al. 1997; Swope et al. 1999). Thus activation of other neurotransmitter or modulatory receptors that are coupled to specific second-messenger systems may lead to alteration of the activity of specific nAChR subtypes.

Previous studies show that prostaglandin E2 (PGE2), an inflammatory mediator that also modulates neuronal excitability, and arachidonic acid (AA), a PG precursor, can modulate nAChR activity in chick sympathetic and parasympathetic neurons (Tan et al. 1998; Vijayaraghavan et al. 1995). Studies of AA further suggest that the modulation may be subtype-specific. In chick ciliary ganglion neurons, AA preferentially inhibits alpha 7-containing nAChRs versus the alpha 3alpha 5beta 4 receptors (Vijayaraghavan et al. 1995), whereas it enhances the activity of heterologously expressed nAChR receptors comprised of rat alpha 7 subunits and other rat alpha /beta subunit combinations (Nishizaki et al. 1998). Such studies suggest that at the level of macroscopic current analysis, subtype-specific modulation of nAChRs may provide additional mechanism(s) of synaptic tuning at cholinoceptive sites. Likewise, the coordinate modulation of nAChRs at pre- and postsynaptic locations may constitute further levels of regulation.

Based on biophysical and pharmacological characteristics (i.e., conductance, kinetics, opening probability, rate of desensitization, and agonist and antagonist sensitivity), chick sympathetic neurons express three to seven distinguishable nAChR channel subtypes (Moss and Role 1993; Moss et al. 1989; Yu and Role 1998a,b). The subtypes expressed, as well as their distribution, change during embryonic development and synaptogenesis and are thought to be influenced by both input and target interactions (Devay et al. 1994, 1999; Moss and Role 1993; Yang et al. 1998). Different nAChR subtypes may be further modified by post-translational mechanisms.

This report examines differential modulation of nAChR subtypes by PGE2. We tested whether the modulation of nAChRs by nanomolar PGE2 is selective for different nAChR subtypes at developing synapses, and we examined the functional impact of PGE2 modulation at synapses where both pre- and postsynaptic nAChRs are expressed. PGE2 has different effects on the four nAChR subtypes characteristic of the later developmental stage tested, and these effects are independent of direct interactions as changes in channel function were observed in cell-attached patches with PGE2 applied to the remainder of the cell. One of the four nAChR subtypes is enhanced in opening probability (Po) and in open duration, while the activity of the three other subtypes is depressed. The net effect of PGE2 is sufficient to change the profile of nAChR activity from a predominantly large and fast channel subtype to a smaller one with increased Po and opening duration, resulting in significant changes in synaptic transmission. Our report provides evidence for a possible mechanism of selective activation and/or suppression of synaptic pathways mediated by specific nAChR subtype(s) at both pre- and postsynaptic sites. Such modulatory mechanisms may be involved in synaptic plasticity by serving as a switch to turn on and off specific synaptic pathways.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Sympathetic neuron cultures

Cultures of embryonic day (ED) 14 chick sympathetic neurons were prepared as described previously (Role 1984). Briefly, lumbar sympathetic ganglion (LSG) chains were removed from chick embryos. LSG neurons were dispersed by trituration after trypsin treatment (100 µg/ml, 30 min), plated at a density of 0.75 chain/dish in 35-mm polyornithine-coated culture dishes, and maintained in vitro at 37°C for 3-5 days with culture media containing 5% chick embryo extract (CEE) before recording. The culture medium was Eagle's minimal essential medium (GIBCO) supplemented with 10% heat-inactivated horse serum, 5% CEE, 500 µg/ml penicillin, 50 µg/ml streptomycin, 2.4 mM glutamine, and 0.5 µg/ml nerve growth factor.

Dorsal spinal cord explant and sympathetic neuron synaptic co-cultures

Cultures of ED14 chick LSG neurons were prepared as described in the preceding text and maintained in vitro for 2 day with culture media containing 2% CEE. Explants of the dorsal spinal cord region (mid thoracic to upper lumbar) that contain sympathetic preganglionic neurons (visceral motor neurons or VMNs) were then prepared from ED9 chick embryos as described (Gardette et al. 1991) and added to dispersed sympathetic neurons in 0.5 ml 2% CEE medium to facilitate rapid settling and attachment. During this initial incubation in minimal volume and for the remainder of the co-culture period, culture dishes were maintained in a hydrated, gas-permeable environment. After 14- to 18-h incubation, additional medium was added to a final volume of 1.5 ml. Under these conditions, preganglionic projections emerge from the explants within hours and contact sympathetic (LSG) neurons within 1-2 days and have formed numerous synapses by the time of recording (3-4 days).

Electrophysiological recordings

Cells were visualized on a phase-contrast inverted microscope (Zeiss IM). Membrane currents were recorded by the patch-clamp technique (Hamill et al. 1981). nAChR single-channel currents gated by acetylcholine (ACh, 10-20 µM) were recorded in cell-attached patches from ED14 chick sympathetic neurons. Unless specified otherwise, the patches were held at +40 mV, resulting in a net cross membrane potential of about -95 mV within the patch as the resting membrane potential of the sympathetic neurons is approximately -55 mV. To determine channel conductance, holding potentials of +20 and +60 mV were applied for 1 min each at the beginning of the recording. Macroscopic and synaptic currents were recorded by the whole cell configuration with cells held at -60 mV. Recordings were made at room temperature except for synaptic recordings where cultures were heated to 32-37°C to enhance spontaneous synaptic activity. The extracellular solution contains (in mM) 145 NaCl, 3 KCl, 2.5 CaCl2, and 10 HEPES, pH = 7.4. Glucose (10 mM) was also added for synaptic recordings to enhance spontaneous synaptic activity. To block action potential-dependent activity, 1 µM tetrodotoxin (TTX) was added. The intracellular solution includes (in mM) 3 NaCl, 150 KCl, 1 MgCl2, 1 EGTA, 10 HEPES, 5 MgATP, and 0.3 NaGTP, pH = 7.2. Voltage-clamp recordings were performed with a List EPC-7 patch-clamp amplifier (Medical System). Membrane currents with a cutoff frequency of 10 kHz were recorded continuously with a Sony VCR through a digital recorder interface (VR100B, Instrutech). Kimax-51 glass capillaries were used for patch and puffer pipettes and were pulled using a List L/M-3P-A 2 stage vertical puller (Medical System). A Narishige microforge was used for fire polishing recording pipette tips. To reduce pipette capacitance in single-channel recordings, pipette tips were coated with Sigmacote (Sigma). All recording solutions were filtered through 0.2-µm-pore-diameter membranes (Nalgene and Gelman Sciences) before use.

Drugs and drug application

ACh chloride, choline chloride, and (-)-nicotine (hydrogen tartrate salt) were purchased from Sigma. alpha -Bungarotoxin (alpha BgTx) was purchased from Molecular Probes, PGE2 from Cayman Chemical, and TTX from Calbiochem. During recordings, cultures were continuously perfused with normal extracellular solution by gravity perfusion. For bath application of drugs, a second gravity perfusion device containing drug solution was used. For focal application of drugs, a puffer pipette (~1µm pore diameter) containing the drugs was placed under the microscope close to the cell of interest. Drugs were applied at known concentrations by pressure-driven focal ejection with a Picospritzer (General Valve). For single-channel studies, PGE2 was applied to the extrapatch membrane for 10 min following a 10-min control. For macroscopic and synaptic current recordings, nAChR agonists were applied by focal application, while other drugs were applied through bath application. The typical focal drug application paradigm for macroscopic current recordings consisted of applying the drug in brief pulses (0.5-2 s) with 4-5 min of washout intervals. For synaptic current recordings, the application paradigm was to apply the agonists for 1 min followed by a minimum of 10-min washout period. For experiments using alpha BgTx, the tissues were pretreated with the drug for >= 10 min prior to assay.

Data collection and analysis

Single-channel currents were filtered at 2 kHz with a 4-pole Bessel filter (DC Amplifier/Filter, Warner Instrument) and acquired at 20 kHz using a Pentium computer (Micron) with pCLAMP 6 software (Axon Instruments) through a DigiData 1200 A/D interface (Axon Instruments). Ten minutes of records were sampled from control, during PGE2 treatment, and after washout respectively. Detection and analysis of single-channel events (<= 5 amplitude classes) were performed with pCLAMP6 software (FETCHAN and pSTAT). Events <250 µs were ignored due to system resolution. Amplitude, duration, and Po of each channel class were determined by FETCHAN analysis. Po is the product of mean open duration (tau open) times opening frequency (Po = tau open × fopen). Channel conductance was determined by slope of the linear fit of a three-point current-voltage relationship at +20, +40, and +60 mV holding potentials. For macroscopic and synaptic currents, signals were filtered at 1 kHz and acquired at 5 kHz. Peaks of macroscopic currents were determined by FETCHAN measurement. For spontaneous synaptic currents, amplitude, rise time, decay time constant, and frequency of mEPSCs were measured with custom software written by C. F. Stevens and A. Kyrosis in Axobasic (Axon Instruments). Data were expressed as means ± SE. Student's paired or unpaired t-test was performed where appropriate to determine statistical significance.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

There are four nAChR subtypes in chick sympathetic neurons

The single-channel studies reported are based on recordings from 100 cell-attached patches in cultured sympathetic neurons. To test whether modulation of nAChR channels by PGE2 might be subtype-specific, we examined the effects of PGE2 applied to the extra-patch membrane on ACh-elicited single-channel activity in ED14 sympathetic neurons. As previously shown (Moss and Role 1993; Moss et al. 1989; Yu and Role 1998a,b), later stage (ED14-18) chick sympathetic neurons express multiple nAChR subtypes, with principle conductance values consistent with four classes (~14, ~23, ~36, and ~54 pS; Fig. 1A). More than one subtype can be detected in the same patch, and assays examining the effects of PGE2 on multiple classes are confined to patches containing all four classes (n = 6).



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Fig. 1. Prostaglandin E2 (PGE2) modulates neuronal nicotinic acetylcholine receptor (nAChR) subtypes in a subtype-specific manner. A-C: examples of single-channel records obtained from 1 cell-attached patch containing all 4 nAChR subtypes. Due to low opening frequency of nAChRs, channel openings (downward deflections) of 4 different nAChR subtypes are either cut and pasted together in a sequential order for demonstration purpose (top) or cut and grouped together to show the effects of PGE2 on each subtype (bottom). Dashed lines mark the amplitude of channel openings of different nAChR subtypes, with the corresponding conductance values indicated on the left. In control (A), 4 nAChR subtypes (14, 23, 36, and 54 pS) are apparent within the patch, with the 36 pS as the predominant channel type. PGE2 (10 nM, B), applied to the extrapatch membrane, significantly increases the activity of the 23 pS channel type by increasing both the opening frequency and opening duration of this subtype and decreases the activity of the other 3 nAChR subtypes, such that the 23 pS channel type becomes the predominant channel type. On washout of PGE2 (C), activity of each nAChR subtype gradually returns to control. Patch pipette contained 10 µM ACh, and was held at +40 mV (about -95 mV in net cross membrane potential within the patch). D: effects of PGE2 on channel-opening frequency (left) and open duration (right) of each nAChR subtype from the same patch. PGE2 differentially affects the opening frequency and open duration of each nAChR subtype.

The 36 pS subtype is the most obvious class as it opens the longest (mean = 1.83 ± 0.22 ms) and at relatively high-frequency (0.12 ± 0.03/s). The 14 pS subtype appears to open most frequently (0.48 ± 0.09/s), but most of the openings are brief (mean = 0.78 ± 0.09 ms). The 23 and 54 pS subtypes are similar in mean opening duration (1.12 ± 0.23 and 1.20 ± 0.08 ms, respectively), but open less frequently (0.09 ± 0.02 and 0.07 ± 0.02/s, respectively). There are occasional openings of larger conductance channels (>= 70 pS). Such events occur at very low frequency (<1% of total events) and, hence, were not included in further analyses. These data show that at this developmental stage, the 36 pS subtype is the predominant channel followed by the 14 pS and 54 pS subtypes, with the 36 pS subtype contributing >40% to the ensemble current. The 23 pS subtype accounts for the least amount of current (~10%), due to its relatively low frequency of opening and smaller conductance.

PGE2 inhibits three of the four nAChR subtypes

The effects of and recovery from PGE2 treatment of different nAChR subtypes were determined in each of six patches with all four subtype classes. Exposure of the extrapatch membrane to 10 nM PGE2 significantly changed the activity of nAChR channels within the patch membrane. Analysis of a representative set of recordings from a single patch is shown in Fig. 1. Figure 2 summarizes results of all six patches tested. PGE2 inhibits three of the four nAChR subtypes. The Po of the 14, 36, and 54 pS nAChR subtypes was greatly reduced by PGE2 (Figs. 1, B and D, and 2A). The decrease in Po ranges from 57 (14 pS) up to 86% (36 pS). These modulatory effects of PGE2 on neuronal nAChRs are subtype-specific, as the channel kinetics of 14, 36, and 54 pS nAChRs are differentially affected (Figs. 1D and 2A). PGE2 decreases the opening frequency of the 14 pS subtype (0.28 ± 0.07 vs. 0.57 ± 0.12/s in control, P < 0.001), while the open duration is unchanged (0.70 ± 0.07 vs. 0.86 ± 0.10 ms in control, P > 0.05). The inhibition of the 36 and 54 pS subtypes involves reductions in both opening frequency and open duration (frequency: from 0.13 ± 0.03 to 0.04 ± 0.01/s for 36 pS and from 0.06 ± 0.02 to 0.01 ± 0.00/s for 54 pS; duration: from 1.73 ± 0.32 to 1.03 ± 0.09 ms for 36 pS, and from 1.24 ± 0.07 to 0.63 ± 0.12 ms for 54 pS; P < 0.001 for all values; Fig. 2A). Changes in channel Po, opening frequency, and open duration induced by PGE2 for each nAChR subtype are plotted together in Fig. 2B. It shows that extrapatch-applied PGE2 down-modulates three of the four nAChR subtypes routinely detected in cell-attached patches within somatic or proximal dendritic regions of sympathetic neurons. The decreased activity differs in detail among subtypes in that the opening frequency, Po, and/or open duration are differentially affected. On washout of PGE2, activity of these nAChR subtypes returns toward control levels within 5-10 min (Fig. 1C).



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Fig. 2. Summary effects of extrapatch PGE2 on channel-opening probability, opening frequency and opening duration of each nAChR subtype within the patches (n = 6). A: mean values of opening probability [P(open)], opening frequency, and duration of all nAChR subtypes in control and following PGE2 treatment are plotted separately. PGE2 increases the opening probability (Po) of the 23 pS subtype and decreases that of the other three subtypes. Note that the changes in channel kinetics produced by PGE2 are also subtype-specific: the opening frequency and opening duration are mostly affected by PGE2 for the 14 and 23 pS, respectively, while both are significantly decreased by PGE2 for the 36 and 54 pS subtypes. * Statistical significance vs. control (P <=  0.05). B: effects of PGE2 on Po, opening frequency, and opening duration of all nAChR subtypes expressed as changes from control, demonstrating the contrasting effects of PGE2 on the Po, opening frequency, and opening duration of the 23 pS nAChR subtype vs. the 3 other subtypes.

PGE2 upregulates one of the nAChR subtypes

The most striking aspect of the subtype-specific modulation by PGE2 is the selective increase in the activity of the 23 pS channel subtype (Figs. 1B and 2A). Po increased more than three times on average (3.4 ± 0.7, n = 6). As a consequence of the enhanced activity of the 23 pS class and the concurrent decreased activity of the other identified channel subtypes following PGE2 treatment, the 23 pS conductance class replaces the 36 pS as the predominant nAChR subtype. Overall, the contribution of the 23 pS channel to the total current changes from <10 to >50%. The enhancement of the 23 pS channel is due not only to an increase in channel-opening frequency (0.16 ± 0.03 vs. 0.11 ± 0.03/s in control, n = 6, P < 0.05; Fig. 2A), but also to a significant increase in channel mean open duration (from 1.24 ± 0.34 to 3.22 ± 0.75 ms; n = 6, P < 0.001). On removal of PGE2 from the extrapatch membrane, activity of the 23 pS nAChR subtype within the patch partially returns to the control level within 5-10 min (Fig. 1C).

Figure 2B summarizes all analyses of PGE2 modulation-induced changes in channel Po, opening frequency, and open duration. Channel Po, opening frequency, and open duration all increase significantly with PGE2 treatment for the 23 pS nAChR subtype, whereas they all decrease for the other three nAChR subtypes. Thus PGE2 significantly alters the profile of the nAChR channels such that the 36 pS nAChR subtype is replaced by the 23 pS subtype as the predominant channel type contributing to the ensemble current.

Properties of the PGE2-upregulated 23 pS nAChR subtype

The increased activity of the 23 pS subtype by PGE2 was examined in more detail. In chick sympathetic neurons, both somatic nicotinic receptor responses and presynaptic nAChR-mediated facilitation include alpha BgTx-sensitive components. Both the pre- and postsynaptic alpha BgTx-sensitive components are eliminated by prior treatment of neurons with alpha 7-specific antisense oligonucleotides but not by non-alpha 7 oligonucleotides (McGehee et al. 1995; Yu and Role 1998b), consistent with a specific contribution of alpha 7 nAChRs. However, the concentrations of alpha BgTx required to block these responses (IC50 of 10-70 nM) (McGehee et al. 1995; Yu and Role 1998b) are much higher than those reported for alpha 7 homomeric channels expressed in Xenopus oocytes and for type IA currents in hippocampal neurons (Alkondon and Albuquerque 1993; Couturier et al. 1990). Such comparisons have led to the suggestion that pre- and postganglionic neurons in chick sympathetic chains may express alpha 7-containing nAChR subtype(s) that are distinct from homomeric alpha 7 receptors. As a class of nAChRs with conductance near 23 pS is absent following alpha BgTx as well as alpha 7 subunit-specific antisense treatment, Yu and Role (1998b) suggest that this nAChR subtype may include alpha 7 subunits. To further examine the properties of the 23 pS nAChR, we tested whether single-channel and macroscopic currents of this subtype can be gated by the alpha 7 nAChR-selective agonist choline (Alkondon et al. 1997; Papke et al. 1996).

THE 23 PS SUBTYPE IS THE PREDOMINANT NACHR GATED BY CHOLINE. Outside-out patches were excised from sympathetic neurons and held at -60 to -100 mV. In patches that responded to choline application (7 of 14), high concentrations of choline (1-10 mM) activated predominantly a single conductance subtype (23 pS, Fig. 3B) in contrast to ACh or nicotine, which elicited a variety of opening events with different conductance and open duration (Fig. 3A, and not shown). A plot of the current-voltage relationship of the choline-activated currents reveals a single, ohmic conductance of 23.5 pS, and a reversal potential near 0 mV, as expected of nAChRs (Fig. 3C). Fitting of dwell time histograms of choline-gated channel openings (Fig. 3D) reveals open time kinetics that match those of the 23 pS ACh-gated channel observed in this and prior studies (tau 1 and tau 2 = 0.9 and 4.9 ms for choline-gated channel vs. 1.1 and 4.9 ms for the 23 pS nAChR subtype) (also see Yu and Role 1998b, Appendix). At the concentrations of choline used, a few openings of larger amplitude were seen, comprising <5% of the total openings, consistent with the low efficacy of choline in the activation of non alpha 7-containing nAChR subtypes as reported previously (Alkondon et al. 1997; Papke et al. 1996).



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Fig. 3. The 23 pS nAChR subtype is gated by the alpha 7 containing-nAChR-selective agonist choline. A and B: example of single-channel records of nAChRs gated by ACh and choline, respectively. ACh in a cell-attached patch (holding potential = +40 mV) activates 4 subtypes of nAChRs (A, left). A, right: the channel openings of the 23 pS subtype activated by ACh at different cross membrane potentials. B: at the same membrane potentials, choline activates in an outside-out patch channel openings similar to the ACh-activated 23 pS subtype both in amplitude and in open duration. C: current-voltage relationships of the ACh-gated 23 pS channel and the choline-gated channel. Amplitudes of the 23 pS ACh-gated channel and the choline-gated channel as determined by amplitude histograms at varying membrane potentials are plotted against the membrane potentials, revealing that the choline-gated channel reverses near 0 mV and has the same conductance as the ACh-gated channel (23.5 vs. 23.7 pS for ACh).D: open dwell time analysis shows that the choline-gated channel has a similar open dwell time distribution as the ACh-gated 23 pS subtype (tau 1 and tau 2 = 0.9 and 4.9 ms respectively for choline vs. 1.1 and 4.9 ms for ACh). Solid lines are multiple exponential fits of the channel open dwell time distributions, with the underlying exponential components depicted by dashed lines, and the tau  of each component by its peak, respectively. E: the choline-gated channel is modulated in the same way as the ACh-gated 23 pS subtype is by PGE2. In cell-attached patches (holding potential = +40 mV), the Po and mean open duration of the choline-gated channel are increased by 1 nM PGE2 (right; Po: 295.3 ± 74.8% increase; open duration: 122.4 ± 32.6% increase; n = 2), like those of the ACh-activated 23 pS subtype (left; Po: 340.6 ± 73.3% increase; open duration: 160.5 ± 60.6% increase; n = 6).

PGE2 MODULATES THE CHOLINE-ACTIVATED CHANNEL IN A SIMILAR MANNER AS THE 23 PS NACHR SUBTYPE. We next compared the channel-opening characteristics and modulation by PGE2 of choline-gated 23 pS channels to that of the 23 pS, ACh-gated nAChRs. In cell-attached patches, the choline-gated 23 pS currents have brief opening duration and low Po like that of the 23 pS, ACh-gated channel. Treatment with PGE2 (1 nM) increases the Po of the choline-gated channel mainly due to an increase in mean opening duration (from 0.5 ± 0.0 ms in control to 1.1 ± 0.1 ms with PGE2 treatment, n = 2; Fig. 3E). Likewise, only the 23 pS, ACh-gated nAChR subtype increases in Po and open duration with PGE2 treatment. These data support our proposal that the 23 pS, choline-gated channels that are enhanced in activity by PGE2 are the same nAChR subtype as the 23 pS subtype gated by ACh and deleted by alpha 7 antisense (Yu and Role 1998b).

CHOLINE GATES A SLOW-DECAYING, alpha BGTX-SENSITIVE MACROSCOPIC CURRENT. To further compare the choline-activated currents with the alpha BgTx-sensitive currents previously characterized by Yu and Role (1998b), we examined the macroscopic currents elicited by choline. Choline (10 mM) evokes a small, slow-decaying macroscopic current in sympathetic neurons (Fig. 4A), similar to the slow currents underlying an alpha 7-dependent component described by Yu and Role (1998b). At -80 mV holding potential, the average peak response is 70 ± 6 pA (n = 10, Fig. 4B). In cells that have been continuously exposed to a low concentration of choline (7 µM), 10 mM choline elicits a smaller response (45 ± 7 pA, n = 6, P < 0.05 vs. control; Fig. 4A), consistent with the reported desensitizing effect of prior exposure to choline (Alkondon et al. 1997; Papke et al. 1996).



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Fig. 4. Choline activates macroscopic currents that are inhibited by alpha -bungarotoxin (alpha BgTx). In most cells tested, 10 mM choline evokes a slow-decaying macroscopic current response (A, control). In cells that have been continuously exposed to a low concentration of choline (7 µM), 10 mM choline produces a smaller response (A, choline 7 µM). The current response to choline appears to be dose-dependent, as 1 mM choline evokes significantly smaller responses (B; 1 mM: 30 ± 4 pA, n = 4; 10 mM: 70 ± 6 pA, n = 10, P < 0.05). The choline-activated macroscopic currents are inhibited by alpha BgTx (250 nM; C and D; with alpha BgTx: 36.3 ± 1.3% of control; washout: 66.5 ± 0.8% of control; n = 3).

The slow choline-evoked macroscopic currents are inhibited by high concentrations of alpha BgTx (250 nM, 15- to 20-min prior treatment; Fig. 4, C and D), consistent with prior observation that high concentrations of alpha BgTx were needed to inhibit the proposed alpha 7-dependent component of ACh-gated currents in chick sympathetic neurons (Yu and Role 1998b). During treatment with alpha BgTx, responses to choline are reduced to 36.3 ± 1.3% of control (n = 3, P < 0.001). The residual component is likely due to the activation of non alpha 7-containing nAChRs by high concentrations of choline, as choline activates some openings other than those of the 23 pS subtype. After 15-20 min washout of alpha BgTx, the choline responses recover slightly (66.5 ± 0.8% of control, n = 3). These results demonstrate that choline activates a slow-decaying, alpha BgTx-sensitive macroscopic current, matching the alpha BgTx-sensitive, alpha 7-containing nAChR current previously reported (Yu and Role 1998b). Taken together, the single-channel and macroscopic current data are consistent with our proposal that the 23 pS nAChR subtype is an alpha 7-containing nAChR.

Modulation of pre- and postsynaptic nAChRs by PGE2

Our single-channel analyses demonstrate that PGE2 modulates somatic nAChRs, changing the subtype and activity profiles. Such alterations of channel activity may have significant functional impact not only on postsynaptic nAChRs that mediate synaptic transmission, but also on presynaptic nAChRs at axonal terminals that modulate synaptic transmission (see Jones et al. 1999; Role and Berg 1996; Wonnacott 1997). Thus we examined the net impact of PGE2 modulation at synapses from visceral motoneuron and sympathetic neuron co-culture (VMN-LSG synapses), where both pre- and postsynaptic nAChRs are expressed (McGehee et al. 1995; Yang et al. 1998).

FUNCTIONAL ASSESSMENT OF THE PRE- AND POSTSYNAPTIC NACHRS. In innervated sympathetic neurons, miniature excitatory postsynaptic currents (mEPSCs) with amplitudes ranging from 5.5 pA (lowest mEPSC detection threshold) to 30 pA occur frequently in the presence of 1 µM TTX (Fig. 5A). As previously observed, nicotine (50-500 nM) increases the frequency of mEPSCs but elicits minimal macroscopic current (postsynaptic) responses (<10 pA) (McGehee et al. 1995). We assessed the functional states of the post- and presynaptic nAChRs by examining the amplitude versus frequency of mEPSCs, respectively.



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Fig. 5. PGE2 reduces the amplitude and changes the characteristics of miniature excitatory postsynaptic currents (mEPSCs). A-C: example traces of mEPSCs recorded from visceral motoneuron and sympathetic neuron co-culture (VMN-LSG) synapses in control (A), during PGE2 treatment (B), and after washout (C). PGE2 (1 nM) reduces the amplitude and increases the decay time course of mEPSCs (B). On washout of PGE2, mEPSCs gradually returned to control (C). D: PGE2 reduces the amplitude of mEPSCs (control: 13.5 ± 2.2 pA; PGE2: 7.5 ± 1.2 pA; washout: 10.1 ± 2.3 pA; n = 4). - - -, the detection threshold for mEPSCs. E: effect of PGE2 on mEPSC decay time constant (tau ). The decay tau  in both histograms can be fitted with 2 components (tau 1 ~ 2 ms, tau 2 ~ 4 ms). In control, most mEPSCs (92%) had a shorter decay tau  (top). Treatment with PGE2 shifted many mEPSCs (43%) to the longer decay tau  (bottom). Insets: representative fast and slow mEPSCs scaled to the same amplitude to demonstrate the difference in their decay.

PGE2 inhibits mEPSCs

Treatment with PGE2 (1 nM) decreases the average amplitude of mEPSCs under control recording conditions (from 13.5 ± 2.2 to 7.5 ± 1.2 pA, n = 4, P < 0.05; Fig. 5, B and D). This result is consistent with an inhibition of postsynaptic nAChRs. On washout, the amplitude of mEPSCs gradually recovers to near control levels (10.1 ± 2.3 pA; Fig. 5, C and D).

The strong inhibition by PGE2 of the mEPSC amplitude (Fig. 5D) is consistent with the predominance of the PGE2-susceptible 36 pS channels under control conditions at postsynaptic sites. The remaining spontaneous synaptic events detected in the presence of PGE2 are also longer in duration (Fig. 5B). The average decay time constant of mEPSCs shifts from 1.6 ± 0.03 ms in control to 3.2 ± 0.15 ms following PGE2 treatment (n = 1,000 and 131 events, respectively, P < 0.001; Fig. 5E). Since only the 23 pS subtype is enhanced by PGE2 in activity and open duration, both the decrease in amplitude and the increase in duration of mEPSCs detected under PGE2 are consistent with a decreased participation of larger conductance nAChR subtypes in spontaneous synaptic transmission, and an increased contribution of the longer duration, 23 pS subtype to mEPSCs following treatment with PGE2.

The reduction in mEPSC amplitude following exposure to PGE2 is accompanied by a significant decrease in the number of mEPSCs detected under control recording conditions (0.6 ± 0.2 vs. 3.4 ± 0.8 event/5 s in control, n = 4, P < 0.001; Fig. 6B), consistent with a presynaptic as well as a postsynaptic component of modulation by PGE2. However, the observed decrease in mEPSC amplitude appears to be sufficient to account for the quantitative change in mEPSC frequency observed. By reducing the mEPSC amplitude near the detection threshold level (Fig. 5D), PGE2 could cause a significant decrease in the number of mEPSCs detected due to an increase in the number of missed events. If the detection threshold is increased proportionally near the average amplitude of mEPSCs under control conditions, the number of events that would be missed (80%) is quantitatively the same as the decrease in the number of mEPSCs detected under PGE2 (82 ± 5%, n = 4). Thus, although we cannot exclude a presynaptic component, the effects of PGE2 on baseline synaptic transmission can be accounted for by purely postsynaptic mechanisms.



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Fig. 6. Nicotine-induced synaptic facilitation persists under PGE2 treatment. A-C: example frequency histograms of mEPSCs recorded from VMN-LSG synapse(s) in 1 neuron under baseline conditions and during nicotine-induced presynaptic facilitation (Nic, 0.5 µM), in control (A), with PGE2 treatment (1 nM; B), and after washout (C). A: in control, nicotine induces presynaptic facilitation, as evidenced by a robust increase in mEPSC frequency during its application. B: under PGE2 treatment, the nicotine-induced presynaptic facilitation persists although there is a large reduction in the frequency of mEPSCs in baseline conditions. C: on washout of PGE2, both the frequency of baseline mEPSCs and the nicotine-induced presynaptic facilitation return to control. D: average frequency of mEPSCs in baseline conditions and during nicotine application, in control and during PGE2 treatment (n = 4). Nicotine-induced presynaptic facilitation persists under PGE2 treatment. E: nicotine-induced presynaptic facilitation expressed as changes of frequency from baseline conditions. PGE2 appears to enhance the presynaptic facilitation induced by nicotine (9.3 ± 1.0-fold increase vs. 4.7 + 1.4-fold increase in control, n = 4, P < 0.05). However, this interpretation is constrained as changes in mEPSC amplitude and hence event detection threshold with PGE2 confound the analysis of a presynaptic effect.

Nicotine-induced synaptic facilitation persists under PGE2 treatment

Under control conditions, nicotine induces robust presynaptic facilitation, increasing the frequency of mEPSCs in a reproducible and statistically significant manner (16.2 ± 1.9 during nicotine vs. 3.4 ± 1.6 event/5 s in baseline condition, n = 4, P < 0.001; Fig. 6A). In contrast to the apparent decrease in the number of mEPSCs detected under control conditions and in the presence of PGE2, exposure to nicotine induced a clear increase in the number of mEPSCs detected at PGE2-treated synapses (9.9 ± 0.9 during nicotine vs. 0.6 ± 0.3 event/5 s in baseline condition, n = 4, P < 0.001; Fig. 6, B and D). Although the rate of rise in the initial phase of nicotine facilitation appears to be slower in the presence of PGE2 as shown in Fig. 6B, this effect was not observed in other synapses. If one examines the nicotine-induced changes in mEPSC frequency by expressing them as fold increases over baseline mEPSC frequency, the nicotine facilitation appears to be greater in PGE2-treated (9.3 ± 1.0-fold increase) than at non-PGE2-exposed synapses (4.7 ± 1.4-fold increase, n = 4, P < 0.05; Fig. 6E). This interpretation is constrained as changes in mEPSC amplitude and hence in event detection threshold with PGE2 confound the analysis of a presynaptic effect. In fact, the observed changes in mEPSCs that may result from pre and/or postsynaptic effects of PGE2 make the comparison of the effects of nicotine on transmitter release ± PGE2 more problematic. To quantify the effect of PGE2 on nicotine facilitation, we compared the effects of PGE2 on the number of mEPSCs detected and compared the values at baseline with those during nicotine facilitation. The effect of PGE2 on the number of detected mEPSCs during nicotine facilitation (39 ± 3% decrease) is significantly smaller than the effect of PGE2 on the number of mEPSCs at baseline condition (82 ± 5% decrease, n = 4, P < 0.05). Likewise, the "net" increase in the number of detected mEPSCs during nicotine facilitation appears to be similar in the presence or absence of PGE2 (Fig. 6D). The net effects of PGE2 on the apparent spontaneous synaptic activity thus includes a significant depression of baseline but not of the nicotine-stimulated mEPSC frequency. On washout, the baseline synaptic activity recovers and nicotine-induced facilitation returns to control level (Fig. 6, C and E).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The major findings of the present study are: 1) the modulation of somatic nAChR channels in cell-attached patches of sympathetic neurons by PGE2 is mediated by an indirect (second-messenger type) mechanism, as PGE2 is effective when applied to the extrapatch membrane; 2) the modulation is subtype-specific; 3) PGE2 modulation on the activity of nAChRs at synaptic sites per se is subtype-specific; and 4) PGE2 does not inhibit nicotine-elicited presynaptic facilitation. Thus the combined effect of pre- and postsynaptic nAChR modulation by PGE2 is an increase in frequency of mEPSCs of smaller amplitude and prolonged duration. Such modulation would be expected to increase the proportion of small synaptic events and decrease the contribution of faster, larger conductance postsynaptic nAChRs. As such, the modulation of nAChRs by PGE2 may constitute an important mechanism for fine-tuning synaptic transmission in the ganglia.

The diversity in subunit composition of neuronal nAChRs suggests the notion that nAChRs may differ in their susceptibility to modulation by intra- and extracellular mechanisms in a subtype-specific manner (see Lukas 1995; McGehee and Role 1995; Role 1992). This hypothesis has received considerable support from a variety of experimental applications. In chick ciliary ganglia, arachidonic acid (Vijayaraghavan et al. 1995) and pituitary adenylate cyclase-activating polypeptide (Pardi and Margiotta 1999) preferentially inhibit the activity of alpha BgTx-sensitive nAChR subtypes compared with other nAChR subtypes. Arachidonic acid has the opposite effect on alpha 7-containing and alpha /beta -type nAChR complexes produced by heterologous expression of rat nAChR subunit cRNAs (Nishizaki et al. 1998). However, it is difficult to compare the prior studies of arachidonic acid with the current work on PGE2 modulation in view of the widely different concentrations of eicosanoids tested (µM arachidonic acid vs. nM PGE2).

The present study reveals that a common metabolic product of arachidonic acid, PGE2, is an effective, subtype-specific modulator of neuronal nAChRs at nanomolar concentrations. The specificity of the modulation is supported by several observations. First, PGE2 inhibits three of the four nAChR subtypes detected at this developmental stage (ED14; 14, 36, and 54 pS). Inhibition of the larger conductance subtypes confirms our previous observations at earlier stages of development (Tan et al. 1998). PGE2 also selectively enhances the activity of the 23 pS subtype. This observation, at first, appears to contradict the inhibition of the 25 pS channel by PGE2 that we previously observed in the earlier developmental stage (Tan et al. 1998). However, subtypes and subunit composition of nAChRs undergo significant changes during embryonic development in chick sympathetic neurons (Devay et al. 1994; Moss and Role 1993; Yu and Role 1998a). In the previous study by Tan et al., sympathetic neurons of early embryonic stage (mostly ED11) were used. The 25 pS channel in these neurons corresponds to the ~25-30 pS subtype that is abundantly expressed at early developmental stages. Evidence from many studies converges to suggest that this subtype may include both alpha 3 and beta 2 subunits (Listerud et al. 1991; Yu and Role 1998a,b). In the present study, sympathetic neurons of later embryonic stages were used (>= ED14). The 23 pS nAChR subtype that appears at the later development stages may contain the alpha 7 subunit, as suggested by previous and present studies (Yu and Role 1998b; also see appendix in Yu and Role 1998a). Moreover, the subtype-specificity of PGE2 modulation is supported by the observation that the inhibitory modulation by PGE2 is manifest in distinct changes of channel-opening probability, frequency, and open duration of each nAChR subtype. Thus the predominant effect of PGE2 on the 14 pS nAChR subtype is a decrease in frequency of opening. Inhibition of the 36 pS and 54 pS nAChR subtypes by PGE2 is evident both in a significant decrease in event frequency and a decrease in dwell time in the open state. In contrast, the predominant effect of PGE2 on the 23 pS nAChR subtype is an increase in open duration, although significant enhancement of opening frequency is also observed. By inhibiting the larger conductance subtypes and increasing the activity of a smaller conductance subtype, PGE2 significantly alters the cell's macroscopic responses to ACh.

The unique enhancement of the activity and opening duration of the 23 pS nAChR subtype by PGE2 stimulated more detailed examination. Prior studies of this subtype of nAChR channels with alpha 7-specific antagonist alpha BgTx and with alpha 7-targeted antisense oligonucleotides (Yu and Role 1998b), as well as the current observations with alpha 7 nAChR-selective agonist choline, are consistent with the proposal that the 23 pS nAChRs include the alpha 7 subunit. First, block by alpha BgTx and treatment with alpha 7-specific antisense oligonucleotide functionally deletes nAChRs with conductance and kinetic properties equivalent to those of the 23 pS subtype that is enhanced by PGE2 (Yu and Role 1998b). Second, choline gates nAChR channels of the same conductance, reversal potential, and kinetics as the nAChR subtype that is "up-modulated" by PGE2. Modulation by PGE2 of the 23 pS nAChR subtype and that of the choline-gated nAChR subtype are similar in detail: PGE2 predominantly enhances the opening duration of both nAChRs. Finally, choline evokes the same slow-on, slow-off type of macroscopic current elicited by ACh and blocked by alpha BgTx (Yu and Role 1998b). As such, we suggest that the 23 pS nAChR subtype that is uniquely up-modulated by PGE2 is equivalent to the nAChR class previously proposed as "alpha 7 containing" (Yu and Role 1998b).

The modulation of nAChRs by PGE2 is likely to be mediated by specific membrane receptor(s) for PGE2 that are coupled to changes in intracellular signal(s). This proposal is based on our observations that nAChRs are modulated by nanomolar levels of PGE2 in the cell-attached patch configuration. In this configuration, the nAChRs under study within the patch are not directly exposed to PGE2. The effects of nM PGE2 appear to be independent of kinase activity and may be mediated by PGE2-induced increases in intracellular Ca2+ (W. Tan and L. W. Role, unpublished observations). These proposed signaling mechanisms are consistent with those known for high-affinity (EP1 type) PGE2 receptors (Ankorina-Stark et al. 1997; Ito et al. 1991). Modulation of nAChRs by intracellular Ca2+ has been demonstrated in studies of nAChRs in guinea pig submucosal neurons (Glushakov et al. 1999) and of recombinant alpha /beta nAChRs in Xenopus oocytes (Crabtree et al. 1997; Girod et al. 1999).

Subtype-specific modulation of nAChRs by PGE2 has significant functional impact on synaptic transmission at developing sympathetic synapses. During PGE2 treatment, there is a significant decrease in the average amplitude of spontaneous synaptic currents and a reduction in the number of mEPSCs detected at baseline. The decrease in mEPSC amplitude following PGE2 treatment is consistent with the observed decrease in activity of three of the four classes of somatodendritic nAChRs. The reduction in baseline mEPSC frequency is likely due to the decrease in amplitude of mEPSCs by PGE2 with respect to the detection threshold, such that a significant proportion of the events are below the detection threshold. Indeed, the observed decreases in mEPSC amplitude is sufficient to account quantitatively for the change in mEPSC frequency. In contrast to the reduced mEPSC frequency at baseline, many mEPSCs are detected during nicotine stimulation under PGE2 treatment. This observation is consistent with the idea that the apparent reduction in mEPSC frequency is largely due to PGE2 inhibition of postsynaptic, rather than presynaptic, nAChRs.

The lengthening of the time constant of mEPSCs during PGE2 treatment is consistent with an enhancement of the relative contribution of smaller amplitude, longer duration postsynaptic nAChRs to synaptic activity, as PGE2 selectively enhances the activity and open duration of the somatic 23 pS nAChR subtype. These data are consistent with an overall effect of PGE2 to "switch" the profile of participant synaptic nAChRs from fast, relatively large currents to slow and smaller ones. It is interesting to note that under control conditions, the contribution of the 23 pS nAChRs to synaptic currents is relatively minor. A possible interpretation of the increased contribution of the postsynaptic 23 pS nAChRs following nicotine treatment is that previously "silent" nAChRs are unmasked by nicotine and/or PGE2 exposure, analogous to glutamate receptors (for review, see Malenka and Nicoll 1997).

In contrast to the predominantly inhibitory effect of PGE2 on somatic nAChR channels, PGE2 may not inhibit the nicotine-induced synaptic facilitation that is mediated by presynaptic nAChRs. Previous studies using alpha 7-selective nAChR antagonist alpha BgTx and alpha 7-targeted antisense oligonucleotide treatment suggest that the presynaptic nAChRs at visceral motor-sympathetic ganglion synapses include alpha 7-containing nAChRs (McGehee et al. 1995). As presynaptic facilitation is also elicited by choline (unpublished data) and apparently is not inhibited by PGE2, the presynaptic, alpha 7-containing nAChRs may be similar to the postsynaptic, 23 pS nAChR subtype.

In conclusion, PGE2 modulates nAChRs at pre- and postsynaptic sites in a subtype-specific manner, resulting in oppositely directed modulatory effects. Such differential modulation of nAChRs by nM PGE2 may be relevant physiologically, as similar concentrations of PGE2 are found in cerebrospinal fluid in both animals and humans (Yergey et al. 1989) and are synthesized locally by central and peripheral neurons (Bishai and Coceani 1992; Tan et al. 1998). Depending on the subunit composition of nAChRs at a particular synapse, PGE2 may thus produce significant changes in the profile of synaptic transmission (e.g., see Nishizaki et al. 1999a,b; Oyaizu and Narahashi 1999; also see Broide and Leslie 1999; Levin and Simon 1998).


    ACKNOWLEDGMENTS

We thank J. Turner for editorial assistance and Drs. R. Girod, M. Jareb, and P. Flood for critical reading of the manuscript.

This work was supported by National Institutes of Health Grants DA-00391 (C. Du) and DA-09366 (L. W. Role) and by NIH Program Project Grant NS-29832 (L. W. Role).


    FOOTNOTES

Address for reprint requests: L. W. Role, Dept. of Anatomy and Cell Biology, Center for Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University, 1051 Riverside Dr., Kolb Research Annex Room 807, New York, NY 10032 (E-mail: lwr1{at}columbia.edu).

Received 24 August 2000; accepted in final form 15 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society