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
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ABSTRACT |
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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 7-selective
agonist choline, and choline-gated currents are inhibited by
-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,
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.
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INTRODUCTION |
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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
7-containing nAChRs
versus the
3
5
4 receptors (Vijayaraghavan et al.
1995
), whereas it enhances the activity of heterologously
expressed nAChR receptors comprised of rat
7 subunits and other rat
/
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.
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METHODS |
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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.
-Bungarotoxin (
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
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 (
open) times
opening frequency (Po =
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.
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RESULTS |
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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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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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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 BgTx-sensitive
components. Both the pre- and postsynaptic
BgTx-sensitive components
are eliminated by prior treatment of neurons with
7-specific
antisense oligonucleotides but not by non-
7 oligonucleotides
(McGehee et al. 1995
; Yu and Role 1998b
), consistent with a specific contribution of
7 nAChRs. However, the
concentrations of
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
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
7-containing nAChR subtype(s) that are distinct from homomeric
7
receptors. As a class of nAChRs with conductance near 23 pS is absent
following
BgTx as well as
7 subunit-specific antisense treatment,
Yu and Role (1998b)
suggest that this nAChR subtype may
include
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
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
(
1 and
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
7-containing nAChR
subtypes as reported previously (Alkondon et al. 1997
;
Papke et al. 1996
).
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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 7 antisense (Yu and Role
1998b
).
CHOLINE GATES A SLOW-DECAYING,
BGTX-SENSITIVE MACROSCOPIC
CURRENT.
To further compare the choline-activated currents with the
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
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
).
|
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.
|
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.
|
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).
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DISCUSSION |
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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
BgTx-sensitive nAChR subtypes compared with other nAChR subtypes.
Arachidonic acid has the opposite effect on
7-containing and
/
-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
3 and
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
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
7-specific antagonist
BgTx and with
7-targeted antisense
oligonucleotides (Yu and Role 1998b
), as well as the
current observations with
7 nAChR-selective agonist choline, are
consistent with the proposal that the 23 pS nAChRs include the
7
subunit. First, block by
BgTx and treatment with
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
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 "
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
/
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 7-selective nAChR antagonist
BgTx and
7-targeted
antisense oligonucleotide treatment suggest that the presynaptic nAChRs
at visceral motor-sympathetic ganglion synapses include
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,
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
).
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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).
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FOOTNOTES |
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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.
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REFERENCES |
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