Department of Physiology and Institute for Neuroscience, Northwestern University Medical School, Chicago, Illinois 60611
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ABSTRACT |
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Song, Wen-Jie,
Tatiana Tkatch, and
D.
James Surmeier.
Adenosine Receptor Expression and Modulation of Ca2+
Channels in Rat Striatal Cholinergic Interneurons.
J. Neurophysiol. 83: 322-332, 2000.
Adenosine is
a potent regulator of acetylcholine release in the striatum, yet the
mechanisms mediating this regulation are largely undefined. To begin to
fill this gap, adenosine receptor expression and coupling to
voltage-dependent Ca2+ channels were studied in cholinergic
interneurons by combined whole cell voltage-clamp recording and
single-cell reverse transcription-polymerase chain reaction.
Cholinergic interneurons were identified by the presence of choline
acetyltransferase mRNA. Nearly all of these interneurons (90%,
n = 28) expressed detectable levels of
A1 adenosine receptor mRNA. A2a and
A2b receptor mRNAs were less frequently detected.
A3 receptor mRNA was undetectable. Adenosine rapidly and
reversibly reduced N-type Ca2+ currents in cholinergic
interneurons. The A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine completely blocked the effect of
adenosine. The IC50 of the A1 receptor
selective agonist 2-chloro-N6-cyclopentyladenosine was 45 nM, whereas
it was near 30 µM for the A2a receptor agonist CGS-21680.
Dialysis with GDPS or brief exposure to the G protein (Gi/o) alkylating agent N-ethylmaleimide
also blocked the adenosine modulation. The reduction in N-type currents
was partially reversed by depolarizing prepulses. A membrane-delimited
pathway mediated the modulation, because it was not seen in
cell-attached patches when agonist was applied to the bath. Activation
of protein kinase C attenuated the adenosine modulation. Taken
together, our results argue that activation of A1 adenosine
receptors in cholinergic interneurons reduces N-type Ca2+
currents via a membrane-delimited, Gi/o class G-protein
pathway that is regulated by protein kinase C. These observations
establish a cellular mechanism by which adenosine may serve to reduce
acetylcholine release.
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INTRODUCTION |
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Cholinergic interneurons are key regulators of
striatal function (Wooten 1990). Parkinson's disease,
for example, can be treated either by trying to replace the lost
dopamine or by antagonizing cholinergic neurotransmission. Cholinergic
signaling is normally terminated by the rapid hydrolysis of
acetylcholine (ACh) by acetylcholine esterase (AChE). Although
cholinergic fibers and terminals are evenly distributed in the rodent
striatum (Kemp and Powell 1971
; Phelps et al.
1985
; cf. Graybiel et al. 1986
), AChE is nearly absent from the striosomal compartment (Herkenham and Pert
1981
). It is unclear whether other mechanisms are in place to
abbreviate cholinergic neurotransmission with this functionally
distinctive region (Gerfen 1992
; Graybiel
1990
).
One potential regulator of cholinergic signaling in the striosomal
compartment is adenosine. Several observations are consistent with this
possibility. First, 5'-nucleotidase, an ecto-enzyme that metabolizes
AMP to adenosine, is enriched in striosomes (Schoen and Graybiel
1992). Second, cholinergic interneurons co-release ACh and ATP
(Richardson et al. 1987
). ATP is rapidly metabolized by
ecto-ATPases and ectoADPases to AMP in the extracellular space (Brundege and Dunwiddie 1997
). Adenosine generated by
5'-nucleotidase metabolism of AMP is capable of modulating neuronal
function by activating G-protein-coupled receptors (Palmer and
Stiles 1995
). To date, four such adenosine receptors have been
cloned: A1, A2a, A2b, and A3 (Fink et
al. 1992
; Mahan et al. 1991
; Stehle et
al. 1992
; Zhou et al. 1992
). Activation of
A1 receptors has been reported to inhibit N-type
Ca2+ currents, whereas activation of
A2 receptors potentiates P/Q-type Ca2+ currents (Gross et al. 1989
;
Mogul et al. 1993
; Mynlieff and Beam
1994
; Scholz and Miller 1991
; Umemiya and
Berger 1994
; Zhu and Ikeda 1993
). These changes
in transmembrane Ca2+ flux are thought to
underlie the ability of A1 receptors to inhibit and A2 receptors to enhance synaptic transmission
(Brundege and Dunwiddie 1997
).
Similar mechanisms may regulate cholinergic synaptic transmission in
the striatum. Pharmacological assays show that activation of
A1 receptors inhibits striatal ACh release
(Brown et al. 1990; Jin et al. 1993
;
Kirkpatrick and Richardson 1993
). Although neurons are
capable of releasing adenosine itself (Brundege and Dunwiddie 1997
), the conversion of released ATP to adenosine in the
extracellular space is critical to the A1
receptor-mediated inhibition of ACh release (Richardson et al.
1987
). RNA for A1 adenosine receptors (the receptors linked to presynaptic inhibition) has been localized to
large, presumed cholinergic interneurons in the striatum (Dixon et al. 1996
). On the other hand, A2a
receptor-selective agonists have been reported to either enhance
(Brown et al. 1990
; Kirkpatrick and Richardson
1993
) or have no affect on ACh release (Jin and Fredholm
1997
; Jin et al. 1993
). Attempts to localize
A2a receptor mRNA have either concluded that
cholinergic interneurons do not express A2a
receptors (Fink et al. 1992
; Schiffmann et al.
1991
) or express very low levels (Dixon et al.
1996
; Svenningsson et al. 1997
).
This study was undertaken to answer two questions. First, what
adenosine receptors do identified striatal cholinergic interneurons express? Previous attempts to answer this question have relied on
relatively insensitive in situ hybridization techniques and have failed
to unequivocally identify the transmitter phenotype of the neurons
examined. To overcome these limitations, single-cell, reverse
transcription-polymerase chain reaction (scRT-PCR) techniques (Cauli et al. 1997; Song and Surmeier
1996
; Surmeier et al. 1996
) were used to
determine how the expression of known adenosine receptor mRNAs was
coordinated within identified cholinergic interneurons. The second
question posed was how activation of adenosine receptors expressed by
cholinergic interneurons couple (if at all) to voltage-dependent Ca2+ channels known to control neurotransmitter release? To
ensure that the effects of exogenously applied ligands were mediated solely by receptors expressed by the neuron under examination, acutely
isolated cholinergic interneurons were studied. Isolated neurons were
voltage clamped to determine the impact of adenosine receptor
activation on Ca2+ channel function. Our results revealed
that cholinergic interneurons express detectable levels of
A1, A2a, and A2b receptor mRNA.
However, only A1 receptors had clearly demonstrable effects
on Ca2+ channels in our preparation. Activation of
A1 receptors inhibited N-type Ca2+ channels
through a membrane-delimited, Gi/o protein pathway that was
sensitive to protein kinase C and transmembrane voltage. These observations establish a cellular mechanism by which ATP released from
synaptic terminals may serve to reduce ACh release.
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METHODS |
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Acute-dissociation procedure
Striatal neurons from juvenile and young adult (>3-6 wk) rats
were acutely dissociated using procedures similar to those described previously (Song and Surmeier 1996; Song et al.
1998
). In brief, rats were anesthetized with methoxyflurane and
decapitated; brains were quickly removed, iced, and then blocked for
slicing. Blocked tissue was cut in 400-µm slices with a Microslicer
(Dosaka, Kyoto, Japan) while bathed in a high sucrose solution (in mM:
250 sucrose, 2.5 KCl, 1 Na2HPO4, 2 MgSO4, 2 CaCl2, 11 glucose,
15 N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid] (HEPES), pH 7.4, 300-305 mOsm/l). Slices were then incubated for 1-6 h at room temperature (20-22°C) in a
NaHCO3-buffered saline bubbled with 95%
O2-5% CO2 (in mM: 126 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 26 NaHCO3 1.25 NaH2PO4, 1 pyruvic acid,
0.2 ascorbic acid, 0.1 NG-nitro-L-arginine, 1 kynurenic
acid, and 10 glucose; pH 7.4 with NaOH, 300-305 mOsm/l). All reagents
were obtained from Sigma Chemical (St. Louis, MO). Slices were then
removed into a low Ca2+ (100 µM),
HEPES-buffered salt solution (in mM: 140 Na isethionate, 2 KCl, 4 MgCl2, 0.1 CaCl2, 23 glucose, and 15 HEPES; pH 7.4, 300-305 mOsm/l). With the aid of a
dissecting microscope, regions of the dorsal striatum were dissected
and placed in an oxygenated Cell-Stir chamber (Wheaton, Millville, NJ)
containing HEPES-buffered Hank's balanced salt solution (HBSS, Sigma)
and pronase (Sigma protease Type XIV, 1-3 mg/ml). This solution was
held at 35°C. After 30-40 min of enzyme digestion, tissue was rinsed
three times in the low-Ca2+, HEPES-buffered
saline and mechanically dissociated with a graded series of
fire-polished Pasteur pipettes. The cell suspension was then plated
into a 35-mm Lux Petri dish containing HEPES-buffered HBSS saline. The
dish was then transferred to the stage of an inverted microscope. After
allowing the cells to settle, the solution bathing the cells was
changed to an HEPES-buffered saline.
Whole cell recordings
Whole cell recordings of Ba2+ currents
through Ca2+ channels employed standard
techniques (Song and Surmeier 1996). Electrodes were
pulled from Corning 7052 glass and fire-polished before use. The
internal solution consisted of (in mM) 180 N-methyl-D-glucamine (NMG), 40 HEPES, 4 MgCl2, 0.1-5 1,2 bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), 12 phosphocreatine, 2 Na2ATP, 0.2 Na3GTP, and 0.1 leupeptin (pH 7.2-3 with
H2SO4, 265-270 mOsm/l. The
pH of NMG solutions was measured with a Corning model 476570 probe. The external solution consisted of (in mM) 135 NaCl, 20 CsCl, 1 MgCl2, 10 HEPES, 0.001 TTX, 2 BaCl2, and 10 glucose (pH 7.3 with NaOH, 300-305
mOsm/l). Ba2+ was used instead of
Ca2+ as charge carrier to minimize current
rundown. All reagents were obtained from Sigma except for a low metals
grade sulfuric acid (Fluka, Ronkowa, NY). Recordings were obtained with
an Axon Instruments 200 patch-clamp amplifier, controlled and monitored
with a PC running pCLAMP (ver. 6.0) with a 125-kHz interface (Axon
Instruments, Foster City, CA). Electrode resistances were typically
2-4 M
in the bath. After seal rupture, series resistance (4-10
M
) was compensated (70-90%) and periodically monitored. Recordings
were made only from large neurons (>14 pF) that had short (<75 µm) proximal dendrites; the identity of these neurons as cholinergic interneurons was verified with scRT-PCR (see Single-neuron
RT-PCR). The adequacy of voltage control was assessed
after compensation by examining tail currents generated by strong
depolarization. Incomplete or discontinuous decay back to the baseline
was taken as evidence of poor space clamp, and the cell was discarded.
Cell-attached patch recording
Recordings were obtained with an internal solution consisting of
(in mM) 110 BaCl2, 10 HEPES (pH 7.4 with TEA
hydroxide). Electrodes were made as for whole cell recordings except
that they were coated with silicone elastomer (Sylgard). After seal formation, the transmembrane potential was nominally zeroed by bathing
the cell in an isotonic K+ solution
containing (in mM) 140 K gluconate, 1 MgCl2, 5 ethylene glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 10 HEPES, and 5 glucose (pH 7.4 with KOH, 300-305 mOsm/l). Transmembrane currents were
evoked by stepping the electrode to
20 mV from a holding potential of
+80 mV.
Pharmacological methods
Receptor ligands and second-messenger reagents were made up as
concentrated stocks in water or dimethyl sulfoxide and stored at
80°C. Adenosine receptor ligands
2-chloro-N6-cyclopentyladenosine (CCPA), CGS21680 hydrochloride
(CGS), 8-cyclopentyl-1,3-dimethylxanthine (CPT), adenosine, and the
muscarinic receptor agonist oxotremorine methiodide (oxo-M) were
obtained from RBI (Natick, MA) or Tocris (Northpoint, UK).
Phorbol-12-myristate-13-acetate (PMA) and 4
-phorbol were obtained
from Sigma. The calcium channel blocker
-conotoxin GVIA (
-CgTx)
was obtained from Peninsula Labs (Belmont, CA) or Calbiochem (San
Diego, CA). Aliquots were thawed and diluted the day of use. Final
dilutions were made in external media containing 0.01-0.1% cytochrome
C when using
-CgTx. The involvement of Gi/o proteins was studied using N-ethylmaleimide (NEM; Sigma).
Solutions were applied with a gravity-fed "sewer pipe" system. The application capillary (~150 µm ID) was positioned a few hundred micrometers from the cell under study. Solution changes were effected by altering the position of the array with a DC drive system controlled by a microprocessor-based controller (Newport-Klinger, Irvine, CA). Solution changes were complete within <1 s.
Statistical methods
Data were analyzed with AxoGraph (Axon Instruments, ver. 2.0)
and SYSTAT (SPSS, Chicago, IL). Box plots were used for graphic presentation of the data because of small sample sizes (Tukey 1977). The box plot represents the distribution as a box with the median as a central line and the hinges as the edges of the box
(the hinges divide the upper and lower halves of the distributions in
half). The inner fences (shown as a line originating from the edges of
the box) run to the limits of the distribution excluding outliers
(defined as points that are >1.5 times the interquartile range beyond
the interquartiles); outliers are shown as asterisks or circles.
Single-neuron RT-PCR
As we have reported previously (Song and Surmeier
1996; Song et al. 1998
), after recording, cells
were lifted up into a stream of control solution and aspirated into the
patch electrode by negative pressure. Electrodes contained ~5 µl of
sterile recording solution (see above). Some cells were harvested
without recording, with electrodes filled with diethyl pyrocarbonate
(DEPC)-treated water. The capillary glass used for making electrodes
was autoclaved and heated to 150°C for 2 h. Sterile gloves were
worn during the procedure to minimize RNase contamination. After
aspiration, the electrode was broken and contents ejected into a 0.5-ml
Eppendorf tube containing 5 µl DEPC-treated water, 0.5 µl RNAsin
(28,000 U/ml), and 0.5 µl dithiothreitol (DTT; 0.1 M). One microliter oligo(dT) (0.5 µg/µl) was added before the mixture was heated to
70°C for 10 min and incubated on ice for 1 min. Single strand cDNA
was synthesized from the cellular mRNA by adding SuperScript II RT (1 µl, 200 U/µl), buffer (4 µl, 5 × First Strand Buffer: 250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl2), RNAsin
(0.5 µl), DTT (1.5 µl, 0.1 M), and mixed dNTPs (1 µl, 10 mM). The
reaction mixture (20 µl) was transferred to a 42°C water bath and
incubated for 50 min. The reaction was terminated by heating to 70°C
for 15 min. The mixture was then placed on ice. The RNA strand in the RNA-DNA hybrid was then removed by adding 1 µl RNase H (2 U/µl) and
incubating for 20 min at 37°C. All reagents except for RNAsin (Promega, Madison, WI) were obtained from Life Technologies (GIBCO BRL,
Grand Island, NY).
The cDNA from the reverse transcription (RT) of RNA in single striatal
neurons was subjected to polymerase chain reactions (PCR) to detect the
expression of mRNAs coding for choline acetyltransferase (ChAT)
and adenosine receptors. PCR amplification was carried out with a
thermal cycler (MJ Research, Watertown, MA) with thin-walled plastic
tubes. Reaction mixtures contained 2-2.5 mM MgCl2, 0.5 mM
of each of the deoxynucleotide triphosphates, 0.8-1 µM
primers, 2.5 U Taq DNA polymerase (Promega), 5 µl
10 × Buffer (Promega) and 4 µl of the cDNA template made from
the single cell RT reaction. After a 5-min denaturing step to 95°C, a
common thermal cycling program was executed for all primer sets: 94°C
for 1 min, 56°C for 1 min, 72°C for 1.5 min. Forty-five PCR cycles
were followed by a 10-min extension at 72°C. The ChAT mRNA
(Brice et al. 1989) was identified using a pair of
primers flanking a splicing site near the 3' terminus of the coding
region; the primers and conditions of this reaction have been published
previously (Yan and Surmeier 1996
). The primers for
A1 receptor cDNA (Genbank accession No. M64299)
(Mahan et al. 1991
) were 5'-AAC CTG AGT GTG GTA GAG CAA
GAC-3' (nucleotides 748-771) and 5'-A GTC CTC AGC TTT CTC CTC TGG G-3'
(nucleotides 1263-1285). This set yielded a product of 538 bp. The
primers for A2a receptor (Genbank accession No. S47609)
(Fink et al. 1992
) were 5'-AG ATC ATC CGA ACCCAC GTC CTG-3' (nucleotides 961-983), and 5'-A CTC TGA AGA CCA TGA GGA AGCTC-3' (nucleotides 1270-1293). This set yielded a product of 333 bp. The primers for A2b receptor (Genbank accession No.
M91466) (Stehle et al. 1992
) were 5'-AAT AAG AGC TGC TGC
CCT GTG AAG-3'(nucleotides 594-617), and 5'-GCT CAG ACT GAA AGT TGA
CTG TCC-3' (nucleotides 1077-1100). This set yielded a product of 507 bp. The primers for A3 receptor (Genbank accession No.
M94152) (Zhou et al. 1992
) were 5'-ATG TCC TGT GTG CTT
CTG GTCTTC-3' (nucleotides 577-600), and 5'-CTC TCT GAA GCC AGT CAG
ATT CTG-3' (nucleotides 949-972). This set yielded a product of 396 bp. PCR products were visualized after separating by electrophoresis in
1.5-2% agarose gels and staining with ethidium bromide.
Representative products were sequenced and found to match published
sequences. Serial dilution experiments were performed using twofold
dilutions of half the total cellular cDNA derived from the RT reaction
(Barnauskas et al. 1999
; Song et al.
1998
; Tkatch et al. 1998
).
Procedures designed to minimize the chances of cross-contamination were
followed to carry out the PCR reactions (e.g., Cimino et al.
1990). Negative controls for contamination from extraneous and
genomic DNA were run for every batch of neurons. To ensure that genomic
DNA did not contribute to the PCR products, neurons were aspirated and
processed in the normal manner except that the reverse transcriptase
was omitted. Contamination from extraneous sources was checked by
replacing the cellular template with water. Both controls were
consistently negative in these experiments.
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RESULTS |
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Striatal cholinergic interneurons express primarily A1 and A2a adenosine receptor mRNAs
After acute-dissociation of striatal tissue, large, presumptive cholinergic interneurons can readily be distinguished from medium spiny projection neurons by size (Fig. 1A). Large cells with a few primary dendrites (often 2 or 3) and whole cell capacitance >14 pF were chosen for recording. RT-PCR analysis of these neurons revealed that all expressed detectable levels of ChAT mRNA (n = 28; Fig. 1B, right panel).
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To determine how the expression of adenosine receptor subtypes was
coordinated in cholinergic interneurons, RT-PCR experiments were
performed. As a positive control for PCR primers and amplification protocol, whole striatal mRNA was screened for adenosine receptors. As
shown in Fig. 1B (left panel), all four adenosine
receptor mRNAs were detected in the striatum. The amplicons were of the predicted size and sequence, verifying the selectivity of the amplification. Next, single cholinergic interneurons were examined. To
maximize preservation of mRNA, neurons were aspirated without recording. Of 28 ChAT interneurons profiled using one-fifth of the
cellular cDNA as a template, 90% had detectable levels of A1 adenosine receptor mRNA (Fig. 1B).
On the other hand, A2a adenosine receptor mRNA
was detected in only 27% of the interneurons tested (n = 33) using a similar protocol. To determine whether this reflected low
transcript detectability or differential expression, serial dilution
experiments were performed (Song et al. 1998;
Tkatch et al. 1998
). The distribution of detection
thresholds for A2a receptor mRNA in a sample of
20 cholinergic interneurons is shown in Fig. 1C. The
progressive increase in detection probability with increasing cDNA
fraction suggests A2a mRNA was present in low
abundance. Using a simple detection protocol with one-fifth of the
total cellular cDNA, A2b adenosine receptor mRNA
was found in an even smaller subset of identified interneurons (3/13).
A3 receptor mRNA was not detected. Although
quantitative analyses were not attempted for A2b
mRNA, our interpretation of these results is that the low detection
probability reflected low mRNA abundance (Song et al.
1998
; Tkatch et al. 1998
).
Adenosine inhibits Ca2+ currents by activating A1 receptors
Electrophysiological analysis was restricted to large (>14 pF)
neurons that previous work had shown were cholinergic interneurons (Yan et al. 1997). RT-PCR analysis of a subset of
neurons analyzed here (n = 12) confirmed their
expression of ChAT. In these interneurons, adenosine rapidly and
reversibly decreased Ba2+ currents evoked by
depolarizing voltage steps (Fig. 2). The
median reduction in peak Ba2+ current produced by
10 µM adenosine was 23% (n = 29). The reduction also
was frequently accompanied by an alteration in current kinetics (Fig.
2A) and a small rightward shift in the current-voltage
relationship (Fig. 2B). The application the
A1 receptor agonist CCPA (100 nM) also reversibly
reduced the current. At the same concentration (100 nM), the
A2a receptor agonist CGS-21680 did not detectably alter Ba2+ currents (n = 6, data
not shown), suggesting that A2a receptors were
not coupled to somatodendritic Ca2+ channels.
This notion was further supported by experiments using the
A1 receptor antagonist CPT. As shown in Fig.
3, CPT (1 µM) virtually eliminated the
effects of adenosine (10 µM). Removing the antagonist restored the
ability of adenosine to modulate currents. Similar results were seen in
all cells tested (n = 8, P < 0.05, Mann-Whitney U test, see inset Fig.
3C). Dose-response experiments with
A1- and A2a-selective
agonists also supported this identification. As shown in Fig.
3D, the A1-selective agonist CCPA
reduced Ba2+ currents with an
IC50 of 45 nM. In contrast, the
A2a-selective agonist CGS-21680 was much less
effective, having an IC50 near 30 µM. These
results suggest that adenosine inhibits Ca2+
channels by activating A1 receptors and that
A2a receptors, although expressed, are not
coupled to somatodendritic Ca2+ channels in this
preparation.
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NEM-sensitive G proteins mediate the A1 receptor action
To test for the involvement of G proteins, the impact of adenosine
was examined with electrodes filled with 2 mM GDPS (0 GTP). The
nonhydrolysable GDP
S competes with endogenous GTP for the nucleotide
binding site on G
proteins, locking G proteins in an inactive state
(Eckstein et al. 1979
). Intracellular replacement of GTP
with GDP
S almost completely eliminated the response to adenosine,
suggesting that G proteins are involved in this process. The median
modulation in cells dialyzed with GDP
S was 7% of that in control
cells recorded the same day (n = 5, P < 0.05, Mann-Whitney U test). Previous electrophysiological
studies have found that A1 receptors are coupled
to pertussis toxin (PTX)-sensitive G proteins
(Gi/o-class) (Scholz and Miller
1991
; Zhu and Ikeda 1993
). To test whether the
A1 receptor effects in cholinergic interneurons also involved PTX-sensitive G proteins, the sulfhydryl alkylating agent, N-ethylmaleimide (NEM), was used. NEM has been shown
to disrupt coupling of PTX-sensitive G proteins to
Ca2+ channels (Shapiro et al.
1994
). Unlike PTX, NEM acts quickly, allowing a positive
control to be taken for the same cell. As shown in Fig.
4, adenosine (10 µM) and CCPA (100 nM)
both reduced peak currents. A brief (2 min) application of NEM (50 µM) reduced the responses to both adenosine (n = 5)
and CCPA (n = 3). Figure 4D shows the box
plots of the percent modulation by adenosine and CCPA before and after
the application of NEM in five experiments (CCPA was tested in 3 cells). NEM reduced both the adenosine and the CCPA modulation to
~20% of the control value, suggesting that the
A1 receptor modulation was mediated by
Gi/o-class G proteins.
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A1 receptors inhibit N-type Ca2+ currents
Previous studies in several types of neuron have shown that
A1 adenosine receptors inhibit N-type
Ca2+ channels (Gross et al. 1989;
Mynlieff and Beam 1994
; Umemiya and Berger
1994
; Zhu and Ikeda 1993
). Striatal cholinergic
interneurons have been shown to express five pharmacologically distinct
types of Ca2+ channels, including N-type
(Yan and Surmeier 1996
). To determine whether
A1 receptors in cholinergic interneurons also
target N-type channels, the ability of
-CgTx GVIA to occlude the
modulation was tested. As shown in Fig.
5, the application of
-CgTx GVIA (1 µM) eliminated the modulation of whole cell
Ba2+ currents by adenosine. Shown in Fig.
5A is a time course from one of these experiments where peak
current evoked by a voltage step to 0 mV is plotted as a function of
time. Representative current traces are shown in Fig. 5B.
Initially, the application of adenosine reduced peak currents. As
expected, the application of
-CgTx dramatically reduced peak
currents. Subsequently, adenosine had little or no effect. A box plot
summarizing the modulation by adenosine after the block of N-type
channels (n = 4) is shown in Fig. 5A, inset.
On average, >80% of the current reduction produced by adenosine was
of N-type.
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A1 receptor modulation is membrane-delimited
In other cells, A1 receptor activation of
Gi proteins leads to a reduction in cytosolic
cAMP levels by inhibiting adenylyl cyclase (Linden 1991;
Zink et al. 1995
). However, previous work has failed to
reveal any effect of cytosolic cAMP on voltage-dependent Ca2+ channels in cholinergic interneurons
(Yan et al. 1997
). On the other hand, G protein-coupled
receptors can inhibit Ca2+ channels through a
membrane-delimited pathway involving G protein
subunits
(Herlitze et al. 1996
; Ikeda 1996
). One
characteristic of this type of modulation is rapid onset kinetics. To
test whether the A1 modulation of
Ca2+ channels was fast enough to be consistent
with this sort of mechanism, onset kinetics were measured at low and
high agonist concentrations. In these protocols, a short (30 ms)
depolarizing step to
20 mV was repeated once a second (faster rates
led to N-type current inactivation), and CCPA was used instead of
adenosine to minimize potential activation of other receptor subtypes.
When applied at a high concentration (10 µM), CCPA rapidly reduced
the current (Fig. 6A). The
onset of the modulation was typically biexponential with a principal
time constant near 1-2 s (Fig. 6B). At a lower agonist
concentration (0.1 µM), the onset kinetics were slower (median,
3.2 s; n = 4). A box plot summary of the onset
time constants at high (10 µM) and low (0.1 µM) agonist
concentrations in four experiments is shown in Fig. 6B
(inset). These onset kinetics are close to the range of
those reported for membrane delimited signaling pathways in other cells
(Hille 1994
).
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A more direct test of a membrane-delimited pathway is to bath apply
agonist when recording in the cell-attached configuration. In this
recording configuration, receptors outside the patch of membrane in the
electrode are incapable of modulating Ca2+
channels inside the patch through a membrane-delimited pathway. In
principle, channels can only be affected in this configuration by
soluble second messengers (Hille 1994). In all the
interneurons studied (n = 5), bath application of
adenosine failed to alter Ba2+ currents in
cell-attached macropatches (data not shown), in spite of the fact that
application of adenosine to adjacent interneurons recorded in the whole
cell configuration produced a robust modulation of currents. These
results further support the hypothesis that A1
receptors modulate N-type Ca2+ channels through a
membrane-delimited, G protein pathway.
Another commonly reported feature of this type of signaling pathway is
voltage dependence (Bean 1989; Hille
1994
). That is, the modulation of currents produced by receptor
activation appears to lessen at very depolarized membrane potentials.
Rather than examining the response to strong depolarization per se,
this property is routinely tested by examining the impact of the
modulator on currents evoked by a standard test pulse before and after
a strong (e.g., +100 mV) conditioning step. As shown in Fig.
7A, adenosine produced a
robust modulation of Ba2+ currents evoked from
negative holding potentials. However, when Ba2+
currents were evoked shortly after depolarizing the membrane to +100 mV
for 30 ms (Fig. 7B), the effect of adenosine was
dramatically reduced. On average, the depolarizing step reduced the
modulation of adenosine (10 µM) to 40% of control values (Fig.
7B, inset).
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A1 receptor modulation is disrupted by activation of protein kinase C (PKC)
Adenosine inhibition of Ca2+ channels has
been shown to be disrupted by activation of PKC in rat cortical,
hippocampal, as well as sensory neurons (Swartz 1993).
Muscarinic modulation of Ca2+ channels in
cholinergic interneurons is disrupted by activation of PKC (Yan
et al. 1997
). To test whether the A1
receptor modulation possesses a similar sensitivity, PKC was activated
by bath application of PMA. Before PMA treatment, both adenosine and
oxo-M reduced evoked Ba2+ currents (Fig.
8, A and B). After
PMA (500 nM) exposure, adenosine had substantially less of an impact on
currents, as did oxo-M (Fig. 8, A and D). PMA
appeared to specifically disrupt a component accompanying kinetic
alteration (cf., Fig. 8, B and C). After PMA
treatment, the effects of both adenosine and oxo-M were reduced to
~30% of control modulation. A box plot summarizing the results from
this and three other experiments is shown in Fig. 8D.
Application of the inactive phorbol analogue, 4
-phorbol (500 nM),
was without effect on both adenosine and oxo-M modulation
(n = 6, Fig. 8D), arguing that the PMA
effects were mediated by PKC activation.
|
The ability of PKC to disrupt both the A1 receptor and m2 receptor modulation suggests that they share common signaling elements. If this were the case, coactivation of the receptors should result in a subadditive modulation. To test this hypothesis, adenosine and oxo-M were co-applied. As shown in Fig. 9, in the presence of oxo-M, adenosine had little effect on depolarization-evoked Ba2+ currents. The median adenosine modulation in the presence of oxo-M was around 10% of the control modulation (see Fig. 9A, inset).
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DISCUSSION |
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Cholinergic interneurons express A1 adenosine receptor mRNA
Large cholinergic interneurons could readily be visualized in the
dissociated preparation and subsequently identified by RT-PCR detection
of ChAT mRNA. Ninety percent (25/28) of ChAT neurons had detectable
levels of mRNA for the A1 adenosine receptor
using one-fifth of the total cellular cDNA in the detection reaction. This percentage undoubtedly would have risen to near 100% had a larger
fraction of the total cellular cDNA been used in the detection
reaction. The inference that A1 adenosine
receptor expression was ubiquitous is consistent with ability of
adenosine to inhibit Ca2+ currents in every cell
tested. In addition to A1 adenosine receptor mRNA, a substantial subset of interneurons (27%) had detectable levels
of A2a receptor mRNA. Serial dilution experiments
suggested that the abundance or detectability of this mRNA was low in
cholinergic interneurons. No evidence was found for a subset of
interneurons in which A2a mRNA abundance was
high. In light of these results, the most parsimonious interpretation
of our results is that A2a mRNA is present in all
cholinergic interneurons, but at relatively low levels. Based on
experiments in medium spiny neurons where A2a
receptor mRNA is readily detected (Song and Surmeier, unpublished observations), it is unlikely that the difficulty in detection was a
consequence of inefficient reverse transcription or amplification. This
interpretation is also consistent with previous in situ hybridization studies in which A1 receptor mRNA was more
readily detected in cholinergic interneurons than
A2a receptor mRNA (Dixon et al. 1996; Fink et al. 1992
; Schiffmann et al.
1991
; Svenningsson et al. 1997
). Although
semiquantitative, single-cell studies were not attempted with
A2b receptor mRNA, the most parsimonious
interpretation of its infrequent detection is that it too is present in
all cholinergic interneurons but at low levels.
Activation of A1 receptors reduced N-type Ca2+ currents through a fast, membrane delimited, voltage-sensitive pathway
Several lines of evidence suggest that the effects of adenosine on
Ca2+ currents in cholinergic interneurons were
mediated by A1 receptors. Beyond the virtual
ubiquity of A1 receptor mRNA, the effects of adenosine were mimicked by nanomolar concentrations of the
A1 receptor-selective agonist CCPA, but not by
similar concentrations of the A2a-selective
agonist CGS-21680. More detailed analysis of these agonists revealed
nearly a thousand-fold difference in the IC50s
for CCPA and CGS-21680 in modulating Ba2+
currents. Furthermore, the A1 receptor-selective
antagonist CPT blocked the effects of adenosine. Third, the effects of
adenosine were attenuated by brief exposure to NEM, which is known to
disrupt signaling through Gi/o proteins
(Shapiro et al. 1994). In contrast to
A2a receptors, A1 receptors
couple to intracellular signaling elements through
Gi/o proteins (Linden 1991
;
Palmer and Stiles 1995
).
Although A1 receptors are capable of
inhibiting adenylyl cyclase (Linden 1991; Zink et
al. 1995
), their effects on Ca2+currents
were characteristic of a direct inhibition mediated by G protein
-subunits (Herlitze et al. 1996
; Ikeda
1996
). As in autonomic ganglion neurons, the reduction in
evoked currents was largely occluded by the N-type channel-selective
antagonist
-CgTx GVIA. The modulation was rapid, having a time
constant of a few seconds and was not seen in cell-attached patches
when the agonist was applied outside the recorded patch. Both
observations suggest a membrane-delimited signaling pathway. Last, as
in other
subunit-mediated modulations of N-type channels, the
inhibition of currents was accompanied by alteration in current
kinetics that resembled changes seen in other cell types. This
modulation was reversed by strong depolarization. Although a
contribution by A2 adenosine receptors cannot be
completely excluded, the broad outlines of this modulation are similar
to those described in other cell types following activation of
A1 adenosine receptors (Gross et al.
1989
; Mynlieff and Beam 1994
; Scholz and
Miller 1991
; Umemiya and Berger 1994
; Zhu
and Ikeda 1993
).
A1 receptor modulation was also PKC sensitive, much like that of the muscarinic autoreceptor
The features of the A1 receptor
modulation of Ca2+ currents are very similar to
those of muscarinic m2/m4 receptors in cholinergic interneurons
(Yan and Surmeier 1996). Activation of both m2/m4 and
A1 receptors evoked a rapid, membrane delimited
inhibition of N-type Ca2+ currents that was
reversed by strong depolarization. In addition, both modulations were
reversed by activation of PKC (Swartz 1993
; Zhu
and Ikeda 1994
). The similarities in these pathways suggest that they may have common signaling elements. In agreement with this
hypothesis, muscarine almost completely occluded the effects of
A1 receptor activation. At present, it is unclear
whether the occlusion is a consequence of shared target channels or
more proximal signaling elements. Regardless, these observations
suggest that A1 receptors should influence
cellular function in a manner similar to that of muscarinic autoreceptors.
Like D5 dopamine receptors, A2a adenosine receptors do not appear to couple to Ca2+ channels
Although cholinergic interneurons express low levels of
A2a adenosine receptor mRNA, we found no evidence
that these receptors (if present) couple to voltage-dependent
Ca2+ channels. In other cell types,
A2a receptors couple to Gs
proteins, leading to the stimulation of adenylyl cyclase and protein
kinase A (PKA) (Brundege and Dunwiddie 1997).
A2 adenosine receptor activation has been
reported to enhance P-type Ca2+ currents,
presumably through a PKA-dependent mechanism (Mogul et al.
1993
; Umemiya and Berger 1994
). In striatal
medium spiny neurons, stimulation of adenylyl cyclase and PKA modulates
voltage-dependent Ca2+ currents (Surmeier
et al. 1995
). It is possible that the enzyme digestion or the
dissociation protocol employed in our experiments disrupted the ability
of A2a receptors to couple to
Ca2+ channels. These receptors may, for example,
be present in distal dendrites that are lost during the dissociation.
Nevertheless, the conclusion that A2a receptors
expressed by cholinergic interneurons do not couple to somatic/proximal
dendritic Ca2+ channels is in agreement with
previous work using a similar preparation. In particular, activation of
D5 dopamine receptors or dialysis with cAMP
analogues fails to modulate Ca2+ currents in
cholinergic interneurons, in spite of the fact that these manipulations
result in the modulation of GABAA channels through a PKA-dependent mechanism (Yan and Surmeier
1997
). The reasons for the apparent discrepancy are unclear.
Cholinergic interneurons express Ca2+ channel
1 subunits known to be targets of PKA (Yan et al.
1997
). However, PKA may not be appropriately anchored to
phosphorylate these channels in cholinergic interneurons (Gao et
al. 1997
; Klauck et al. 1996
).
Inhibition of N-type Ca2+currents provides a cellular mechanism for the effects of adenosine on ACh release
How A1 receptor inhibition of N-type
Ca2+ channels will affect synaptic integration
and spike generation in cholinergic interneurons is unclear. N-type
Ca2+ channels are found throughout the dendritic
membrane of most types of neuron (Westenbroek et al.
1992). Reductions in dendritic Ca2+
currents could attenuate augmentation of excitatory synaptic events by
voltage-dependent conductances (Bernander et al. 1994
; Kim and Connors 1993
). The A1
modulation should also attenuate dendritic Ca2+
entry during back propagation of somatic spikes (Spruston et al.
1995
).
The consequences of A1 receptor modulation of
N-type Ca2+ channels in synaptic terminals are
more easily inferred. Ca2+ entry through N-type
Ca2+ channels has been shown to be a major
determinant of transmitter release in a variety of neurons
(Dunlap et al. 1995). In cholinergic interneurons,
A1 receptor activation has been shown to reduce ACh release (Brown et al. 1990
; Jin et al.
1993
; Kirkpatrick and Richardson 1993
). In all
likelihood, this reduction in ACh release is dependent on
A1 receptor-mediated inhibition of N-type
Ca2+ currents. The phenomenological similarities
and shared signaling elements in the A1 and m2/m4
muscarinic autoreceptor pathways reinforce this conclusion. Both
receptors appear to be part of a negative feedback system; with
terminal muscarinic m2/m4 receptors being stimulated by released ACh
and terminal A1 receptors being stimulated by
adenosine generated by the metabolism of co-released ATP
(Richardson et al. 1987
). The negative feedback
regulation of ACh release through A1 receptors
should be particularly strong in the striosomes given this
compartment's prominent expression of 5'-nucleotidase (Schoen
and Graybiel 1992
). However, A1 receptor inhibition of N-type Ca2+ channels also provides
a mechanism for heterosynaptic inhibition of ACh release.
Activity-dependent elevations in extracellular adenosine may result
from the metabolism of transported cAMP or the direct release of
adenosine (Bonci and Williams 1996
; Brundege and
Dunwiddie 1997
; Harvey and Lacey 1997
). ATP may
also be released from corticostriatal glutamatergic terminals in an
activity-dependent manner (Brundege and Dunwiddie 1997
),
providing yet another source of adenosine capable of inhibiting ACh release.
In contrast, our results do not provide an explanation for the ability
of A2a receptor agonists to increase ACh release
(Brown et al. 1990; Kirkpatrick and Richardson
1993
). Although cholinergic interneurons express
A2a receptor mRNA, agonists of these receptors had no obvious effect on Ca2+ channels linked to
transmitter release. It is possible that A2a receptor-mediated activation of PKA directly facilitates ACh release (Kondo and Marty 1997
). Given the promise of
A2a receptor antagonists in treating Parkinson's
disease (Ferre et al. 1997
; Kanda et al. 1998
; Richardson et al. 1997
), determining the
functional impact of these receptors on cholinergic interneurons is an
important task.
In summary, our results demonstrate that cholinergic interneurons express both A1 and A2 adenosine receptor mRNAs. Our results also demonstrate that A1 adenosine receptor activation of Gi/o proteins results in the inhibition N-type Ca2+ channels through a rapid, membrane delimited signaling pathway that is sensitive to strong depolarization and protein kinase C. This signaling pathway provides a cellular mechanism for the A1 receptor inhibition of striatal ACh release.
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ACKNOWLEDGMENTS |
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We thank Drs. Z. Yan, Gytis Baranauskas, and J. Flores-Hernandez for assisting in some of the experiments and Dr. L. Dudkin for technical help. Much of this work was performed at the University of Tennessee, Memphis, TN.
This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-34696 to D. J. Surmeier and a Parkinson's Disease Foundation fellowship and Grant 11170232 from the Japan Ministry of Education, Science, Sports and Culture to W.-J. Song.
Present address of W.-J. Song: Dept. of Electronic Engineering, Faculty of Engineering, Osaka University, Suita, Osaka 565-0871, Japan.
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FOOTNOTES |
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Address for reprint requests: D. J. Surmeier, Dept. of Physiology/Northwestern University Institute for Neuroscience, Northwestern University Medical School, 320 E. Superior St., Chicago, IL 60611.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 5 February 1999; accepted in final form 30 September 1999.
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REFERENCES |
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