Department of Biological Sciences and Neurobiology Research Center, University at Albany, State University of New York, Albany, New York 12222
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ABSTRACT |
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Zhang, Chunyi and
John T. Schmidt.
Adenosine A1 and Class II Metabotropic Glutamate Receptors
Mediate Shared Presynaptic Inhibition of Retinotectal
Transmission.
J. Neurophysiol. 82: 2947-2955, 1999.
Presynaptic inhibition is one of the major control mechanisms
in the CNS. Previously we reported that adenosine A1 receptors mediate
presynaptic inhibition at the retinotectal synapse of goldfish. Here we
extend these findings to metabotropic glutamate receptors (mGluRs) and
report that presynaptic inhibition produced by both A1 adenosine
receptors and group II mGluRs is due to Gi protein coupling
to inhibition of N-type calcium channels in the retinal ganglion cells.
Adenosine (100 µM) and an A1 (but not A2) receptor agonist reduced
calcium current (ICa2+) by 16-19% in
cultured retinal ganglion cells, consistent with their inhibition of
retinotectal synaptic transmission (30% amplitude of field potentials). The general metabotropic glutamate receptor (mGluR) agonist 1S,3R-1-amino-cyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD, 50 µM) and the selective group II mGluR receptor agonist
(2S,2'R,3'R)-2-(2',3'-dicarboxy-cyclopropyl)glycine (DCG-IV, 300 nM)
inhibited both synaptic transmission and
ICa2+, whereas the group III mGluR agonist
L-2-amino-4-phosphono-butyrate (L-AP4)
inhibited neither synaptic transmission nor
ICa2+. When the N-type calcium channels were
blocked with
-conotoxin GVIA, both adenosine and DCG-IV had much
smaller percentage effects on the residual 20% of
ICa2+, suggesting effects mainly on the
N-type calcium channels. The inhibitory effects of A1 adenosine receptors and mGluRs were both blocked by pertussis toxin, indicating that they are mediated by either Gi or Go. They
were also inhibited by activation of protein kinase C (PKC), which is
known to phosphorylate and inhibit Gi. Finally, when
applied sequentially, inhibition by adenosine and DCG-IV were not
additive but occluded each other. Together these results suggest that
adenosine A1 receptors and group II mGluRs mediate presynaptic
inhibition of retinotectal synaptic transmission by sharing a pertussis
toxin (PTX)-sensitive, PKC-regulated Gi protein coupled to
N-type calcium channels.
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INTRODUCTION |
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Synaptic transmission is strongly modulated by
neurotransmitter control of the calcium channels mediating vesicle
release (Sanchez-Prieto et al. 1996). Often these
receptors furnish autoregulation of release by responding to
transmitters released from the same terminal, such as glutamate and
adenosine, and by coupling directly via G-proteins to inhibition of
calcium channels (Chavis et al. 1994
; Clapham
1994
; Swartz and Bean 1992
; Trombley and
Westbrook 1992
; Yawo and Chuhma 1993
;
Zhou et al. 1995
). These modulations by adenosine and
metabotropic glutamate receptors are of interest not only because of
their ability for fast, second-to-second reshaping of transmission
(Swartz et al. 1993
), but also because of their implication in controlling synaptic plasticity (Pekhletski et al. 1996
) and in controlling neurodegeneration caused by excess glutamate release (Nicoletti et al. 1996
).
In the visual system, the retinal ganglion cells contain adenosine and
have adenosine A1 (but not A2) receptors on their terminals in optic
tectum (Blazynski et al. 1989; Braas et al.
1987
; Goodman et al. 1983
; Wan and Geiger
1990
). In a previous study, we showed that retinotectal
synaptic transmission in goldfish is mainly mediated by N-type calcium
channels and is inhibited by activation of presynaptic adenosine A1
receptors (Zhang and Schmidt 1998
). Blocking A1
receptors increased synaptic transmission, indicating the presence of
tonic adenosine inhibition. The inhibition was mediated by pertussis
toxin (PTX)-sensitive G-proteins and was interrupted by C-kinase
activation. However, there have been no systematic studies addressing
the effect of adenosine receptors on calcium channels in this synapse.
The presynaptic effects of metabotropic glutamate receptors (mGluRs) in
the visual system have also not been thoroughly explored in spite of
the potential effects on the transmission of visual information and on
visual plasticity. In hippocampus (Baskys and Malenka
1991; Desai et al. 1994
), cerebellum
(Chavis et al. 1994
), cortex (Burke and Hablitz
1994
), olfactory bulb (Trombley and Westbrook
1992
), and striatum (East et al. 1995
;
Swartz et al. 1993
), activation of either class II or
class III metabotropic receptors presynaptically modulates glutamate
release, giving rise to an autoregulation of release. The retinotectal
projection is also known to use glutamate as its transmitter
(Kageyama and Meyer 1989
; Langdon and Freeman
1987
; Schmidt 1991
). Although one paper
(Rothe et al. 1994
) examined the effect of metabotropic receptor activation on calcium channels in ganglion cells, the authors
used a nonselective agonist that did not distinguish which class of
metabotropic receptors produced inhibition and which produced
augmentation of the calcium currents; nor did they relate the effects
to presynaptic inhibition.
In the present study, we use both the isolated nerve-tectum preparation and cultured goldfish retinal ganglion cells for parallel studies on presynaptic inhibition and calcium channel modulation, and report that activation of A1 adenosine receptors and group II metabotropic glutamate receptors both inhibit N-type calcium channels to produce the presynaptic inhibition. The parallel effects of adenosine A1 and class II metabotropic receptors also prompted us to investigate whether they shared a common mechanism. Both were sensitive to activation of protein kinase C (PKC) and to PTX treatment, indicating coupling via the same Gi-protein to the N-type calcium channels. Moreover, inhibition by one occluded the inhibition by the other, verifying that some part of the mechanism is common to both.
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METHODS |
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Field potential recordings
The recording of extracellular synaptic potentials elicited by
electrical stimulation of optic nerve are described in detail in the
previous paper (Zhang and Schmidt 1998). The dissection of the in vitro nerve-tectum preparation, the flow chamber, and the
electronic equipment were exactly as in the previous report.
Cell culture
Calcium current recording experiments were performed on cultured
retinal ganglion cells of goldfish (Carassius auratus). The general cell-culture procedures have been previously reported (Schmidt et al. 1991). Briefly, the optic nerves of
goldfish were crushed 10 days before cell culture to facilitate axonal
regeneration. After dark adaptation, the eyes were removed and the
retinas dissected out. The retina was blotted on filter paper to remove
the photoreceptor layer, then treated with bacterial neutral protease
(Sigma, 0.4 mg/ml) for 30 min while bubbling with
O2. After washing three times with cold
dissection buffer containing 1% BSA, trypsin protease inhibitor, and
kynurenate, the retinas were triturated in culture medium with DNAse
(0.25 mg/ml) and protease inhibitor (15 µg/ml). The cell suspension
was distributed to coverslips coated with laminin (25 µg/ml) in 35-mm
culture dishes. The L15 culture medium was supplemented with 10% fetal
calf serum and 0.1 mg/ml gentomycin (all from Sigma). Cultured cells
were generally recorded during the first 7-10 days.
Recording of membrane calcium currents
Calcium currents were recorded from ganglion cells using the
whole cell patch-clamp technique. The current signal was amplified with
PC-One Patch Clamp Amplifier (Dagan Corporation, Minneapolis, MN) and
was digitized and stored in a 386 computer with Data Translation 2801A
A/D board and software written by John Dempster (available from Dagan).
The ganglion cells were identified by their slightly oval shape,
relatively large nuclei, and conspicuous nucleoli (Ishida and
Cohen 1988). In 1-wk-old cultures, the ganglion cells usually
were 12-18 µm diam and had a long axon extending from one pole of
the multipolar cell body.
The culture dish was used as a recording chamber. Cells were
continuously superfused with a solution containing the following (in
mM): 88 NaCl, 30 TEA-Cl, 10 glucose, 22.5 HEPES, 1 MgCl2, 2.7 KCl, and 5 CaCl2. The solution was oxygenated by bubbling with pure O2. The superfusion was gravity driven
at a flow rate of ~1 ml/min. In all experiments where calcium current
was recorded, tetrodotoxin (0.5 µM, Sigma) blocked the sodium
currents, and tetraethylammonium (TEA) inhibited potassium currents.
Ionic currents were recorded with glass microelectrodes pulled from
1.5-mm glass capillaries (TW150F-4, World Precision Instruments,
Sarasota, FL), and fire-polished on a heated filament. The pipette
solution contained the following (in mM): 80 CsCl, 10 TEA-Cl, 10 EGTA, 10 glucose, 25 HEPES, 1 MgCl2, 0.5 CaCl2, 2 ATP, 0.1 GTP, and 20 creatine phosphate
with 50 U/ml creatine phosphokinase. The DC resistance of the pipettes
was 4-6 M in bath solution. The low free calcium in this pipette
solution (estimated at 10 nM) was to prevent calcium-driven rundown of
the calcium current or contamination by
Ca2+-dependent K+ or
Cl
currents. After obtaining a gigaohm seal, we
compensated for series resistance (7-10 M
) and electrical
capacitance. Unless otherwise stated, the holding potential was set at
90 mV. Membrane calcium current was evoked by applying a depolarizing
pulse of 90 mV ×100 ms, except in experiments where the
current-voltage relationship was studied. The rundown of calcium
current, monitored in preliminary experiments, averaged <3% per hour
(n = 5), a much longer period of time than that
required for testing the effect of a drug on calcium current. Thus
rundown was not a problem under the present recording conditions.
For purposes of testing for adenosine- and mGluR-mediated effects on
input resistance and membrane potential, we replaced the external TEA
with NaCl and omitted TTX, and we used an intracellular solution based
on KMeSO3 replacement of CsCl and TEA-Cl, with other components the same. This is labeled "normal" solution
conditions to distinguish from solutions (above) that isolate calcium
currents. The cells were recorded in current clamp with the whole cell
patch technique and tested with 50 pA × 100 ms current steps to
determine input resistance in the presence or absence of adenosine and
(2S,2'R,3'R)-2-(2',3'-dicarboxy-cyclopropyl)glycine (DCG-IV).
Application of drugs by superfusion
A SF-77B Perfusion Fast-Step (Warner Instrument, Hamden, CT) was used to switch between different perfusion solutions within a second. For concentration-response curves, the drug was started at its lowest concentration and switched to the next higher concentration when the preceding concentration reached its plateau effect, which occurred in <2 min.
Data analysis
Numerical data reported in the text are means ± SE. Groups of 10 current traces were collected, leak subtracted, and averaged with the computer program for each measurement; 2-3 such measurements were made before drug application, at the plateau of drug action and after drug wash out. Values measured after wash out were averaged with the predrug values for use as a control. Comparison of means was performed with either Student's t-test or one-way ANOVA as appropriate. A P value <0.05 was regarded as statistically significant.
Drugs used
Adenosine and tetradecanoyl phorbol acetate (TPA) were obtained
from Sigma. Cyclohexyladenosine (CHA) and
N6-[2-(3,5-dimethoxyphenyl)-2-(2methylphenyl)-ethyl]
adenosine (DPMA) were obtained from Research Biochemicals (Natick, MA).
L-2-Amino-4-phosphonobutyrate (L-AP4),
1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), and
(2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) were
obtained from Tocris Cookson (Ballwin, MO). Lactam, TTX, and
-conotoxin GVIA (
-Ctx GVIA) were obtained from Calbiochem (La
Jolla, CA).
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RESULTS |
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Inhibitory effect of adenosine A1 receptors
We previously reported that adenosine presynaptically inhibits
retinotectal synaptic transmission by acting on A1 adenosine receptors
(Zhang and Schmidt 1998). The effect was presynaptic as
judged by increased paired-pulse facilitation. Examples of this
inhibition are shown in Fig.
1A: the field potential
elicited by stimulation of the optic nerve and recorded in the retinal terminal layer of goldfish tectum was inhibited by adenosine and by the
selective A1 receptor agonist CHA but not by the selective A2 receptor
agonist DPMA. In addition, the adenosine inhibition was fully reversed
by the selective A1 antagonist
8-cyclopentyl-1,3-dipropylxanthine. These results suggested
that this effect was due to a G-protein-mediated inhibition of N-type
calcium channels controlling vesicle release from retinal terminals
(Zhang and Schmidt 1998
).
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Although it is not possible to record calcium currents from retinal
terminals, we could use the whole cell patch technique to record them
from retinal ganglion cells in cultures. First, to rule out the
possibility that adenosine activation of K channels (Trussel and
Jackson 1987) could mediate the presynaptic inhibition, we
recorded under "normal" solution conditions (see
METHODS), to test for effects on input resistance and
membrane potential. Adenosine (100 µM) produced no significant
decrease in input resistance (
1.206 ± 4.63%, mean ± SE, n = 6), and no consistent change in membrane potential. Next, we recorded retinal ganglion cells in voltage
clamp under conditions that isolate calcium currents. Consistent with
the presynaptic inhibition results above, the calcium currents recorded
in retinal ganglion cells were significantly inhibited by adenosine and
by the A1 agonist CHA, but not by the A2 agonist DPMA (see Fig.
1B). The calcium currents averaged 303 ± 17 pA
(n = 84), in the range of those (250-1,300 pA)
reported by Bindokas and Ishida (1996)
. The
inhibition produced by adenosine was concentration dependent (Fig.
2B), with half-maximal
inhibition below 10 µM. This concentration-response relationship was
very similar to that seen previously for the inhibition of synaptic transmission (Zhang and Schmidt 1998
). The maximal
inhibition was 15.9 ± 2.6% (n = 6, P < 0.01). The A1 receptor agonist CHA, at the
concentration that maximally inhibited the field potentials (50-100
nM), inhibited the calcium currents by 19.4 ± 3.5%
(n = 10, P < 0.001). (Although
the inhibition produced by CHA is slightly larger than that produced by
adenosine, the difference is not statistically significant,
P = 0.46.) In contrast, the A2 receptor agonist
DPMA (100 nM) did not significantly inhibit calcium current (Figs.
1B and 2A).
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Similar inhibition by metabotropic glutamate receptors
In parallel to the adenosine study above, we tested the effects of
activating metabotropic glutamate receptors (mGluRs) both on
retinotectal synaptic transmission and on calcium currents in ganglion
cells. Metabotropic glutamate receptors are classified into several
groups based on pharmacological profiles and on molecular cloning
(Abe et al. 1992; Okamoto et al.
1994
; Saugstad et al. 1994
; see also
review by Pin and Duvoisin 1995
).
In the present study of presynaptic inhibition, we tested the general
mGluR agonist 1S,3R-ACPD, the group II mGluR agonist DCG-IV, and the
group III agonist L-AP4, all of which have been linked to
presynaptic inhibition in other systems. As shown in Fig.
3A, the general agonist,
1S,3R-ACPD (50 µM), which is somewhat more active at group II mGluRs
than at other mGluRs, inhibited the field potential by 17.90 ± 1.97% (n = 10, P < 0.001).
DCG-IV, which at 300 nM has been demonstrated to be a selective group II agonist (Hayashi et al. 1993; Ishida et al.
1993
), inhibited the field potentials by 23.3 ± 2.54%
(n = 6, P < 0.001). In
contrast, the specific group III agonist L-AP4 did not
inhibit synaptic transmission, but at 25 µM actually produced a
slight increase (6.07 ± 2.68%, n = 4, ns) in
field potential amplitude. As with adenosine, the inhibition produced
by DCG-IV was presynaptic, as indicated by the increased paired-pulse
facilitation (data not shown).
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In whole cell patch recordings of retinal ganglion cells under normal solution conditions, DCG-IV (500 µM) did not produce any decrease in cell input resistance (+2.54 ± 0.82%, n = 4) or any consistent change in membrane potential that could account for the presynaptic inhibition. Under voltage-clamp conditions that isolated calcium currents, however, DCG-IV potently inhibited calcium currents, consistent with its presynaptic inhibition of retinotectal synaptic transmission. 1S,3R-ACPD produced a smaller inhibition of calcium current, whereas L-AP4 had no effect. Sample recordings are shown in Fig. 3B, and the summary data are shown in Fig. 4A. DCG-IV at 300 nM inhibited the calcium current by 20.4 ± 3.0% (n = 6, P < 0.01). At 50 µM, 1S,3R-ACPD inhibited the calcium current by 12.3 ± 5.1% (n = 4, P < 0.05). In contrast, L-AP4 at 25 µM did not inhibit the calcium current (103 ± 1.6% of control; n = 3, P > 0.05). The inhibitory effect of DCG-IV was concentration dependent, as shown in Fig. 4B, with an IC50 of <100 nM. The results indicate a selective inhibition by group II mGluRs of both synaptic transmission and calcium currents.
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Voltage dependence of adenosine and DCG-IV inhibitions
We next explored whether the inhibition produced by adenosine and DCG-IV was due to a reduced amplitude of peak ICa2+ or due to a shift of the current-voltage (I-V) curve to more depolarized membrane potentials, both of which could contribute to presynaptic inhibition by reducing calcium influx during the action potential. As shown in Fig. 5, it seemed that both mechanisms were involved in the inhibitory effects of adenosine and DCG-IV. In the presence of adenosine (100 µM) or DCG-IV (300 nM), the I-V curve of the calcium current was shifted upward and rightward, indicating both a decreased maximum current and a need for a greater depolarizing voltage to open the channels.
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Adenosine and DCG-IV effects are mainly on N-type calcium channels
There are several types of calcium channels in goldfish retinal
ganglion cells, as differentiated by the use of selective toxins
(Bindokas and Ishida 1996). In our previous paper, we
demonstrated that the synaptic transmission at goldfish retinotectal
synapses is predominantly mediated by N-type calcium channels (100%
inhibition by
-Ctx GVIA, 0% by
-AgaTx IVA). Here
-Ctx GVIA at
0.5-1.0 µM reduced the peak calcium current from 300.5 ± 40 pA
to 56.6 ± 13 pA, a decrease of 82.9 ± 2.03%
(n = 8, P < 0.01, Fig.
6A), comparable with that
observed by Bindokas and Ishida (1996)
. Adenosine (100 µM) then produced only a much smaller percentage inhibition of the
remaining calcium current (5.3 ± 1.2%, n = 4, see Fig. 6B for sample records and Fig. 6D for
summarized data). Similarly, DCG-IV after
-Ctx GVIA produced only a
small 4.9 ± 0.8% inhibition (n = 3, see Fig.
6C for sample records and Fig. 6D for summarized data). These findings demonstrate that the inhibitory effects of both
adenosine and DCG-IV are mainly on the N-type calcium channels.
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Common mediation by PTX-sensitive G proteins
Adenosine receptors and mGluRs belong to the superfamily of G protein-coupled receptors. Our previous paper has shown that adenosine-induced presynaptic inhibition of retinotectal synaptic transmission was sensitive to PTX. We thus tested whether the inhibitory effects of adenosine and DCG-IV on ganglion cell calcium current could be blocked by PTX. As shown in Fig. 7A, preincubation of ganglion cells with PTX (1 µg/ml, 4 h) did not in itself cause any noticeable decrease in the amplitude of the calcium currents (293 ± 26 pA vs. 303 ± 17 pA in controls), but it consistently resulted in a virtual elimination of the inhibition produced both by adenosine and by DCG-IV. The decrease in calcium current produced by 100 µM adenosine and by 300 nM DCG-IV in PTX-treated cells was 1.5 ± 0.3% (n = 5) and 3.3 ± 0.7% (n = 5), respectively, significantly smaller than those produced in normal, untreated cells (Fig. 7B). These results indicate that the inhibitory effects of both adenosine and DCG-IV on calcium currents were mediated by PTX-sensitive inhibitory G proteins.
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Common uncoupling by protein kinase C (PKC)
PKC modulates cellular functions by phosphorylating a large array
of cellular proteins, including membrane receptors, G proteins, and ion
channels (Nishizuka 1986). Because we previously found that PKC activation uncoupled adenosine from presynaptic inhibition of
field potentials, we tested whether PKC activation by TPA (a phorbol
ester) or by (
)7-octylindolactam V (lactam), a nonphorbol PKC
activator, also uncoupled both adenosine and DCG-IV from the inhibition
of calcium currents in ganglion cells. Pretreatment of ganglion cells
with TPA (1 µM) or lactam (300 nM) for 30 min greatly decreased the
inhibitory effects of adenosine and DCG-IV (see Fig.
8 for sample records). The inhibition
produced by 100 µM adenosine was 1.4 ± 0.28%
(n = 5, P > 0.05) and 3.4 ± 0.8% (n = 7, P > 0.05) in the
presence of TPA (1 µM) and lactam (300 nM), respectively (vs.
15.9 ± 2.6% inhibition in untreated cells). The corresponding
values for DCG-IV inhibition were 5.1 ± 1.3% (n = 3, P > 0.05) and 3.3 ± 0.3%
(n = 5, P > 0.05), respectively. TPA
and lactam alone each had no significant effect on the membrane calcium
current. Thus activation of PKC greatly decreased the inhibitory
actions of both adenosine and DCG-IV.
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Occlusion between adenosine receptor and mGLuR effects
Because the inhibitory effects on the calcium current produced by adenosine and DCG-IV were both similarly modulated by PKC and sensitive to prior PTX treatment, it seems likely that they share a common mechanism of action. On the other hand, PTX knocks out both Go and Gi, so they might work through different G proteins. To test this possibility, we performed occlusion experiments in which the application of adenosine was followed by DCG-IV, or in the reverse sequence (Fig. 9). The results showed that the inhibitory effects of adenosine and DCG-IV were not additive. No matter which agonist was applied first, the application of the second (with continued presence of the 1st) always produced a far smaller percent inhibition than that produced by the agonist when applied alone. Thus the inhibitions produced by adenosine and DCG-IV occluded each other, indicating that they share a common mechanism of action and act on the same population of N-type calcium channels.
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DISCUSSION |
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Main findings of the present study
The present study compared the inhibitory effects of adenosine A1 receptors and mGluRs both on retinotectal synaptic transmission and on calcium currents in retinal ganglion cells. The results showed 1) that activation of adenosine A1 receptors and the group II mGluRs both inhibit retinotectal synaptic transmission in goldfish, 2) that this inhibition can be attributed to the inhibition of calcium current through the N-type calcium channels that were previously shown to mediate transmission, and 3) that the two inhibitory pathways share a common mechanism of action because they are both blocked by PTX inactivation of G-proteins, they are both uncoupled by C-kinase activation, and their effects occlude each other.
The results, taken together, suggest that the inhibition of calcium
currents accounts for the mechanism of presynaptic inhibition by both
adenosine and mGluRs. This is based on several lines of evidence. The
first is the fact that neither adenosine nor DCG-IV decreased input
resistance or hyperpolarized the retinal ganglion cells, thereby ruling
out a strong activation of K channels as an alternative method of
producing presynaptic inhibition. Second, there were many parallels
between the effects on presynaptic inhibition and on calcium channels,
including the same subclass of receptors (A1 adenosine and group II
type of mGluRs), the same concentration dependence for agonists such as
adenosine, CHA, and DCG-IV, and the same sensitivities to both PTX and
PKC. Finally, the decreases in calcium current are sufficiently large,
given the third or fourth power relationship between calcium
concentration and transmitter release, to produce much larger
percentage decreases in transmission. In a closer parallel, this
exaggerated, nonlinear effect on transmission was also seen when N- or
P-type subpopulations of calcium channels were blocked with selective
toxins in mammalian CNS (Wheeler et al. 1994). Thus the
calcium channel modulation is both large enough, and the only currently
viable mechanism that can account for the presynaptic inhibition
produced by adenosine A1 and group II mGluRs in this projection.
Because these are the first findings in the visual system, this study
extends the autoregulatory modulation of transmission to the goldfish
retinotectal synapses where it may play a role both in shaping visual
processing and in altering visual plasticity. We return to this latter
point after discussing the components of the mechanism below.
Heterogeneity of calcium channels
By the use of -conotoxins and
-agatoxins, at least four
types of calcium channels, namely N, L, P, Q, have been identified as
contributing to transmitter release in the CNS (Dunlap et al. 1995
; Reuter 1996
; Wheeler et al.
1994
), although their relative contributions differ at
different synapses. The goldfish retinotectal projection seems to be
unique in CNS in its apparently exclusive reliance on N-type calcium
channels. Although T-, L-, and N-type calcium channels are present in
the goldfish retinal ganglion cells (Bindokas and Ishida
1996
), we previously found that the N-type channel blocker
-Ctx GVIA completely blocked retinotectal synaptic transmission,
whereas the P-type blocker
-agatoxin IVA had no effect, and the
L-type blocker nifedipine slightly augmented transmission (Zhang
and Schmidt 1998
). Our present study provides evidence that the
N-type calcium channels are the main targets of the inhibitory effects
of both adenosine A1 receptors and the group II mGluRs, because after
the N-type channels were blocked with
-Ctx GVIA, both receptors
produced much smaller percentage inhibitions of the small, remaining
calcium current. The small effect most likely represents residual
unblocked N-type current, as cost precluded using
-Ctx GVIA above
0.5 µM in all but one case. In this regard, the goldfish retinal
ganglion cells may prove to be a useful model for the study of calcium
channel modulation by various neurotransmitters as well as
pharmacological agents.
Inhibition mediated by inhibitory G proteins
Because the number of G protein subtypes is far less than the
number of neurotransmitters, many neurotransmitters may utilize the
same G proteins in their signal transduction pathways. The PTX-sensitive Go and Gi
proteins mediate most of the inhibitory effects on calcium channels, as
they clearly do here. In neuroblastoma cell lines, the PTX-sensitive
Go and Gi proteins both
converge on the N-type calcium channels, but Go
is capable of mediating only certain neurotransmitter effects and not
others, which must therefore be ascribed to Gi
(Taussig et al. 1992).
The present study showed that adenosine A1 receptors and the group II
mGluRs may share the same PTX-sensitive G protein in producing
inhibition of N-type calcium channels. This is suggested by the high
degree of occlusion of the two inhibitory effects, and also by the
ability of PKC to uncouple the inhibition. Only Gi is known to be inactivated by PKC
phosphorylation (Katada et al. 1985), and group II
mGluRs are known to inhibit adenylate cyclase by acting through
Gi (Nakanishi 1994
;
Pin and Duvoisin 1995
). Finally in sympathetic ganglia,
PTX-sensitive inhibition, but not the separate PTX-insensitive
inhibition of synaptic transmission, was modulated by PKC activation
(Zhang et al. 1996
), indicating an action of PKC at the
G-protein.
PKC modulation of the inhibition by adenosine receptors and mGLuRs
In the previous study, we observed that the inhibitory action of
adenosine A1 receptors on retinotectal synaptic transmission could be
blocked by activation of PKC with phorbol esters. The present study
extended that finding by showing a similar block of the inhibition of
calcium current following PKC activation with either the phorbol ester
TPA or the nonphorbol activator lactam. The latter shows that this is
not a result of non-PKC action of phorbols. In addition, the inhibitory
action of the group II mGluRs on N-type calcium channels are similarly
modulated by PKC activation. This PKC modulation is common to the
inhibitory actions of many transmitters, especially those mediated by
PTX-sensitive G-proteins (Shapiro et al. 1994;
Swartz 1993
; Zhu and Ikeda 1994
) and opens the process to control by other second-messenger pathways, providing a more flexible control of synaptic transmission. In the
previous paper (Zhang and Schmidt 1998
), we showed that
PKC modulation of this inhibition may play a significant role in
stabilizing the regenerating projection; responses at the immature
synapses habituate extremely rapidly because of this inhibition, and
PKC activation completely removes this habituation.
Heterogeneity of mGLuRs and inhibition of synaptic transmission
In contrast to group I mGluRs, which are coupled to phospholipase
C, group II and III mGluRs share similar signal transduction mechanisms, including the inhibition of adenylate cyclase (Pin and Duvoisin 1995). Previous studies have shown that activation of both group II and III mGluRs produce presynaptic inhibition of
synaptic transmission, whereas group I mGluRs may augment transmitter release (Sanchez-Prieto et al. 1996
; our unpublished
results). Takahashi et al. (1996)
reported that
L-AP4, a group III agonist, inhibited presynaptic calcium
current in the giant synapse (calyx of Held) of the rat (P-type calcium
channels). However, in our present study, the group III mGluRs neither
inhibited transmission in the goldfish retinotectal synapse, nor
inhibited calcium current in cultured retinal ganglion cells. The lack
of group III mGluR effect in the present study presumably was due to
the absence of group III receptors in ganglion cells because group II
and III mGluRs work through the same G-protein
(Gi). Although no study has investigated the
expression of mGluRs in goldfish retinal ganglion cells, studies in
other central neurons indicated that only a small percentage of neurons
express all three groups of mGluRs (for example, see Chen and
van den Pol 1998
).
Functional implications of presynaptic inhibition
Findings from the present study extend our understanding on the
physiology of retinotectal synaptic transmission and have implications
for shaping of receptive fields, prevention of excitatory neurotoxicity, and control of plasticity. Excessive release of excitatory amino acids may result in neural damage, so it is of obvious
importance that their release is controlled. Presynaptic inhibition of
release by the same transmitter (autoregulation) or by other
transmitters (heterologous regulation) is therefore an important
mechanism for neural protection. One important finding made in our
previous study (Zhang and Schmidt 1998) was that
endogenous adenosine levels even in excised tectum are sufficient to
inhibit transmission at the retinotectal synapse.
The present study demonstrated that the adenosine A1 receptor-mediated
as well as the group II mGluR-mediated inhibition was due to
depressing the N-type calcium channels, which have previously been
shown to mediate synaptic transmission in the goldfish retinotectal synapses (Zhang and Schmidt 1998). Inhibition of calcium
channels can be used either to depress excitatory inputs or to increase excitability by depressing inhibitory inputs (Stefani et al.
1994
). Because we looked only at the excitatory retinal input,
we do not know whether there is a similar control of inhibition in
tectum. Such mGluRs on inhibitory terminals can effectively alter
processing by suppressing recurrent inhibition selectively at the
active cells, thereby enhancing their signal relative to surrounding areas where lateral inhibition is unabated (Nakanishi
1994
). Conversely, we earlier showed that there is a recurrent
cholinergic circuit for presynaptic augmentation of retinal inputs to
tectum (King and Schmidt 1991
). Thus there are strong
mechanisms to ensure the dominance of the strongest visual input at any
one time. Autoregulation of excitatory inputs by mGluRs and adenosine
A1 receptors can also help to explain the property of habituation to
repeated visual presentations that is found in tectal but not lateral
geniculate neurons (Niida et al. 1980
; Oyster and
Takahashi 1975
).
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ACKNOWLEDGMENTS |
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We thank Drs. Gregory Lnenicka and Suzannah Tieman for useful comments on the manuscript and M. Buzzard for technical assistance.
This work was supported by National Eye Institute Grant EY-03736 to J. T. Schmidt.
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FOOTNOTES |
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Address for reprint requests: J. T. Schmidt, Neurobiology Research Center, State University of New York, 1400 Washington Ave., Albany, NY 12222.
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 16 April 1999; accepted in final form 17 August 1999.
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REFERENCES |
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