quitous in brain, is termed ionotropic
because it gates an ion channel directly. We found that an
AMPA receptor can also modulate a G-protein to gate an ion
channel indirectly. Glutamate applied to a retinal ganglion cell
briey suppresses the inward current through a cGMP-gated
channel. AMPA and kainate also suppress the current, an effect
that is blocked both by their general antagonist CNQX and also
by the relatively specic AMPA receptor antagonist GYKI-
52466. Neither NMDA nor agonists of metabotropic glutamate
receptors are effective. The AMPA-induced suppression of the
cGMP-gated current is blocked when the patch pipette in-
cludes GDP- -S, whereas the suppression is irreversible when
the pipette contains GTP- -S. This suggests a G-protein me-
diator, and, consistent with this, pertussis toxin blocks the
current suppression. Nitric oxide (NO) donors induce the cur-
rent suppressed by AMPA, and phosphodiesterase inhibitors
prevent the suppression. Apparently, the AMPA receptor can
exhibit a metabotropic activity that allows it to antagonize
excitation evoked by NO.
Key words: AMPA; glutamate; ionotropic; metabotropic; G-
protein; retina; rat
Glutamate, the major excitatory transmitter in brain, activates a
class of receptors termed ionotropic because they directly gate
ion channels (Nakanishi, 1992; Seeburg, 1993; Hille, 1994; Riedel,
1996; Pass, 1998). Recently, an ionotropic glutamate receptor of
the subclass that binds AMPA was discovered in cortical homog-
enates to also have a metabotropic function: it activates a
G-protein to suppress adenylyl cyclase (Wang et al., 1997). We
wondered whether the AMPA receptor has other metabotropic
functions and whether these might include indirect gating of ion
channels. We thought to probe for a metabotropic effect in a
system suited for subsequent investigation of its role in neural
integration.
We chose the retinal ganglion cell because the effects of stim-
ulating its dendritic AMPA receptors can be investigated in a
slice preparation (Aizenman et al., 1988; Mittman et al., 1990;
Cohen et al., 1994; Leinders-Zufall et al., 1994; Taylor et al.,
1995; Zhang et al., 1995; Coleman and Miller, 1998; Matsui et al.,
1998) and because the role in neural integration could then be
investigated in the intact retina in vitro. Certain ganglion cells
express a cGMP-gated channel that causes an inward current
when nitric oxide (NO) stimulates guanylyl cyclase to raise
[cGMP] (Ahmad et al., 1994). The natural source of NO is
probably a class of amacrine cell that stains intensely for NADPH
diaphorase (NO synthase) (Sandell, 1985; Sager, 1986). Reason-
ing that a current stimulated by one signal (NO) ought to be
antagonized by another, we tested the AMPA receptor and
discovered that in retinal ganglion cells it can activate a G-protein
to suppress the cGMP-gated current.
MATERIALS AND METHODS
Preparation and recording. Slices from adult rat retina were cut at 200 m
(Werblin, 1978) and viewed on a Zeiss (Oberkochen, Germany) upright
microscope with differential interference contrast optics ( 40 water-
immersion objective). Ganglion cells were identied in the slice by their
position and size. Membrane currents were recorded in the whole-cell
conguration (Hamill et al., 1981) using a patch-clamp amplier (Axo-
patch 200A; Axon Instruments, Foster City, CA) linked to a computer.
The voltage-clamp procedures were controlled by the pClamp software
(Axon Instruments). Data were low-pass ltered (four-pole Bessel type)
with a cutoff frequency of 5 kHz and then digitized at 10 kHz by an
analog-to-digital interface. All experiments were performed at room
temperature (2325°C).
Solutions and drugs. The control Ringers solution contained (in m
M
):
NaCl, 135; KCl, 5; CaCl
2
, 1; MgCl
2
, 1; HEPES, 10; and glucose, 10. The
solution was adjusted with NaOH to pH 7.4 and bubbled with oxygen.
CoCl
2
(1 m
M
), picrotoxin (100
M
), and strychnine (1
M
) were also
added to block synaptic transmission. The recording pipette contained
(in m
M
): CsCl, 140; CaCl
2
, 1; EGTA or BAPTA, 5; HEPES, 10; and
Mg-ATP, 2. The solution was adjusted with CsOH to pH 7.4. Pipette
resistance was 7 M .
Test substances were applied through the bath [8-bromo-cGMP,
8-bromo-cAMP, 8-p-chlorophenylthio-cGMP, CNQX, GYKI-52466,
-methyl-4-caboxyphenylglycine (MCPG),
-cyclopropyl-4-phospho-
nophenylglycine (CPPG), 1-methyl-3-isobutylxanthine (IBMX), zap-
rinast, methylene blue, or sodium nitroprusside], via pressure ejection
for 1 sec from a puffer pipette (glutamate, AMPA, kainate, NMDA,
L
-2-amino-4-phosphonobutyrate [
L
-AP-4], or 1S,3R-1-aminocyclopen-
tane-trans-1,3-dicarboxylic acid [trans-(1S,3R)-AC PD]), or via the
patch pipette (cGMP, GTP- -S, GDP- -S, pertussis toxin, or cholera
toxin). MCPG and CPPG were purchased from Tocris Cookson (Ball-
win, MO). Other chemicals were from Sigma (St. Louis, MO).
RESULTS
Recording from ganglion cells in the slice preparation of adult rat
retina, we conrmed the observations of Ahmad et al. (1994) that
certain ganglion cells express a cGMP-gated current. When a
membrane-permeant analog of cGMP (8-bromo-cGMP) was
Received Dec. 16, 1998; revised Feb. 1, 1999; accepted Feb. 3, 1999.
This work was supported by National Institutes of Health Grant EY00828. We
thank N. Vardi, S. Zigmond, P. Liebman, D. Manning, S. Nawy, and A. Kaneko for
comments; R. Smith, M. Freed, L. Haarsma, J. Demb, and J. Tanaka for technical
advice; and S. Watanabe for technical advice on the slice preparation.
Correspondence should be addressed to Dr. Fusao Kawai, c/o Dr. Peter Sterling,
123 Anatomy/Chemistry Building, Department of Neuroscience, School of Medi-
cine, University of Pennsylvania, Philadelphia, PA 19104-6058.
Copyright © 1999 Society for Neuroscience 0270-6474/99/192954-06$05.00/0
The Journal of Neuroscience, April 15, 1999, 19(8):29542959 bath-applied at 1 m
M
to a ganglion cell voltage-clamped at 50
mV, there was a sustained inward current (217
6 pA; mean
SEM) (Fig. 1a). This current disappeared in normal Ringers
solution. The same current was obtained with another membrane-
permeant analog of cGMP [1 m
M
8-p-chlorophenylthio-cGMP,
which strongly resists hydrolysis by phosphodiesterase (PDE)] in
the bath and also when the recording pipette contained cGMP
(Fig. 1d). A permeant analog of cAMP was ineffective (n
4).
The cGMP-gated current was observed in approximately half of
the cells (37 of 73).
When the sustained cGMP-gated current was induced by
8-bromo-cGMP, a puff of 100
M
glutamate evoked a biphasic
response (Fig. 1a). First, there was a transient inward current, as
expected for direct gating of an ionotropic receptor; then, there
was a brief reduction of the sustained inward current (41
7%;
n
5) (Fig. 1a). The same biphasic response was evoked by
AMPA (10100
M
) (Fig. 1b) and by the AMPA receptor agonist
kainate (10
M
; n
3). The amplitude of the second phase
(reduced inward current) was similar for 100
M
AMPA and 10
M
kainate (38
6 and 34
11%, respectively, with 8-bromo-
cGMP). The amplitude of the second phase was also similar for
100
M
by AMPA and 100
M
glutamate (54
7 and 55
8%,
respectively, with cGMP). However, when the sustained inward
current was induced by 8-p-chlorophenylthio-cGMP, 100
M
AMPA did not reduce the second phase of the response (3
1%;
n
3). The reduction by AMPA of the inward current was faster
(Fig. 1e, thick arrow) when the sustained current was induced by
cGMP rather than by 8-bromo-cGMP. The latency to maximal
reduction after the AMPA puff was 4
2 sec for cGMP and 11
3 sec for 8-bromo-cGMP.
To identify which type of glutamate receptor reduced the
inward current, we applied various agonists and antagonists of the
ionotropic receptors. CNQX (100
M
), an antagonist of both
AMPA and kainate subtypes, diminished the effect of AMPA
(n
4) (Fig. 1c). Furthermore, GYKI-52466 (100
M
), a specic
antagonist of the AMPA receptor (Donevan and Rogawski,
1993), also diminished the effect of kainate (n
3). Therefore,
kainate receptors are probably not involved. The AMPA effects
observed here, both the conventional transient inward current
and the novel reduction of the sustained inward current, seem to
desensitize rather little, as reported by others for AMPA re-
sponses of bipolar and ganglion cells in mammalian retina (Co-
hen et al., 1994; Sasaki and Kaneko, 1996). Finally, NMDA,
applied as a 100
M
puff, evoked a monophasic inward current but
did not reduce the inward current (n
4) (Fig. 2a). Thus,
whatever causes this brief reduction of the inward current, it is
apparently triggered specically by the AMPA receptor.
To test whether a metabotropic glutamate receptor (mGluR)
might reduce the inward current, we applied mGluR agonists.
These included
L
-AP-4 (n
4) (Fig. 2b) and ACPD (n
3).
Although these compounds evoke large currents in ON bipolar
cells (Nawy and Jahr, 1990; Shiells and Falk, 1990; Yamashita and
Wa¨ssle, 1991; Tian and Slaughter, 1994) at the concentration used
(100
M
), they did not affect the inward cGMP-gated current.
Furthermore, mGluR antagonists MCPG and CPPG, both ap-
plied at 300
M
, did not affect the AMPA-evoked reduction of the
steady inward current (n
4). We conclude that the reduction by
AMPA of the sustained inward current is not caused by activation
of an mGluR.
If the reduction by AMPA of the sustained inward current
Figure 1. Stimulation of an AMPA receptor reduced a sustained inward
current caused by cGMP. a, b, Puffer application of 100
M
glutamate for
1 sec (thin arrows) or 100
M
AMPA induced a fast transient inward
current. Superfusion with 1 m
M
8-bromo-cGMP (bars) activated a sus-
tained inward current that was reduced (thick arrows) by puffer applica-
tion of glutamate or AMPA. c, Application of 100
M
AMPA failed to
reduce the 8-bromo-cGMP-induced sustained current in the solution
containing 100
M
CNQX. d, Intracellular dialysis with 1 m
M
cGMP
caused a sl