1Departments of Ophthalmology and
Physiology,
Bieda, Mark C. and
David R. Copenhagen.
Sodium action potentials are not required for light-evoked release of
GABA or glycine from retinal amacrine cells. Although most CNS
neurons require sodium action potentials (Na-APs) for normal
stimulus-evoked release of classical neurotransmitters, many types of
retinal and other sensory neurons instead use only graded potentials
for neurotransmitter release. The physiological properties and
information processing capacity of Na-AP-producing neurons appear
significantly different from those of graded potential neurons. To
classify amacrine cells in this dichotomy, we investigated whether
Na-APs, which are often observed in these cells, are required for
functional light-evoked release of inhibitory neurotransmitters from
these cells. We recorded light-evoked inhibitory postsynaptic currents
(IPSCs) from retinal ganglion cells, neurons directly postsynaptic to
amacrine cells, and applied TTX to block Na-APs. In control solution,
TTX application always led to partial suppression of the light-evoked
IPSC. To isolate release from glycinergic amacrine cells, we used
either bicuculline, a GABAA receptor antagonist, or
picrotoxin, a GABAA and GABAC receptor
antagonist. TTX application only partially suppressed the glycinergic
IPSC. To isolate release from GABAergic amacrine cells, we used the
glycine receptor blocker strychnine. TTX application only partially
suppressed the light-evoked GABAergic IPSC. Glycinergic and GABAergic
amacrine cells did not obviously differ in the usage of Na-APs for
release. These observations, in conjunction with previous studies of
other retinal neurons, indicate that amacrine cells, taken as a class,
are the only type of retinal neuron that uses both Na-AP-dependent and
-independent modes for light-evoked release of neurotransmitters. These
results also provide evidence for another parallel between the
properties of retinal amacrine cells and olfactory bulb granule cells.
Most vertebrate CNS neurons require sodium action
potential (Na-AP) production for stimulus-evoked fast classical
neurotransmitter release. Many classes of neurons in sensory structures
release fast classical neurotransmitters via graded potentials instead of Na-APs.
The use of Na-APs versus graded potentials divides neurons into two
separate categories. Previous studies have demonstrated that these two
categories of neurons have consistently different physiological
properties and also probably have different information processing
properties (Juusola et al. 1996 In the retina, photoreceptors, horizontal cells, and bipolar cells are
graded potential neurons, whereas ganglion cells require Na-AP
production for evoked neurotransmitter release. The position of
amacrine cells in this classification is still unclear. Although many
amacrine cells produce Na-APs (Barnes and Werblin
1986 The goal of this study was to assess the role of Na-APs in mediating
light-evoked release of classical neurotransmitters from amacrine
cells. Because amacrine and ganglion cells are the only retinal cell
classes that have active sodium channels during light stimulation, only
these cells will be directly affected by TTX. To measure the release of
inhibitory neurotransmitters by amacrine cells, we recorded the
ON inhibitory postsynaptic current (IPSC) in ganglion cells
by voltage clamping ganglion cells near 0 mV, the reversal potential
for glutamate-gated synaptic currents (Mittman et al.
1990 Retinal slices (200-300 µm) were prepared from tiger
salamander (Ambystoma tigrinum) eyes following UCSF and NIH
guidelines. After forming the eyecup (see Coleman and Miller
1989 The extracellular solution consisted of (in mM) 112 NaCl, 2 KCl, 2 CaCl2, 1 MgCl2, 5 hemisodium HEPES, and 25 glucose, pH 7.6 with NaOH. The internal solution consisted of (in mM)
107 CsMES, 3 MgCl2, 5 EGTA, 5 HEPES, 2 Na2ATP,
and 0.5 NaGTP, pH 7.3-7.4 with CsOH. Whole-cell currents were recorded
with an Axopatch 1D and acquired with PULSE (HEKA electronik GmbH;
Lambrecht, Germany). Most ganglion cells had transient EPSCs in
response to light, with transient ON-OFF cells
predominating. Voltages were corrected for junction potential and
electrode offset. After initiation of whole cell recording, we let
cells dialyze for ~10 min before recording the baseline for
experiments. We used whole-bath perfusion of a ~300-µl chamber with
solution flow at ~2 ml/min to exchange solutions.
A red LED was used to provide full-field light stimulus (1- or 2-s
duration; 12- to 20-s interstimulus interval). These interstimulus intervals generated stable baseline responses with no evidence of any
slow light-adaptational processes. We typically recorded ~10-15
baseline responses before applying TTX.
All compounds were exclusively purchased from Sigma, except TTX, which
was purchased from Calbiochem and Sigma.
All analyses were conducted with custom written software in the IGOR
PRO (Wavemetrics, Lake Oswego, OR) environment (by Bieda).
Efficacy of TTX blockade
Because TTX is an open channel blocker, we applied relatively
large numbers of successive light flashes (typically ~20) during the
TTX application period. In all cases, the TTX-induced suppression of
the ON IPSC quickly stabilized after the initial
approximately five responses. Our results demonstrate a wide range of
variability in the magnitude of TTX suppression (Figs.
1B;
2, B and D, and 3B). To verify that this
variability was not due to problems with the stock of TTX or the
perfusion system, we typically monitored TTX block of the ganglion cell
sodium current during experiments (data not shown). In all cases, TTX
(1 µm) induced complete, partially reversible block of the sodium
current. Therefore the observed variability in TTX-induced
ON IPSC suppression was not due to problems with TTX
efficacy or the perfusion system.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
). Therefore knowledge of
a neuron's use of Na-APs or graded potentials gives important clues to
physiological and information processing aspects of that neuron.
; Roska et al. 1998
), many of these cells
have anatomic features compatible with graded release, including lack
of an axon and reciprocal synapses with bipolar cells (Dowling
1987
). Paradoxically, cultured GABAergic rat amacrine cells
require Na-AP production for evoked release (Taschenberger and
Grantyn 1995
), whereas cultured GABAergic chick amacrine cells
probably do not (Gleason et al. 1993
). Although a recent
report (Cook et al. 1998
) concluded that glycinergic amacrine cells use both Na-AP-dependent and -independent modes of
light-evoked neurotransmitter release, there is no information on
whether GABAergic transmission requires Na-APs in the intact retina.
). To block Na-APs in the circuit, we applied 1 µm TTX,
which has been shown to block all voltage-gated sodium currents in
amacrine cells (Barnes and Werblin 1986
; Bieda and
Copenhagen, unpublished observations). In brief, we found that release
of GABA or glycine from amacrine cells onto ganglion cells uses both Na-AP-dependent and -independent processes. A brief report of this
work has previously been made (Bieda and Copenhagen
1998
).
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
), we removed the retina and applied hyaluronidase (Sigma
type IV-S, 300-400 units/ml; 2-5 min) to eliminate vitreous. Similar
hyaluronidase treatments do not affect retinal response properties
(Coleman and Miller 1989
; Winkler and Cohn
1985
). All procedures were performed with infrared illumination.
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Fig. 1.
Light-evoked release of inhibitory neurotransmitters from amacrine
cells occurs by both sodium action potential (Na-AP)-independent and
-dependent mechanisms. A: representative experiment.
ON inhibitory postsynaptic currents (IPSCs), reflecting
release from amacrine cells, were recorded from a ganglion cell.
Responses are averages of 5 consecutive traces. Bar indicates period of
light ON. Scale bars: 40 pA, 500 ms. B:
summary results from 9 cells demonstrates wide range of suppression of
ON IPSC charge by TTX application.
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Fig. 2.
Both Na-AP-dependent and -independent modes contribute to
light-evoked glycine release. A: representative
experiment; 200 µm picrotoxin present in all solutions. Markings as
in Fig. 1. Scale bars: 80 pA, 500 ms. B: summary results
of experiments with picrotoxin. Note wide range of suppression.
C: representative experiment with 100 µm bicuculline
present in all solutions. Markings as in Fig. 1. Scale bars: 100 pA,
500 ms. D: results from 4 cells with bicuculline present
continuously. All responses (A and C) are
averages of 5 consecutive traces. Measurements in B and
D are of suppression of ON IPSC charge.
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Fig. 3.
Both Na-AP-dependent and -independent modes contribute to light-evoked
GABA release; 10 µm strychnine present in all solutions.
A: representative experiment. Responses are averages of
5 consecutive traces. Markings as in Fig. 1. Scale bars: 80 pA, 500 ms.
B: summary results of suppression of GABAergic
ON IPSC charge by TTX. Note wide range of suppression.
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RESULTS |
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Figure 1 shows the suppression of the ON IPSC induced by TTX (1 µm) application in control saline. In the experiment shown in Fig. 1A, TTX induced a significant, mostly reversible suppression of the amplitude and charge of the ON IPSC. Block of this IPSC by coapplication of bicuculline (100 µm) and strychnine (1 µm) indicates that it was mediated by GABA and/or glycine receptor activation (Fig. 1A). The summary of experiments demonstrates a wide distribution in the amount of TTX suppression (Fig. 1B). These experiments demonstrate that light-evoked release of inhibitory transmitters from at least some amacrine cells does not require Na-APs.
In tiger salamander, light-evoked ON IPSCs in ganglion
cells usually have both GABAergic and glycinergic components
(Mittman et al. 1990; Zhang et al. 1997
).
Therefore, to explain the variability seen in Fig. 1B, we
hypothesized that glycinergic and GABAergic transmission might have
different dependencies on Na-APs and that different ganglion cells
possess different ratios of glycinergic to GABAergic input. Hence we
studied the role of Na-APs in mediating release of GABA and glycine
separately. A recent study with picrotoxin to block GABA receptors
indicates that release from glycinergic amacrine cells does not require
Na-APs (Cook et al. 1998
). However, because application
of picrotoxin has been found to greatly increase the release of
glutamate by bipolar cells (Dong and Werblin 1998
; Zhang et al. 1997
), the relative role of Na-APs in
mediating glycine release may be distorted under this condition. In
contrast, the GABAA receptor antagonists bicuculline and
SR95531 have been reported to have weak effects on the release of
glutamate (Dong and Werblin 1998
; Zhang et al.
1997
) and therefore may provide more easily interpretable
results. Hence to extend and confirm the results of Cook et al. (1998)
,
we tested the effect of TTX on the glycinergic ON IPSC by
employing either picrotoxin or bicuculline to block GABAergic inputs.
Figure 2 shows experiments examining the effect of TTX on glycinergic
ON IPSCs. In Fig. 2, A and B,
picrotoxin (200 µm) was continuously present to block
GABAA and GABAC receptors. In a representative
experiment, TTX led to a mostly reversible decrease in the charge and
amplitude of the strychnine-sensitive ON IPSC (Fig.
2A). Figure 2B shows significant variability from
cell to cell in the amount of TTX-induced suppression. These results
are consistent with those of Cook et al. (1998). In Fig. 2,
C and D, bicuculline (100 µm) was continuously
present to block GABAA receptors. Application of TTX
induced a small, reversible decrease in the charge and amplitude of the
strychnine-sensitive ON IPSC in a representative experiment
(Fig. 2C). Figure 2D displays the results of four
experiments of this type. Three of four neurons showed ~30%
suppression of the ON IPSC by TTX. However, one neuron showed an increase in the ON IPSC with TTX. Because
GABAergic amacrine cells are mutually inhibitory (Zhang et al.
1997
), augmentation of release by TTX in this experiment
probably reflects a net disinhibition of the glycinergic amacrine cells
synapsing onto the ganglion cell. Taken together, the results of Fig. 2
demonstrate that light-evoked release of glycine can occur from
amacrine cells in the absence of Na-APs.
In Fig. 3, we selectively measured GABA release from amacrine cells by performing experiments in strychnine (10 µm). In some experiments, GABAergic ON IPSCs were quite transient (Fig. 3A) and in others relatively sustained (data not shown). Application of TTX induced a small decrease in the picrotoxin-sensitive ON IPSC in the experiment shown in Fig. 3A. Figure 3B displays summary results indicating a wide distribution in the magnitude of TTX-induced suppression. In total, these results demonstrate that light-evoked GABA release from amacrine cells does not require Na-AP generation.
The ranges of TTX suppression of glycinergic (Fig. 2, B and D) and GABAergic (Fig. 3B) transmission are overlapping and not obviously distinguishable. Therefore these results do not support a model with differential reliance of GABAergic and glycinergic amacrine cells on Na-APs for release of neurotransmitters.
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DISCUSSION |
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We conclude that both GABAergic and glycinergic amacrine cells can
produce light-evoked release of GABA or glycine without employing
Na-APs. Therefore, in the context of previous studies (Dowling
1987), our results indicate that amacrine cells are the only
class of retinal cells that employs both Na-AP-dependent and
-independent release modes. This Na-AP-independent release may occur
by graded potentials and/or production of calcium action potentials.
Also these results demonstrate another significant parallel between the
properties of retinal amacrine cells and olfactory bulb granule cells,
which also comprise a class of Na-AP-producing neurons that do not
require Na-APs for inhibitory neurotransmitter release (Isaacson
and Strowbridge 1998
).
Two aspects of the data are also noteworthy. First, there was a wide
distribution in the amount of TTX-induced suppression of the
ON IPSC. Second, there was no clear difference between the
reliance of GABAergic release on Na-APs and the reliance of glycinergic
release on Na-APs. However, because amacrine cells form mutually
inhibitory networks (Roska et al. 1998; Zhang et al. 1997
), we must be cautious in drawing conclusions about the relative magnitudes of TTX effects. This is particularly true in our
experiments with inhibitory antagonists (Figs. 2 and 3) because these
antagonists have been shown to affect glutamate release (Cook et
al. 1998
; Zhang et al. 1997
) and will affect the
inhibition received by amacrine cells. Therefore the percentage of
release remaining in TTX may not accurately reflect the actual percentage of light-evoked Na-AP-independent release from amacrine cells under normal conditions, a conclusion consistent with the TTX-induced increase in release observed in one cell (Fig.
2D). Nonetheless, the large amount of TTX-insensitive,
light-evoked release implies that Na-AP-independent release is
significant under normal conditions when Na-APs are present.
Because this study measures aggregate release from presumably many amacrine cells onto a single ganglion cell, there are several models to account for our results. First, all amacrine cells may use both Na-AP-dependent and -independent release modes (model I). Second, some amacrine cells may rely solely on Na-AP-dependent release, whereas others may rely solely on Na-AP-independent release (model II). Third, there may be a continuum of reliance on Na-APs for release (model III).
For GABAergic release from cultured amacrine cells, Taschenberger and
Grantyn (1995) find an absolute requirement for Na-APs, whereas the
results of Gleason et al. (1993)
imply that there is no requirement.
Our results are not incompatible with these previous, seemingly
contradictory results. Either the segregation model (model II) or model
III could encompass these disparate conclusions. The finding by
Taschenberger and Grantyn (1995)
that some amacrine cells absolutely
require Na-AP production for evoked GABA release would seem to remove
model I from consideration. However, because their work was performed
in a cultured system, it is possible that the results do not apply to
the normally developed retina in vivo. Therefore these experiments do
not eliminate model I.
Future studies with paired recordings of amacrine cells and ganglion
cells should help determine which, if any, of these models is the best
reflection of amacrine cell processing. However, a recently proposed
model (Cook et al. 1998) implies that the ratio of
Na-AP-dependent to -independent release for a single cell may not be
fixed. Also Bloomfield (1996)
demonstrates that, for wide-field rabbit
amacrine cells, TTX-sensitive channels are critical for propagation of
distal inputs to the soma. These results taken together imply that
Na-APs may play diverse and complex roles in amacrine cell function
extending beyond those included in our set of simplified models.
The implications of our finding that amacrine cells, as a class, use
both Na-AP-dependent and -independent release modes for functional
light-evoked release depends on which of the three models presented
previously is closest to the truth. There are many rationales for why
cells might possess each mode of transmission. For example, Na-APs may
be required for transmission of long-distance surround inhibition
(Cook et al. 1998). Also Na-APs may induce faster, more
transient release under some conditions. Local graded potential-mediated release could allow localized neurotransmitter release, allowing dendrites to act as separate computational
compartments (Shepherd and Koch 1990
). These suggestions
represent only a few of the possibilities for this highly flexible system.
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ACKNOWLEDGMENTS |
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We thank Dr. Tania Vu for helpful comments on the manuscript.
M. Bieda was supported by a Howard Hughes Medical Institute predoctoral fellowship and by a training grant from the National Institutes of Health (NIH). This research was supported by the NIH. Additional support was provided by Research to Prevent Blindness and That Man May See, Inc.
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FOOTNOTES |
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Address for reprint requests: D. R. Copenhagen, Dept. of Ophthalmology, UCSF School of Medicine, Room K-141, Box 0730, San Francisco, CA 94143-0730.
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 22 December 1998; accepted in final form 23 February 1999.
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