Sodium Action Potentials Are Not Required for Light-Evoked Release of GABA or Glycine From Retinal Amacrine Cells

Mark C. Bieda1,2 and David R. Copenhagen1

 1Departments of Ophthalmology and Physiology, UCSF School of Medicine, San Francisco 94143; and  2Stanford Neuroscience Program, Stanford, California 94309


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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). 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.

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; 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.

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). 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

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), 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.

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.



View larger version (27K):
[in this window]
[in a new window]
 
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.



View larger version (24K):
[in this window]
[in a new window]
 
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.



View larger version (21K):
[in this window]
[in a new window]
 
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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society