Modulation of Excitatory Synaptic Transmission by GABAC Receptor-Mediated Feedback in the Mouse Inner Retina

Ko Matsui, Jun Hasegawa, and Masao Tachibana

Department of Psychology, Graduate School of Humanities and Sociology, The University of Tokyo, Tokyo 113-0033, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Matsui, Ko, Jun Hasegawa, and Masao Tachibana. Modulation of Excitatory Synaptic Transmission by GABAC Receptor-Mediated Feedback in the Mouse Inner Retina. J. Neurophysiol. 86: 2285-2298, 2001. In many vertebrate CNS synapses, the neurotransmitter glutamate activates postsynaptic non-N-methyl-D-aspartate (NMDA) and NMDA receptors. Since their biophysical properties are quite different, the time course of excitatory postsynaptic currents (EPSCs) depends largely on the relative contribution of their activation. To investigate whether the activation of the two receptor subtypes is affected by the synaptic interaction in the inner plexiform layer (IPL) of the mouse retina, we analyzed the properties of the light-evoked responses of ON-cone bipolar cells and ON-transient amacrine cells in a retinal slice preparation. ON-transient amacrine cells were whole cell voltage-clamped, and the glutamatergic synaptic input from bipolar cells was isolated by a cocktail of pharmacological agents (bicuculline, strychnine, curare, and atropine). Direct puff application of NMDA revealed the presence of functional NMDA receptors. However, the light-evoked EPSC was not significantly affected by D(-)-2-amino-5-phosphonopentanoic acid (D-AP5), but suppressed by 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) or 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466). These results indicate that the light-evoked EPSC is mediated mainly by AMPA receptors under this condition. Since bipolar cells have GABAC receptors at their terminals, it has been suggested that bipolar cells receive feedback inhibition from amacrine cells. Application of (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), a specific blocker of GABAC receptors, suppressed both the GABA-induced current and the light-evoked feedback inhibition observed in ON-cone bipolar cells and enhanced the light-evoked EPSC of ON-transient amacrine cells. In the presence of TPMPA, the light-evoked EPSC of amacrine cells was composed of AMPA and NMDA receptor-mediated components. Our results suggest that photoresponses of ON-transient amacrine cells in the mouse retina are modified by the activation of presynaptic GABAC receptors, which may control the extent of glutamate spillover.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Complex information processing occurs in the mammalian inner retina, where graded responses conveyed by bipolar cells (Berntson and Taylor 2000; Euler and Masland 2000) are converted to spiking discharges in ganglion cells (Nirenberg and Meister 1997; Stone and Pinto 1992). Amacrine cells participate in this signal conversion by interacting with bipolar, amacrine, and ganglion cells. In the present study, we focused on the characteristics of glutamatergic excitatory synaptic transmission from bipolar to amacrine cells.

The neurotransmitter released from bipolar cells is the excitatory amino acid glutamate (Slaughter and Miller 1983; Tachibana and Okada 1991). Unlike most CNS neurons, it is generally assumed that the release of glutamate is triggered by graded potential changes and not by sodium action potentials (but see Pan and Hu 2000; Protti et al. 2000). It has been shown in bipolar cells isolated from the goldfish retina that the amount of glutamate release increases as the duration of depolarizing pulses is prolonged up to a few hundred milliseconds (Sakaba et al. 1997; von Gersdorff and Matthews 1994).

In many vertebrate CNS synapses, glutamate released from the presynaptic neuron activates both non-N-methyl-D-aspartate (NMDA) and NMDA receptors on the postsynaptic membrane. These receptor subtypes differ largely in their biophysical properties. Non-NMDA receptors generally show much faster kinetics, such as activation, deactivation and desensitization, than NMDA receptors (Colquhoun et al. 1992; Lester and Jahr 1992). Therefore the transfer function of glutamatergic synapses is determined not only by the release rate of glutamate but also by the proportion of non-NMDA and NMDA receptor-mediated components in the postsynaptic neuron.

Several studies have identified the presence of both non-NMDA and NMDA receptors in amacrine and ganglion cells of the rat retina (Aizenman et al. 1988; Hartveit and Veruki 1997; Taschenberger et al. 1995). Both receptors are activated either by light stimulation (Mittman et al. 1990) or by depolarization of a single bipolar cell (Matsui et al. 1998) in the amphibian retina. However, only non-NMDA receptors are activated in response to quantal release of glutamate (in mammals, Taschenberger et al. 1995; Tian et al. 1998; in amphibians, Matsui et al. 1998; Taylor et al. 1995). These results suggest that non-NMDA and NMDA receptors may be segregated spatially at the postsynaptic membrane and that each receptor may play a functionally distinct role in mediating excitatory synaptic transmission from bipolar cells.

Applying patch-clamp techniques to the slice preparation of the mouse retina, we analyzed the properties of the light-evoked excitatory postsynaptic current (EPSC) in ON-transient amacrine cells. We report that the light-evoked EPSC is mediated mainly by the AMPA type of non-NMDA receptors when the GABAC receptor-mediated feedback inhibition to bipolar cells is intact. When GABAC receptors are blocked by a specific antagonist, (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), the feedback inhibition observed in ON-cone bipolar cells is suppressed and the amplitude and time course of the light-evoked EPSC are drastically changed in ON-transient amacrine cells. The changes in ON-transient amacrine cells are ascribed both to the desensitization of AMPA receptors and to the activation of NMDA receptors, which may be caused by the enhanced spillover of glutamate released from bipolar cells. Our results support the idea that interaction between bipolar and amacrine cells is important in shaping the visual signal dynamically.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Retinal slice preparation

Adult (postnatal day >28) C57/BL6 mice were kept at room temperature under a normal day-night cycle of lighting. Animals were dark-adapted for >= 10 min prior to cervical dislocation. The preparation of the retinal slice was made under dim red illumination. Procedures for handling of animals and preparation were met by the guidelines of The University of Tokyo and The Physiological Society of Japan.

The procedures for preparing mouse retinal slices were nearly identical to those for amphibian retinal slices (Matsui et al. 1998; Werblin 1978; Wu 1987). Briefly, the anterior chamber of the enucleated eye was removed in a plastic petri dish filled with extracellular solution, saturated with 95% O2-5% CO2. The ganglion cell layer side of the retina was attached to a piece of filter paper, and both were sliced together into 190- to 200-µm sections. The slices were transferred to a recording chamber and held down by the fine nylon threads fastened on a platinum horseshoe. These procedures were done within 20 min at room temperature.

The recording chamber was mounted on the stage of a microscope equipped with infrared differential interference optics (Eclipse E600-FN; Nikon, Tokyo) in a light-tight Faraday cage. All subsequent manipulations were performed in a dark room. Recordings were done within 3 h after making the slice preparation.

Superfusate

The superfusate, which was bubbled with 95% O2-5% CO2, was continuously fed to the recording chamber (1 ml in volume) at a rate of 2 ml/min with a peristraltic pump and sucked through the outlet tubing by a negative pressure. Both the inlet tubing and the recording chamber were heated to maintain the temperature of the superfusate at 34.5-36.5°C, irrespective of the position within the chamber.

The extracellular control solution consisted of (in mM) 120 NaCl, 3.1 KCl, 2 CaCl2, 1 MgSO4, 23 NaHCO3, 0.5 KH2PO4, and 6 glucose. The pH of the solution saturated with 95% O2-5% CO2 at 36°C was ~7.6. Pharmacological agents were added to the control solution and were bath- or pressure-applied from a puff pipette. Picrotoxin, strychnine, D-tubocurarine, atropine, and 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466), concanavalin A (type V, Con A) were purchased from Sigma (St. Louis, MO). Bicuculline, D(-)-2-amino-5-phosphonopentanoic acid (D-AP5), 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX), cyclothiazide (CTZ), TPMPA, (2S,4R)-4-methylglutamic acid (SYM 2081) were from Tocris (Bristol, UK). Picrotoxin and CTZ were dissolved in dimethyl sulfoxide for stock solutions. The final concentration of dimethyl sulfoxide after dilution with superfusate was always kept <0.1% vol/vol.

Whole cell recordings

Patch pipettes for whole cell recordings were pulled on a horizontal puller (P97, Sutter Instruments, Novato, CA). The electrode resistance was 6-9 MOmega in the extracellular control solution when pipettes were filled with the intracellular solution. For voltage-clamp current recordings from amacrine and bipolar cells, we used the intracellular solution (abbreviated as P1 in the subsequent sections) which consisted of (in mM) 112 CsCH3SO3, 0.5 CaCl2, 5 EGTA, 20 HEPES, 5.5 MgCl2, and 5 ATP disodium salt and 0.25-0.5% Lucifer yellow dipotassium salt, of which the pH was titrated to 7.6 with CsOH. For current-clamp voltage recordings from bipolar cells, we used the intracellular solution (abbreviated as P2) which consisted of (in mM) 135 K-gluconate, 3 KCl, 0.5 EGTA, 10 HEPES, 2.5 MgCl2, 2 ATP disodium salt, and 0.5 GTP and 0.1% Lucifer yellow dipotassium salt, of which the pH was titrated to 7.6 with KOH. Liquid junction potential was corrected for all recordings.

To record from amacrine cells, cells in the proximal edge of the inner nuclear layer (the amacrine cell layer) were selected and whole cell voltage-clamped using an Axopatch 200B (Axon Instruments, Foster City, CA). Bipolar cells were selected from the cells in the middle part of the inner nuclear layer. All cells were initially whole cell voltage-clamped, and some were subsequently recorded in the current-clamp mode. Both the current and the voltage were low-pass-filtered at 100 Hz to 1 kHz, digitized at 2.5-5 times the filtering frequency with a digitizer board (Digidata 1200B, Axon Instruments), and stored in the hard disk of a computer. Control of the membrane potential and the timing of light stimulation, and data acquisition were carried out by the pCLAMP software (Axon Instruments). Fast capacitance compensation was adjusted to cancel the transient current caused by the pipette capacitance. After the whole cell configuration was established, the rapid component of the charging current, presumably due to the charging of the soma (Jackson 1992), was cancelled to compensate (<= 40-50%) for the series resistance (in the range of 10 and 30 MOmega ).

Light stimulation

A cube beam-splitter was placed under the condenser lens of the microscope. The side of the cube beam-splitter was illuminated through a diffuser by a light-emitting diode (LED; emission maximum at 520 nm), and thus the retinal slice was diffusely illuminated from underneath of the recording chamber. The diameter of the illuminated area was 1.1 or 2.1 mm, which covered a large portion of a single slice. The light intensity was measured at the position of the retinal slice preparation using a photodiode (S1133; Hamamatsu Photonics, Hamamatsu, Japan). The light intensity was set to 92.9 or 98.6 lx, which evoked almost saturating responses in amacrine cells. Neither the peak amplitude nor the waveform of the response was changed by further increase in light intensity from 92.9 to 328.6 lx (data not shown). Photo-bleaching seemed to be negligible when 2-s flashes were applied at the intensity of 92.9 lx with the inter-stimulus interval of 30-40 s because the light-evoked responses were stable for >20 min.

Cell morphology

Lucifer yellow was introduced into the cell via a recording patch pipette, and the morphology of the cell was inspected under epifluorescence illumination after the recording. In the present study, we did not attempt to morphologically categorize amacrine cells into subtypes since morphological classification of amacrine cells in the mouse retina has not yet been established (MacNeil and Masland 1998). Furthermore, it was likely that a part of the processes extending in the IPL may have been cut during the preparation of the retinal slices. Thus morphological data were used to distinguish amacrine cells from bipolar and interplexiform cells, which send processes to both plexiform layers. As for bipolar cells, morphological data were used to distinguish between rod bipolar and ON-cone bipolar cells.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Amacrine cells are physiologically classified

Light-evoked responses recorded in the superfusate without pharmacological agents were used to classify amacrine cells. Amacrine cells were whole cell voltage-clamped at the holding potential of -57 mV. Since the equilibrium potential of chloride (ECl) was calculated to be -59 mV when P1 (see METHODS) was used as the pipette solution, most of the current response should be excitatory. The recorded cells were divided into ON-transient, ON-sustained, ON-OFF-transient and OFF-transient types based on the waveform of EPSC in response to diffuse illumination (Fig. 1, A1 and B). The segregation of ON and OFF pathways in the inner plexiform layer (IPL) was confirmed in the mouse retina (Fig. 1A2); i.e., ON-type and OFF-type amacrine cells extend their dendrites in the inner and outer sublaminas of the IPL, respectively (Famiglietti et al. 1977).



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 1. Classification of photoresponses recorded from mouse retinal amacrine cells. A1: a typical current trace (bottom) evoked in an ON-transient amacrine cell by a diffuse light stimulus (top). The cell was voltage-clamped at -57 mV (ECl = -59 mV) without any pharmacological agents in the superfusate. A2: photomontage of the cell stained with Lucifer yellow. The current trace shown in A1 was obtained from this cell. The cell body lies in the proximal edge of the inner nuclear layer (INL), and the processes spread in the inner part of the inner plexiform layer (IPL). No processes are found in the outer nuclear layer (ONL), the outer plexiform layer (OPL) or the ganglion cell layer (GCL). B: responses of other types of amacrine cells encountered. These include the ON-sustained, ON-OFF-transient, and OFF-transient types (Vh = -57 mV). Cells that could not be classified into these categories are grouped together into the miscellaneous type (Misc.). An example is shown here; only transient inhibitory postsynaptic currents (IPSCs) were evoked at the onset and termination of the light stimulus (Vh = -37 mV). Horizontal and vertical scale bars indicate 500 ms and 15 pA, respectively. C: population of physiological subtypes of amacrine cells. Data obtained from 92 cells. It should be noted that this figure may not reflect the real population but may be biased by various factors, such as accessibility to the cell soma, susceptibility to surgical damages and displacement of the cell position.

The population of each cell type is shown in Fig. 1C. A few cells could not be classified into any of the aforementioned types and were grouped together into the miscellaneous type (Misc.). Some of this type showed only the light-evoked inhibitory postsynaptic current (IPSC), which was revealed by shifting the holding potential away from ECl (Fig. 1B, Misc.).

In the vertebrate retina, photoreceptors release glutamate as their neurotransmitter in the dark (Copenhagen and Jahr 1989; Miller and Schwartz 1983; Witkovsky et al. 1997). Glutamate depolarizes OFF bipolar cells by activating ionotropic non-NMDA receptors, whereas it hyperpolarizes ON bipolar cells by activating metabotropic glutamate receptors mGluR6 (Euler et al. 1996; Hartveit 1996; Nakajima et al. 1993). Bipolar cells themselves release glutamate as their neurotransmitter (Slaughter and Miller 1983; Tachibana and Okada 1991). Therefore the modulators of ionotropic glutamate receptors must affect the OFF pathway (photoreceptors right-arrow OFF bipolar cells right-arrow OFF amacrine and ganglion cells) both in the outer plexiform layer (OPL) and in the IPL, whereas these substances must affect the ON pathway (photoreceptors right-arrow ON bipolar cells right-arrow ON amacrine and ganglion cells) only in the IPL. Because we were interested in the synaptic interaction between bipolar and amacrine cells in the IPL, we focused on the ON pathway.

Among ON amacrine cells we analyzed the response properties of the ON-transient type, the most frequently encountered one (Fig. 1C). This type included cells with various morphology, but the major dendritic stratification was confined to the inner region of the IPL (49-91%; the outer border of the IPL was taken as 0%; 77 ± 2%, mean ± SE, n = 33). It has been reported that rod bipolar cells terminate at the inner border of the IPL (Euler et al. 1996). Thus ON-transient amacrine cells are likely to receive cone input from ON-cone bipolar cells. However, rod bipolar cells make chemical synapses onto AII amacrine cells, which provide excitatory input to ON-cone bipolar cells via gap junctions (Kolb and Famiglietti 1974). Therefore we cannot neglect the possibility that ON-transient amacrine cells may receive rod input through this pathway.

Glutamatergic input is isolated by a cocktail of antagonists

Amacrine cells interact with one another through inhibitory synapses mediated by GABA and glycine (Miller et al. 1981). Changing the holding potential revealed the presence of both excitatory and inhibitory inputs to ON-transient amacrine cells (Fig. 2A). However, it was difficult to isolate the excitatory input alone because both excitatory and inhibitory inputs seemed to overlap considerably even in the steady phase of the responses. Thus we did not analyze the I-V relationship of the light-evoked responses recorded in the superfusate without pharmacological agents. It was also difficult to determine whether the synaptic input recorded at Vh = -57 mV (near ECl) as glutamatergic, since certain subtypes of amacrine cells use acetylcholine (ACh) as their transmitter (Masland and Ames 1976).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2. Voltage dependence of the light-evoked synaptic currents of ON-transient amacrine cells. A: light-evoked synaptic currents were recorded at various holding potentials, which are shown on the left. The retinal slice preparation was superfused with the solution containing no antagonists (control). Top: the timing of 2-s light stimulation. Note that both inward and outward currents could be resolved at the holding potential of -27 mV (ECl = -59 mV) and that both currents showed considerable overlap. B: light-evoked excitatory postsynaptic currents (EPSCs) from the same cell as in A recorded in the solution containing a cocktail of the antagonists (bicuculline, strychnine, D-tubocurarine, and atropine; abbreviated as BSCA). Note that no component of the current evoked by light stimulation reversed its polarity at ECl (-59 mV). The peak amplitude (top right) and response charge (bottom right) of the light-evoked EPSC are plotted against the holding potential. The peak amplitude and the response charge were normalized to the values at the holding potential of -57 mV as -1. Each data point represents the data obtained from 2 to 5 cells (means ± SE).

A cocktail of antagonists was applied to block major nonglutamatergic inputs to amacrine cells and to isolate the direct glutamatergic input from bipolar cells. The solution included bicuculline (100 µM), strychnine (1-10 µM), D-tubocurarine (curare; 60 µM), and atropine (2 µM) (abbreviated as the BSCA solution) to block GABAA receptors, glycine receptors, nicotinic ACh receptors, and muscarinic ACh receptors, respectively. As will be discussed later, bicuculline was sometimes replaced with picrotoxin to block GABAA receptors.

A typical light-evoked current of ON-transient amacrine cells in the presence of the antagonists is shown in Fig. 2B. The current evoked by light stimulation reversed its polarity not at ECl but near 0 mV. This indicates that IPSC carried by Cl- was completely blocked in the BSCA solution and that the remaining current was truly excitatory. At the holding potential of -57 mV, diffuse illumination evoked a transient inward current, which was followed by a small sustained inward current that lasted during illumination. Compared with the light-evoked current in the absence of the antagonists (Fig. 2A, the trace at -57 mV), the peak amplitude became slightly larger and the decay rate was accelerated (Fig. 2B). The time course of the excitatory input was expected to be changed by the application of the antagonists because GABA and glycine receptors exist not only in the IPL but also in the OPL. However, it was difficult to locate exactly the sites affected by the antagonists in the complex retinal circuit. In the subsequent sections, we always used the BSCA solution, unless otherwise specified, as control to isolate and focus on the glutamatergic input to ON-transient amacrine cells.

Two measures were taken to quantitate the glutamatergic input to amacrine cells; the peak amplitude of the initial transient and the response charge that was calculated by integrating the current evoked by a 2-s flash. The latter measure was useful when the time course of the light-evoked current was changed without alternation of the former measure after the addition of a pharmacological agent. The peak amplitude of the initial transient was -101.9 ± 15.7 pA and the response charge was 19.8 ± 2.6 pC at the holding potential of -57 mV in the BSCA solution (pooled data are expressed as means ± SE; n = 12). With the inter-stimulus interval of 30-40 s, light stimulation evoked responses with little variation for >20 min in the BSCA solution; SD of the peak amplitude and the response charge normalized to the mean was 0.09 ± 0.02 and 0.11 ± 0.02, respectively (Vh = -57 mV, n = 12).

NMDA receptors contribute little to the light-evoked EPSC

We first examined whether NMDA receptors play any roles in mediating synaptic transmission from bipolar to amacrine cells. Since NMDA receptors are effectively blocked by extracellular Mg2+ at deep negative potentials (Nowak et al. 1984), the light-evoked EPSC of amacrine cells was recorded at various holding potentials (Fig. 2B).

The peak amplitude and response charge of the light-evoked EPSC were measured and plotted against the holding potential (Fig. 2B, right). Both relationships were not obviously outward rectified, suggesting that the light-evoked EPSC may not include the NMDA receptor-mediated component. However, a detailed examination of the EPSC waveform showed that the decay phase was slightly slower at positive potentials than at negative potentials (Fig. 2B). The half decay time of the initial transient of the EPSC at +43 and -57 mV was 28.9 ± 6.5 and 24.8 ± 6.9 ms, respectively (n = 7; P = 0.04, paired t-test). This may be ascribed to the activation of NMDA receptors and/or to the slowing of desensitization rate of non-NMDA receptors at positive potentials (Higgs and Lukasiewicz 1999; Raman and Trussell 1995). A possible contribution of NMDA receptors was investigated further by the following pharmacological experiments.

First, NMDA was applied directly to ON-transient amacrine cells to examine the presence of functional NMDA receptors. The cell was voltage-clamped at -57 mV in the Mg2+-free BSCA solution and NMDA (500 µM) was co-applied with glycine (10 µM) via a puff pipette. The tip of the puff pipette was carefully positioned at a `hot spot' on the surface of the inner half of the IPL. In all six cells tested, NMDA evoked the inward current that could be significantly suppressed by 50 µM D-AP5 (Fig. 3, A and B).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 3. N-methyl-D-aspartate (NMDA) receptors scarcely contribute to the light-evoked EPSC of amacrine cells. A: the current evoked by puff-applied NMDA. One compartment of the theta tube was filled with the solution containing 500 µM NMDA and 10 µM Gly, and the other with the same solution supplemented with 50 µM D(-)-2-amino-5-phosphonopentanoic acid (D-AP5). Both solutions were dissolved in the Mg2+-free BSCA solution. The tip of the theta tube was placed near the surface of the IPL, and each content of the theta tube was ejected independently by a positive pressure. NMDA evoked a prominent response (left), which was suppressed by 50 µM D-AP5 that was included in both the bath and puff solutions (right). The traces are the average of 3 responses for each condition. The holding voltage was -57 mV for A and C. B: summary bar graph of the peak amplitude of responses evoked by NMDA alone (n = 6) and by NMDA with D-AP5 (n = 3). C: light-evoked EPSCs recorded from the same cell as A. Addition of 50 µM D-AP5 to the superfusate had little effect on the light-evoked EPSC (+DAP5). Further addition of 10 µM 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) almost completely blocked the light-evoked EPSC (+DAP5 + NBQX). The traces are the average of 3 responses in each condition. D: the peak amplitude () and response charge () of the light-evoked EPSC in the presence of D-AP5 alone (+DAP5, n = 9) or in the presence of D-AP5 and NBQX (+DAP5 + NBQX, n = 8) were normalized to those in the control superfusate. All recordings here were performed in the Mg2+-free superfusate. In a few recordings, picrotoxin and strychnine were included in the control superfusate instead of the mixture of bicuculline, strychnine, D-tubocurarine, and atropine, but no discernible difference was observed. For this and all subsequent figures, unless otherwise notified, the bar graph (mean ± SE) includes the data obtained from amacrine cells clamped at -57 or -77 mV; *, the value is significantly different from 1 (significant level <5%).

Next, we examined the effect of D-AP5 on the light-evoked EPSC (Fig. 3C). The light-evoked EPSC was recorded from the same cell illustrated in Fig. 3A. Bath application of D-AP5 had little effect on the light-evoked EPSC. The peak amplitude and response charge of the light-evoked EPSCs in the presence of D-AP5 were normalized to those in control, respectively (Fig. 3D). Pooled data from nine cells indicate that neither the peak amplitude (1.02 ± 0.03 of control) nor the response charge (0.89 ± 0.08 of control) was significantly decreased. On the other hand, addition of 5-10 µM NBQX almost completely blocked the light-evoked EPSC (Fig. 3, C and D; the peak amplitude, 0.11 ± 0.02 of control; the response charge, 0.07 ± 0.03 of control; n = 8). The small residual current persisting in the presence of NBQX could be ascribed to the activation of unblocked receptors (such as metabotropic glutamate receptors) or transporters, but we did not further analyze the properties of the minor residual current.

The preceding results (Figs. 2 and 3) indicate that the light-evoked EPSC is mediated mostly by the activation of non-NMDA receptors in the present circumstances (i.e., in the BSCA solution). It has been reported in the amphibian retina that the light-evoked EPSC is also mediated solely by non-NMDA receptors in a certain subtype of ON amacrine cells (Dixon and Copenhagen 1992).

Light-evoked EPSC is mediated by AMPA receptors

Non-NMDA receptors are classified into AMPA and kainate receptors on the basis of pharmacological and electrophysiological characteristics. It has been shown that the synaptic transmission from photoreceptors to morphologically distinct subtypes of OFF bipolar cells is mediated exclusively by kainate receptors in the ground squirrel retina (DeVries 2000; DeVries and Schwartz 1999). We examined whether ON-transient amacrine cells in the mouse retina use kainate receptors for receiving glutamatergic input from bipolar cells.

GYKI 52466 is a noncompetitive antagonist of AMPA receptors, which minimally affects kainate receptors (Donevan and Rogawski 1993; Paternain et al. 1995). The light-evoked EPSC was recorded in the BSCA solution containing 1 mM Mg2+ and 50 µM D-AP5 to suppress any possible contamination of the NMDA receptor-mediated current (Fig. 4A). When 100 µM GYKI 52466 was added to the bath solution, the peak amplitude and the response charge decreased to 0.11 ± 0.03 and 0.12 ± 0.04 of control, respectively (n = 10). As in the previous section, there remained a small residual current in the presence of GYKI 52466. Since the amplitude and response charge of the residual current were comparable to those in the presence of NBQX (Fig. 3C), which blocks both AMPA and kainate receptors, this residual current was not attributable to the activation of kainate receptors. The preceding results indicate that the light-evoked EPSC is mostly mediated by AMPA receptors.



View larger version (8K):
[in this window]
[in a new window]
 
Fig. 4. AMPA receptors mediate the light-evoked EPSC of amacrine cells. A: the light-evoked EPSC was recorded in the BSCA solution containing 1 mM Mg2+ and 50 µM D-AP5 to isolate the non-NMDA receptor mediated EPSC (left). The light-evoked EPSC was almost completely blocked in the presence of 100 µM 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466; +GYKI), a specific blocker of AMPA receptors. The holding voltage was -77 mV for the cell in A. The traces are the average of 3 responses for each condition. B: the peak amplitude () and response charge () of the light-evoked EPSC in the presence of GYKI 52466 (+GYKI, n = 10) were normalized to those in the absence of GYKI 52466.

Cyclothiazide enhances only the peak amplitude of the light-evoked EPSC

AMPA receptors are known to desensitize very quickly during the continuous presence of extracellular glutamate (Jones and Westbrook 1996; Trussell et al. 1993), and thus the desensitization of AMPA receptors may contribute to the shaping of the light-evoked EPSC in ON-transient amacrine cells. CTZ is known to selectively slow the desensitization of AMPA receptors with little effect on kainate receptors (Partin et al. 1993). Therefore we next examined the effect of CTZ on the light-evoked EPSC.

The slice preparation was superfused with the BSCA solution containing 1 mM Mg2+ and 50 µM D-AP5 to isolate the non-NMDA receptor-mediated current. The same solution supplemented with 200 µM glutamate was puff applied to a whole cell voltage-clamped amacrine cell. The glutamate-induced response was significantly increased after the addition of 100 µM CTZ to the superfusate (Fig. 5, A and B). This indicates that CTZ at this concentration effectively permeated into the slice preparation and reached the AMPA receptors of amacrine cells.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5. The effects of cyclothiazide (CTZ) on the glutamate-induced current and the light-evoked EPSC of amacrine cells. A: the current induced by a puff of glutamate (200 µM for 300 ms; timing indicated at the top) was recorded in the BSCA solution containing 1 mM Mg2+ and 50 µM D-AP5. The tip of the puff pipette was placed on the surface of the IPL. The peak amplitude of the response significantly increased in the presence of 100 µM CTZ (right). The traces are the average of 4 responses for each condition. The holding voltage was -57 mV for the cell in A and C. B: summary bar graph of the peak amplitude of glutamate-induced responses in the absence (left) or presence (right) of CTZ (n = 5). C: light-evoked EPSCs recorded from the same cell as A. Addition of CTZ (100 µM) to the superfusate slightly increased the peak amplitude and enhanced the "dip" (indicated by an down-arrow ) of the light-evoked EPSC (+CTZ). The traces are the average of 3 responses for each condition. D: the peak amplitude () and response charge () of the light-evoked EPSC in the presence of CTZ (+CTZ, n = 5) were normalized to those in the absence of CTZ.

Using the same cell, we examined the effect of CTZ on the light-evoked EPSC (Fig. 5C). The effect of CTZ was much weaker on the light-evoked EPSC than the glutamate-induced current. A close examination of the EPSC waveform revealed that the initial peak amplitude was slightly but significantly increased (1.32 ± 0.10 of control, n = 8). However, the response charge was not significantly increased (1.22 ± 0.16 of control, n = 8; Fig. 5D), probably because the subsequent "dip" became prominent (Fig. 5C, +CTZ, down-arrow ).

Immunocytochemical studies have shown that GABAC receptors are localized to bipolar cell terminals (Enz et al. 1996; Wässle et al. 1998), and it has been proposed that amacrine cells provide feedback inhibition to bipolar cells via these receptors (Dong and Werblin 1998; Hartveit 1999; Lukasiewicz and Shields 1998). If such GABAC receptor-mediated feedback loop is functioning between ON bipolar and ON-transient amacrine cells, the application of CTZ may increase the glutamatergic excitatory input to amacrine cells by decreasing the desensitization of AMPA receptors, which may subsequently enhance the inhibitory feedback from amacrine to bipolar cells, resulting in the prominent dip of the amacrine cell response. This possibility was examined in the following sections.

GABAC receptors are present in ON-cone bipolar cells

We first examined whether ON-cone bipolar cells in the mouse retina have functional GABAC receptors. With Lucifer yellow staining, cone bipolar cells could be morphologically distinguished from rod bipolar cells that have club-like axon terminals at the inner edge of the IPL (Euler et al. 1996). Among cone bipolar cells the cells that depolarized in response to light stimulation were identified as ON-cone bipolar cells.

Synaptic transmission in the retinal slice was blocked by replacing Ca2+ and Mg2+ in the extracellular control solution with 3 mM Co2+, and GABAA receptors of bipolar cells were blocked by 100 µM bicuculline. Puff application of 20 µM GABA to the IPL evoked an outward current (4.28 ± 1.06 pA; n = 3) in ON-cone bipolar cells which were voltage-clamped at the holding potential of +3 mV (Fig. 6A, left). In the presence of 50 µM TPMPA, a selective and competitive antagonist of the GABAC receptor (Ragozzino et al. 1996), the GABA-evoked current was significantly reduced to 0.31 ± 0.12 of control (Fig. 6, A, right, and B; n = 3). This result indicates that ON-cone bipolar cells have bicuculline insensitive GABAC receptors, which could be partially but effectively blocked by 50 µM TPMPA (see Euler and Wässle 1998). We next examined the function of GABAC receptors in ON-cone bipolar cells.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 6. GABAC receptors in ON-cone bipolar cells are effectively blocked by (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA). A: ON-cone bipolar cell was whole cell voltage-clamped at the holding voltage of +3 mV. One compartment of the theta tube was filled with the solution containing 20 µM GABA and the other with the same solution supplemented with 50 µM TPMPA. Both the superfusate and puff solutions contained 3 mM Co2+ as divalent cations to block synaptic transmission and 100 µM bicuculline to block GABAA receptors. The tip of the theta tube was placed near the surface of the IPL. When GABA was puff-applied, a prominent response was evoked (left), which was suppressed by 50 µM TPMPA included in both the bath and puff solutions (right). The traces are the average of five responses for each condition. B: the ratio of the peak amplitude was taken between the responses evoked by GABA with bicuculline and the responses evoked by the same solution supplemented with 50 µM TPMPA (n = 3).

TPMPA modulates the light-evoked voltage responses of ON-cone bipolar cells

The light-evoked voltage responses were recorded from ON-cone bipolar cells in the solution without any antagonists. A typical voltage response of ON-cone bipolar cells in the current-clamp mode is shown in Fig. 7A. The pipette was filled with the K+-based solution (P2; see METHODS), and the ECl was calculated to be at -69.7 mV. The resting potential was -49.7 ± 1.6 mV in the dark (n = 3). Light stimulation evoked a sustained depolarization with a gradual sag, and a prominent hyperpolarization was generated at the termination of light stimulation. These properties are essentially similar to those reported in ON-cone bipolar cells of the mouse (Berntson and Taylor 2000) and rat (Euler and Masland 2000) retinas.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 7. Light-evoked voltage responses of ON-cone bipolar cells. A: ON-cone bipolar cells were whole cell clamped in the current-clamp mode with the K+-based pipette solution (P2). In the superfusate without any antagonists (control), a sustained depolarization was evoked during light illumination and a prominent hyperpolarization (the OFF hyperpolarization) was induced at the termination of the light stimulus. In the BSCA solution, a transient hyperpolarization (indicated by black-triangle, BSCA) followed the initial depolarization in the same cell. B: after the application of 50 µM TPMPA, the decay of the initial depolarization was slowed and the following transient hyperpolarization was partially suppressed (+TPMPA). The traces in B are the average of six responses for each condition. C: after the application of 5 µM NBQX and 50 µM D-AP5, the transient hyperpolarization following the initial depolarization was completely abolished (+NBQX + DAP5). However, the OFF hyperpolarization at the termination of the light stimulus persisted. The traces in C are the average of 4 responses for each condition.

In the BSCA solution, the cholinergic and major inhibitory synaptic transmissions would be blocked, whereas the GABAC receptor-mediated transmission should remain intact. Interestingly, application of the BSCA solution evoked a marked transient hyperpolarization following the initial depolarization at the light onset (Fig. 7A, BSCA, black-triangle). The initial depolarization and the subsequent hyperpolarization peaked at 48.2 ± 3.7 and 116.7 ± 7.8 ms after the light onset, and the amplitude was 4.5 ± 1.5 and -6.7 ± 1.6 mV relative to the resting potential in the dark, respectively (n = 5).

As shown in Fig. 7B, the addition of 50 µM TPMPA to the BSCA solution decreased the transient hyperpolarization following the initial depolarization at the light onset. The decrease in amplitude of the transient hyperpolarization was also accompanied by a slowdown of the decay rate of the initial depolarization. Application of TPMPA did not shift the resting potential in the dark significantly (-53.5 ± 1.8 mV in the BSCA solution, -52.6 ± 2.2 mV in the +TPMPA solution; n = 3). The changes in the light-evoked voltage response of ON-cone bipolar cells are likely to enhance the glutamate release from these cells.

It is interesting to note that the transient hyperpolarization was not completely blocked by 50 µM TPMPA. It has been shown that GABAC receptors have a high affinity for GABA and that the potency of the antagonism by TPMPA becomes extremely weak for high concentrations of GABA (Ragozzino et al. 1996). This may explain the incomplete blockade of the transient hyperpolarization in ON-cone bipolar cells.

To further elucidate the origin of the transient hyperpolarization, 5 µM NBQX and 50 µM D-AP5 were added to the BSCA solution to block the synaptic transmission mediated by ionotropic glutamate receptors. The transient hyperpolarization after the initial depolarization at the light onset was completely suppressed and the depolarization was maintained during light stimulation in ON-cone bipolar cells (Fig. 7C). It should be recalled that such treatment almost completely suppressed the light-evoked responses in ON-transient amacrine cells (Fig. 3), i.e., the glutamatergic transmission from bipolar to amacrine cells in the IPL was completely blocked. Therefore it can be assumed that the sustained depolarization in bipolar cells is attributable to the synaptic input from cone photoreceptors to ON-cone bipolar cells via mGluR6 in the OPL. The resting potential of bipolar cells was not significantly changed by the addition of NBQX and D-AP5 (-56.0 ± 4.9 mV in the BSCA solution; -53.4 ± 1.7 mV in the +NBQX + DAP5 solution; n = 2).

The results illustrated in Fig. 7 could be interpreted as follows. As shown in Fig. 2, application of the BSCA solution blocks the inhibitory input to amacrine cells, resulting in the enhancement of excitation in amacrine cells. This may well strengthen the GABAC receptor-mediated feedback inhibition to bipolar cells. Such a sequence would generate the transient hyperpolarization after the initial depolarization in bipolar cells at the light onset (Fig. 7A, BSCA). The dip observed in the light-evoked EPSC of ON-transient amacrine cells in the BSCA solution (Figs. 2-5) is likely to be the consequence of the transient hyperpolarization of ON-cone bipolar cells. The fact that the blockade of excitatory input to amacrine cells by NBQX and D-AP5 completely abolishes the transient hyperpolarization in ON-cone bipolar cells (Fig. 7C, +NBQX + DAP5) further supports the idea that the transient hyperpolarization was induced by the GABAC receptor-mediated feedback from amacrine cells to ON-cone bipolar cells in the IPL. It is also interesting to note that in OFF-cone bipolar cells superfused with the BSCA solution, the termination of light stimulation evoked a strong depolarization, which was followed by a marked transient hyperpolarization (data not shown). These results indicate that the transient hyperpolarization observed in bipolar cells is triggered by the depolarization of bipolar cells irrespective of bipolar cell types or of the temporal sequence of light stimulation (Dong and Werblin 1998; Hartveit 1999). Furthermore, these results suggest that the transient hyperpolarization is induced by the synaptic interaction in the IPL.

In this section, it is demonstrated that the application of TPMPA partially blocks the transient hyperpolarization and prolongs the depolarizing state during light stimulation in ON-cone bipolar cells. It can be assumed that the prolongation of the depolarized state may enhance the release of glutamate from bipolar cells. In the subsequent sections, we examined the effects of the GABAC receptor antagonist on the light-evoked EPSC of ON-transient amacrine cells.

Blockade of GABAC receptors increases excitatory input to amacrine cells

The light-evoked EPSC was recorded from ON-transient amacrine cells in the BSCA solution. Addition of 50 µM TPMPA to the bath solution induced a marked increase in the transient and sustained components of the EPSC (Fig. 8A, +TPMPA). The peak amplitude and response charge of the light-evoked EPSC significantly increased to 1.27 ± 0.13 and 2.13 ± 0.24 of control, respectively (Fig. 8B; n = 10). The increase in the response charge was always larger than the increase in the peak amplitude, reflecting the drastic change in the EPSC waveform.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 8. Blockade of GABAC receptors increases the excitatory input to amacrine cells. A: the light-evoked EPSC was recorded in the BSCA solution (left) from ON-transient amacrine cells. Addition of 50 µM TPMPA (+TPMPA), a specific blocker of the GABAC receptor, increased the transient and sustained components of the EPSC. Addition of 100 µM picrotoxin (+PIC) to the BSCA solution had a weak effect on the light-evoked EPSC. The holding voltage was -57 mV for the cell in A. B: the peak amplitude () and response charge () of the light-evoked EPSC in the presence of TPMPA (+TPMPA, n = 10) or picrotoxin (+PIC, n = 7) were normalized to those in the BSCA solution.

In contrast to the effect of TPMPA, addition of 100 µM picrotoxin to the BSCA solution had a relatively small effect (Fig. 8, A and B, +PIC; the peak amplitude, 1.00 ± 0.02 of control; the response charge, 1.20 ± 0.06 of control; n = 7). The weak effect of the picrotoxin confirms the fact that GABAC receptors in rat and mouse bipolar cells are extremely resistant to picrotoxin (Feigenspan and Bormann 1994; Kaneko et al. 1991).

It seems probable that the release rate of glutamate from bipolar cells is increased after the blockade of GABAC receptors at the bipolar cell terminals by TPMPA. Glutamate may spread over a larger portion of the postsynaptic region of amacrine cells and activate a larger population of glutamate receptors with TPMPA than without TPMPA. In the next section, we re-examined what types of glutamate receptors are responsible for the enhancement of the light-evoked EPSCs after the blockade of GABAC receptors.

NMDA receptors are activated after blockade of GABAC receptors

The light-evoked EPSC was recorded from ON-transient amacrine cells in the presence of 50 µM TPMPA in the Mg2+-free BSCA solution (Fig. 9A). The light-evoked EPSC was reduced by the addition of 50 µM D-AP5; the reduction was prominent especially for the sustained component (Fig. 9A, +DAP5). The peak amplitude and the response charge decreased to 0.86 ± 0.08 and 0.62 ± 0.06 of those in the presence of TPMPA alone, respectively (Fig. 9B; n = 5). Note that the experimental protocols illustrated in Fig. 9, A and B, are identical to those in Fig. 3, C and D, except for the presence of TPMPA. The blocking effect of D-AP5 on the light-evoked EPSC was significantly larger in the presence of TPMPA (a comparison between Figs. 9B and 3D; unpaired t-test; P = 0.047 and 0.042 for the peak amplitude and the response charge, respectively). Addition of 5 µM NBQX to the superfusate blocked almost completely the light-evoked EPSC (Fig. 9A, +DAP5 + NBQX; the peak amplitude and the response charge were 0.04 ± 0.02 and 0.04 ± 0.01 of the condition with TPMPA alone, respectively; n = 5).



View larger version (9K):
[in this window]
[in a new window]
 
Fig. 9. NMDA receptors in amacrine cells are activated after blockade of GABAC receptors. The light-evoked EPSC was recorded in the Mg2+-free BSCA solution supplemented with 50 µM TPMPA (TPMPA). Addition of 50 µM D-AP5 (+DAP5) to the superfusate clearly curtailed the late phase of the transient current and the sustained current. Further addition of 5 µM NBQX (+DAP5 + NBQX) almost completely blocked the light-evoked EPSC. The holding voltage was -77 mV for the cell in A. The traces are the average of 2 responses for each condition. B: the peak amplitude () and response charge () of the light-evoked EPSC in the presence of D-AP5 (+DAP5, n = 5) or in the presence of D-AP5 and NBQX (+DAP5 + NBQX, n = 5) were normalized to those in the presence of TPMPA alone. This result should be compared with that shown in Fig. 3D, in which TPMPA was not included in the superfusate.

Next, we examined the subtype of non-NMDA receptors that mediated the light-evoked EPSC in the presence of TPMPA. The light-evoked EPSC was recorded in the BSCA solution containing 1 mM Mg2+ and 50 µM D-AP5 to suppress the NMDA receptor-mediated component, and 50 µM TPMPA to block GABAC receptors (Fig. 10A). With addition of 100 µM GYKI 52466 to the superfusate, the peak amplitude and the response charge decreased to 0.06 ± 0.01 and 0.05 ± 0.01, respectively (Fig. 10B; n = 6). This result indicates that, of the non-NMDA receptors, light stimulation activates only the AMPA subtype.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 10. The non-NMDA component of the light-evoked EPSC of amacrine cells is mediated by the activation of AMPA receptors in the presence of TPMPA. A: the light-evoked EPSC was recorded in the BSCA solution containing 1 mM Mg2+ and 50 µM D-AP5 to isolate the non-NMDA receptor mediated EPSC and 50 µM TPMPA to block GABAC receptors (TPMPA). The light-evoked EPSC was almost completely blocked by the addition of 100 µM GYKI 52466 (+GYKI), a specific blocker of AMPA receptors. The holding voltage was -57 mV for the cell in A. The traces are the average of 3 responses for each condition. B: the peak amplitude () and response charge () of the light-evoked EPSC in the presence of GYKI 52466 (+GYKI, n = 6) were normalized to those in its absence. C: the non-NMDA component of the light-evoked EPSC in the presence of TPMPA was little affected after 10-min bath application of Con A (0.3 mg/ml, +Con A). D: the peak amplitude () and response charge () of the light-evoked EPSC in the presence of Con A (+Con A, n = 4) were normalized to those in its absence.

This conclusion was further confirmed by the negative effect of Con A, a high-molecular-weight lectin that has been reported to irreversibly reduce desensitization of kainate receptors (Huettner 1990; Partin et al. 1993). Ten minutes' bath application of Con A (0.3 mg/ml) had no effect on the non-NMDA component of the light-evoked EPSC in the presence of 50 µM TPMPA (Fig. 10, C and D; the peak amplitude and the response charge were 0.93 ± 0.06 and 1.00 ± 0.11 of the condition with TPMPA alone, respectively; n = 4). Four of six amacrine cells examined evoked a small response (-2.29 ± 0.75 pA at the holding potential of -57 mV) when 10 µM SYM 2081, a selective agonist of kainate receptors, was puff-applied. This response was enhanced (-5.24 ± 0.83 pA) with 10-min bath application of Con A, indicating that Con A effectively permeated into the slice preparation. These results suggest that, although kainate receptors are present in some amacrine cells (see Brandstätter et al. 1997), kainate receptors contribute little to the generation of the light-evoked EPSC in ON-transient amacrine cells, even when the release rate of glutamate from bipolar cells is enhanced by the blockade of GABAC receptors.

Cyclothiazide significantly enhances the light-evoked EPSC after blockade of GABAC receptors

When the GABAC receptor-mediated feedback was intact, application of CTZ caused a small, significant increase in the peak amplitude of the light-evoked EPSC but not in the response charge (Fig. 5). Now, we re-examined the effect of CTZ on the light-evoked EPSC after the feedback inhibition to bipolar cells was suppressed by TPMPA. The light-evoked EPSC was recorded in the BSCA solution containing 1 mM Mg2+ and 50 µM D-AP5 to isolate the AMPA component and 50 µM TPMPA to block GABAC receptors.

Bath-application of CTZ (100 µM) increased the peak amplitude of the light-evoked EPSC (Fig. 11A). The effect of CTZ on the EPSC waveform was more prominent for longer light stimulation. The duration of light stimulation was changed, and the response charge was calculated for each condition. The response charge was normalized to that evoked by 2-s light stimulation in the absence of CTZ and plotted against the stimulus duration (Fig. 11B). It is evident that the relationship between the duration and the response charge in the presence of CTZ and TPMPA (Fig. 11B, ) is not superimposable after simple scaling of the relationship in the presence of TPMPA alone (Fig. 11B, open circle ).



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 11. The effects of CTZ on the AMPA component of the light-evoked EPSC of amacrine cells after blockade of GABAC receptors. A: the light-evoked EPSC was recorded in the BSCA solution containing 1 mM Mg2+, 50 µM D-AP5, and 50 µM TPMPA (TPMPA). Responses to 100-ms and 2-s illumination are shown. When 100 µM CTZ was added, the transient current increased in peak amplitude (+CTZ, trace with *). The effect on the decay phase was prominent in the current trace induced by 2-s illumination. The holding voltage was -57 mV for A. B: the response charge of the light-evoked EPSC was normalized to the value evoked by 2-s illumination in the presence of TPMPA alone, and plotted against the duration of light stimulation. Data in the presence of TPMPA alone (TPMPA) and in the presence of TPMPA and CTZ (+CTZ) are illustrated with open circle  and , respectively. Each data point represents the data obtained from 3 to 7 cells. C: the peak amplitude () and response charge () of the 2-s light-evoked EPSC after the addition of CTZ (+CTZ, n = 7) were normalized to those in the presence of TPMPA alone. This result should be compared with that shown in Fig. 5D, in which TPMPA was not included in the superfusate.

To compare the effect of CTZ in the presence and absence of TPMPA, we measured the peak amplitude and response charge of the 2-s light-evoked EPSC (Fig. 11C). After the application of CTZ, both the peak amplitude and the response charge increased to 1.73 ± 0.18 and 2.65 ± 0.43, respectively (n = 7). Figure 11C can be compared with Fig. 5D. It should be noted that the only difference between these two experiments was the presence of TPMPA. The degree of enhancement of the light-evoked EPSC by CTZ was significantly larger with TPMPA than without TPMPA for the response charge but not for the peak amplitude (a comparison between Figs. 11C and 5D; unpaired t-test; P = 0.006 for the response charge and P = 0.07 for the peak amplitude).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The mouse is becoming a widely used animal model for the application of transgenic technology, which offers a new set of tools for studying the functions of the nervous system. However, physiological data on the mouse retina have not yet been accumulated, mainly due to technical reasons, such as the small size of eyes and retinal neurons and the vulnerability of neurons of homoiothermal animals. In the present study, we recorded the light-evoked responses from bipolar and amacrine cells in the mouse retinal slice preparation.

Use of a cocktail of the antagonists

In most of the present experiments, bicuculline, strychnine, D-tubocurarine, and atropine were always included in the superfusate. To focus on the synaptic interaction between bipolar and amacrine cells in the IPL, it was important to avoid indirect effects via various synapses. The synaptic transmission from photoreceptors to ON bipolar cells is mediated by metabotropic glutamate receptors and is not susceptible to application of ionotropic glutamate receptor modulators (Hartveit 1996). Therefore we selected the ON pathway and focused on the interaction between ON bipolar cells and ON amacrine cells.

Subtypes of amacrine cells were categorized physiologically based on the excitatory input pattern in response to the diffuse, saturating light stimulus. We did not further categorize the cells into subtypes based on their morphology. The dendritic arborization of amacrine cells may be correlated with the spatial and temporal pattern of both excitatory and inhibitory inputs to the cells. However, differences among morphological subtypes would be minimized by applying the diffuse, saturating light stimulus in the presence of the cocktail of the antagonists. This procedure seemed very useful for extracting the common feature of the synaptic interaction between ON bipolar cells and ON amacrine cells.

Contribution of AMPA and NMDA receptors to the light-evoked EPSC

In many vertebrate CNS synapses, rapid excitatory transmission is mediated by the release of glutamate to AMPA and NMDA receptors. It is generally accepted that AMPA receptors have a lower affinity for glutamate, faster desensitization and deactivation kinetics, and a lower Ca2+ permeability than NMDA receptors, although these properties may differ depending on the subunit composition of receptors (reviewed in Dingledine et al. 1999). Therefore the relative contribution of each receptor type is one of the crucial determinants of the temporal properties of excitatory synaptic transmission.

The present study demonstrated that in ON-transient amacrine cells of the mouse retina, AMPA receptors were mainly activated in response to light stimulation with little contribution of NMDA receptors when the feedback inhibition mediated by GABAC receptors was intact (Figs. 2-4). After the feedback inhibition was suppressed by TPMPA, light stimulation activated both AMPA and NMDA receptors (Figs. 9 and 10).

Our results could be interpreted by a model of receptor segregation, in which AMPA receptors are located at the postsynaptic region immediately beneath each release site, whereas NMDA receptors are located slightly away from the region. Light stimulation evokes glutamate release from bipolar cells and depolarizes amacrine cells. The depolarized amacrine cells in turn inhibit bipolar cells via GABAC receptors. Cells providing this inhibitory feedback are likely to be amacrine cells other than the recorded cell under voltage clamp. The feedback inhibition would limit the release of glutamate from bipolar cells. Application of TPMPA suppressed the activation of GABAC receptors and thus increases glutamate release from bipolar cells (Figs. 6-8). This would lead to a spillover of glutamate, resulting in the activation of NMDA receptors slightly away from the release sites (Fig. 9).

In many CNS synapses, colocalization of non-NMDA and NMDA receptors is suggested by the observation that spontaneous EPSCs are composed of both receptor-mediated components (Bekkers and Stevens 1989). However, it has been suggested that non-NMDA and NMDA receptors may be segregated at postsynaptic sites of retinal third-order neurons because the NMDA receptor-mediated component is absent in spontaneous responses to quantal release of glutamate (in amphibian, Matsui et al. 1998; Taylor et al. 1995; in mammal, Taschenberger et al. 1995; Tian et al. 1998). The idea of spatial segregation of the two receptor subtypes is supported by the fact that NMDA receptors have a much higher affinity for glutamate than non-NMDA receptors (Jonas and Sakmann 1992; Patneau and Mayer 1990). The spillover hypothesis is also supported by the present observation that the GABAC receptor-mediated inhibition can regulate the extent of the NMDA receptor component in the light-evoked EPSC in ON-transient amacrine cells.

Figures 9 and 10 demonstrate that both AMPA and NMDA receptors are activated by light stimulation when GABAC receptors are blocked by TPMPA. However, it should be noted that the degree of blockade of the light-evoked EPSC by D-AP5 varied from cell to cell; the remaining component in the presence of D-AP5 ranged between 0.39 and 0.78 of control for the response charge. This variability may suggest that the ratio between the AMPA and NMDA components differs among subclasses of ON-transient amacrine cells.

Complex effects of CTZ

The result that CTZ was more effective in the presence of TPMPA than in its absence (compare Fig. 5D with 11C) may be interpreted by the following two factors. First, as shown in Fig. 5C, the application of CTZ enhanced the peak amplitude of the light-evoked AMPA receptor-mediated EPSC in the recorded amacrine cells. Such enhancement in the excitatory inputs to amacrine cells may produce a larger inhibitory feedback to bipolar cells. The feedback loop may serve to stabilize and obscure further changes in the circuit, resulting in a minimal effect of CTZ on the response charge of the light-evoked EPSC in amacrine cells (Fig. 5D). After the feedback loop is suppressed by TPMPA, the feedback inhibition no longer acts to curtail the glutamate release from bipolar cells. Therefore in the absence of the feedback loop the application of CTZ not only increases the peak amplitude of the EPSC but also significantly enhances the response charge (Fig. 11C).

Second, our data suggest that the glutamate release from bipolar cells may also be increased by TPMPA (Figs. 7 and 8). Addition of TPMPA in the presence of CTZ caused a small inward shift in the resting current (-1.3 ± 0.9 pA; Vh = -57 mV, n = 5), suggesting that the ambient glutamate concentration level in the dark was not substantially increased by TPMPA. In response to light stimulation, multiple vesicular release may be evoked more frequently in bipolar cells. During prolonged light stimulation, the glutamate concentration in the extracellular space may rise to a high level, resulting in the development of AMPA receptor desensitization. Under this condition, CTZ may affect more efficiently on the EPSC. This assumption is supported by the fact that in other systems, non-NMDA receptors become more desensitized as the release rate of glutamate is higher (Matsui et al. 1998; Trussell et al. 1993).

Alternatively, it may also be possible that CTZ acts presynaptically and directly enhances the transmitter release (Diamond and Jahr 1995). However, it has been reported in goldfish retinal bipolar cells that neither ICa nor exocytosis was affected by CTZ (von Gersdorff et al. 1998).

The relationship between the duration and the response charge in the presence of CTZ and TPMPA (Fig. 11B, ) was not superimposable after simple scaling of the relationship in the presence of TPMPA alone (Fig. 11B, open circle ). This result may be interpreted by the following possibilities. First, in the absence of CTZ, each miniature EPSC may be added nonlinearly due to desensitization of AMPA receptors that is caused by the accumulation of glutamate from multiple successive quantal release, whereas in the presence of CTZ, each miniature EPSC may be added more efficiently. Second, TPMPA insufficiently suppressed the transient hyperpolarization in ON-cone bipolar cells (Fig. 7B), and this residual feedback inhibition may complicate the effect of CTZ on the relationship between the duration of light stimulation and the response charge.

Possible role for other types of GABA receptors

Ionotropic GABAA and GABAC receptors have distinct pharmacological and physiological properties. GABAA receptors are less sensitive to GABA, have the faster activation and deactivation kinetics, and show a stronger steady-state desensitization than GABAC receptors (Feigenspan and Bormann 1994; Ragozzino et al. 1996). Therefore GABAA receptors may participate in mediating the higher frequency component of GABA-mediated negative feedback to bipolar cells. However, it has been reported that bipolar, amacrine, and ganglion cells (in some species, photoreceptors and horizontal cells) express GABAA receptors (Wässle et al. 1998). Therefore it was difficult to examine how GABAA receptors at the bipolar cell terminals contribute to the feedback from amacrine cells.

It has been reported that metabotropic GABAB receptors are also expressed in a variety of cells in the retina (Koulen et al. 1998). Recently, Shen and Slaughter (2001) have shown that both GABAB and GABAC receptors participate in the GABA-mediated negative feedback from amacrine cells to bipolar cells in the tiger salamander retina. However, Koulen et al. (1998) reported that GABAB receptor immunoreactivity was detected not at bipolar cells but at other cells (presynaptic receptors in amacrine and horizontal cells and postsynaptic receptors in amacrine and ganglion cells) in the rat retina. We cannot exclude the possibility that GABAB receptors contribute to the negative feedback circuit between bipolar and amacrine cells in the mouse retina. However, the activation of GABAB receptors may not directly regulate the release rate of glutamate from bipolar cells but possibly control the amount of transmitter release from amacrine cells.

Site of TPMPA action

In contrast to the widely distributed GABAA and GABAB receptors in the retina, GABAC receptors are specifically localized at bipolar cell terminals, although a weak extrasynaptic expression has been observed in the cell body and dendrites of bipolar cells (Wässle et al. 1998). Electron microscopic studies have shown that both cone bipolar and rod bipolar cells receive reciprocal feedback inputs from GABAergic amacrine cells (Vaughn et al. 1981). We found that GABAC receptor could be activated in ON-cone bipolar cells by exogenously applied GABA (Fig. 6). It has been shown that both the GABA-induced current and the spontaneous IPSC of amacrine and ganglion cells are mediated by GABAA receptors because these currents are completely blocked by bicuculline (Boos et al. 1993; Protti et al. 1997; Tian et al. 1998). Therefore in the present study, it can be assumed that TPMPA did not act directly on amacrine cells but specifically affected bipolar cells.

In contrast to GABAC receptors in cold-blooded vertebrates (Lukasiewicz and Shields 1998), GABAC receptors in rat are extremely resistant to picrotoxin (Feigenspan and Bormann 1994). GABAC receptors are composed of rho 1 and rho 2 subunits. It has been shown that the unique methionine residue in the rho 2 subunit of the rat GABAC receptor is responsible for the picrotoxin resistivity (Zhang et al. 1995). This methionine residue is also conserved in the mouse rho 2 subunit (Greka et al. 1998), and GABAC receptors in isolated mouse bipolar cells are resistant to picrotoxin (Kaneko et al. 1991). Recently it has been reported that GABAC receptors are also present in mouse retinal cone terminals but are completely blocked by picrotoxin, which suggests that these receptors may be composed of only rho 1 subunit (Pattnaik et al. 2000). The weak effect of picrotoxin on the light-evoked EPSC in ON-transient amacrine cells (Fig. 8) suggests that GABAC receptors of cone terminals may play a minor role in the synaptic transmission in the OPL, at least under our recording conditions. We did not further subcategorize the GABAC receptor subtypes responsible for the observed negative feedback from amacrine to bipolar cells (see Shen and Slaughter 2001).

Concentration of GABA

GABAC receptors have a high affinity for GABA (EC50 ~ 1 µM), and the potency of the antagonism of TPMPA becomes extremely weak for high concentrations of GABA (Ragozzino et al. 1996). However, TPMPA applied at the concentration of 50 µM effectively affected both the GABA-activated current in ON-cone bipolar cells and the light-evoked responses of bipolar and amacrine cells (Figs. 6-8). This suggests that GABAC receptors may not be exposed to a high concentration of GABA; perhaps ~10 µM range of GABA may gate GABAC receptors of bipolar cells. However, as can be seen in Fig. 8, oscillatory fluctuations of the light-evoked EPSC in amacrine cells were often enhanced on application of TPMPA. The increase in GABA concentration at the synaptic cleft caused by the enhanced excitation of amacrine cells may partially overcome the antagonistic action of TPMPA on GABAC receptors. Such sequence may have caused the damped oscillations of the light-evoked responses in amacrine cells.

OFF hyperpolarization in ON-cone bipolar cells

It is interesting to note that the hyperpolarization of ON-cone bipolar cells observed at the termination of light stimulation (the "off" hyperpolarization) still remained in the presence of TPMPA (Fig. 7B) or of NBQX and D-AP5 (Fig. 7C). This indicates that the off hyperpolarization is not induced by the GABAC receptor-mediated feedback. It is conceivable that the glutamate released from cone photoreceptors at the light termination activates the mGluR6 at the dendrites of ON-cone bipolar cells, which then closes the cGMP-gated channels and hyperpolarize the cells (Euler et al. 1996; Hartveit 1996; Nakajima et al. 1993). The time course of the off hyperpolarization may reflect the time course of glutamate release from cones and the kinetics of the mGluR6 cascade in ON-cone bipolar cells. However, the mechanisms of the generation and shaping of the off hyperpolarization were not examined in the present study.

Function of GABAC receptors

In the present study, we used the diffuse, saturating light stimuli to activate the retinal circuit. Under this condition GABAC receptor-mediated feedback inhibition was likely to be strongly activated. However, the degree of each GABAC receptor-mediated feedback inhibition may be determined locally depending on the spatial and temporal pattern of light stimulation. As demonstrated in our study, the activation of GABAC receptors of bipolar cells regulates the proportion of AMPA to NMDA receptor activation in amacrine cells. The increase in the proportion of NMDA component in the light-evoked EPSC after the suppression of GABAC receptor-mediated feedback can be ascribed both to the activation of NMDA receptors by the spilled-over glutamate and to the desensitization of AMPA receptors by enhanced glutamate release. The functional role of NMDA receptors may be to compensate for the desensitization of AMPA receptors and to reliably transmit graded potential changes of bipolar cells to amacrine cells. Therefore precise localization of the two glutamate receptor subtypes would be important in determining the characteristics of transfer function between the two cells. Our results support the idea that the synaptic interactions between bipolar and amacrine cells must be important for dynamic processing of the visual scene in the retina.


    ACKNOWLEDGMENTS

We thank E. Sumimori for excellent technical assistance. K. Matsui is a research fellow of the Japan Society for the Promotion of Science.

This work was supported by Grants-in-Aid for Scientific Research (12053212) and the Special Coordination Funds for Promoting Science and Technology (NRV Project) from the Ministry of Education, Science, Sports and Culture and from the Japan Society for the Promotion of Science (11480245) to M. Tachibana.


    FOOTNOTES

Address for reprint requests: M. Tachibana, Dept. of Psychology, Graduate School of Humanities and Sociology, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan (E-mail: Ltmasao{at}L.u-tokyo.ac.jp).

Received 8 January 2001; accepted in final form 18 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society