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. 2007 Apr 26;582(Pt 2):569–582. doi: 10.1113/jphysiol.2007.131763

Presynaptic inhibition differentially shapes transmission in distinct circuits in the mouse retina

Erika D Eggers 1, Maureen A McCall 2,3, Peter D Lukasiewicz 1
PMCID: PMC2075342  PMID: 17463042

Abstract

Diverse retinal outputs are mediated by ganglion cells that receive excitatory input from distinct classes of bipolar cells (BCs). These classes of BCs separate visual signals into rod, ON and OFF cone pathways. Although BC signalling is a major determinant of the ganglion cell-mediated retinal output, it is not fully understood how light-evoked, presynaptic inhibition from amacrine cell inputs shapes BC outputs. To determine whether differences in presynaptic inhibition uniquely modulate BC synaptic output to specific ganglion cells, we assessed the inhibitory contributions of GABAA, GABAC and glycine receptors across the BC pathways. Here we show that different proportions of GABAA and GABAC receptor-mediated inhibition determined the kinetics of GABAergic presynaptic inhibition across different BC classes. Large, slow GABAC and small, fast GABAA receptor-mediated inputs to rod BCs prolonged light-evoked inhibitory postsynaptic currents (L-IPSCs), while smaller GABAC and larger GABAA receptor-mediated contributions produced briefer L-IPSCs in ON and OFF cone BCs. Glycinergic inhibition also varied across BC class. In the rod-dominant conditions studied here, slow glycinergic inputs dominated L-IPSCs in OFF cone BCs, attributable to inputs from the rod pathway via AII amacrine cells, while rod and ON cone BCs received little and no glycinergic input, respectively. As these large glycinergic inputs come from rod signalling pathways, in cone-dominant conditions L-IPSCs in OFF cone bipolar cells will probably be dominated by GABAA receptor-mediated input. Thus, unique presynaptic receptor combinations mediate distinct forms of inhibition to selectively modulate BC outputs, enhancing the distinctions among parallel retinal signals.

The timecourse of synaptic signalling is governed by the properties of presynaptic neurotransmitter release and the kinetics and complement of postsynaptic receptors. Previous studies in spinal cord and hippocampus have shown that a balance between the timecourse of inhibition and excitation creates maximal inhibitory modulation of the excitatory signal (Hajos & Mody, 1997; Chadderton et al. 2004; Takahashi, 2005). In the retina, the excitatory synapse between bipolar cells (BCs) and ganglion cells is a critical determinant of the nature of the retinal output. Since BCs receive significant presynaptic inhibitory input onto their axon terminals, this synapse is ideal for exploring how excitation and presynaptic inhibition interact to shape sensory signals.

Mammalian BCs are divided into three major classes at the first synapse in the retina. Cone photoreceptors contact both ON and OFF BCs that respond to increments and decrements of light intensity, respectively. Rod photoreceptors contact rod BCs that respond to light increments. While the excitatory inputs to these parallel rod and cone BC pathways in the outer retina are temporally distinct, attributable to both distinct presynaptic neurotransmitter release and postsynaptic glutamate receptor types (Ashmore & Copenhagen, 1980; Schnapf & Copenhagen, 1982; Cadetti et al. 2005; Li & DeVries, 2006), it is unclear how presynaptic inhibition of BCs in the inner retina shapes their visual output.

BC outputs are shaped by presynaptic inhibitory input from GABAergic and glycinergic amacrine cells (Lukasiewicz & Werblin, 1994; Pan & Lipton, 1995; Dong & Werblin, 1998; Euler & Masland, 2000) onto functionally distinct GABAA, GABAC and glycine receptors on their axon terminals (Euler & Wassle, 1998; Shields et al. 2000; Eggers & Lukasiewicz, 2006a). Agonist application studies suggest that the contributions of GABAA, GABAC and glycine receptors vary with BC class (Euler & Wassle, 1998; Shields et al. 2000; Ivanova et al. 2006), predicting the distinct shaping of their outputs. However, light-evoked inhibition is influenced both by differences in neurotransmitter release and receptor distribution, and little was known about how this differential contribution by inhibitory receptors affected light-evoked inhibition to bipolar cells. We previously showed that distinct receptors differentially shape the peak amplitude (glycine, GABAA) and timecourse (GABAC) of rod BC output (Eggers & Lukasiewicz, 2006b), suggesting that diverse contributions from receptor types could uniquely shape outputs from different BC classes. Additionally, our earlier work suggested that differences in presynaptic inhibition across BC classes shape the visually driven BC outputs to their target ganglion cells (Sagdullaev et al. 2006). However, the synaptic mechanisms responsible for these differences remain unknown because light-evoked inhibition to BCs had not been studied.

Here we characterized how different complements of GABAergic and glycinergic inhibitory receptors produce distinct light-evoked inhibition across BC classes. We found that inhibition within a distinct BC class was determined by unique combinations of GABAC, GABAA and glycine receptors. The timecourse of inhibition across BC classes was well matched to the timecourse of their excitatory inputs that is reported in the literature (Ashmore & Copenhagen, 1980; Schnapf & Copenhagen, 1982; Cadetti et al. 2005; Li & DeVries, 2006). Specifically, large slow GABAC receptor-mediated inputs dominate rod BC light-evoked inhibitory postsynaptic currents (L-IPSCs). Smaller GABAC receptor-mediated inputs combine with fast GABAA receptor-mediated inputs to create shorter decays in ON cone BCs. Glycinergic inhibition dominates the response of OFF cone BCs under dark-adapted conditions, but this inhibition is likely to switch to GABAA receptor-dominated inputs when signalling arises from cone photoreceptor input. Thus, our results suggest that the output of BCs, a major determinant of the retinal output, is shaped by distinct forms of inhibition, adding another level of specificity to visual processing.

Methods

Preparation of mouse retinal slices

Animal protocols were approved by the Washington University School of Medicine Animal Studies Committee. The experimental techniques were similar to those previously described (Eggers & Lukasiewicz, 2006a). Briefly, both wild-type (WT) mice (C57BL/6J strain; Jackson Laboratories; Bar Harbour, ME, USA) and GABACρ1 null mice that lacked functional GABAC receptors in the retina (congenic on a C57BL/6J background) (McCall et al. 2002) were used. For brevity we will refer to the knockout mice as GABACR null, as we have in previous publications (Eggers & Lukasiewicz, 2006a,b). Mice 28–90 days of age were killed using carbon dioxide, their eyes enucleated and the cornea, lens and vitreous removed. The eyecup was incubated for 20 min in dissection and storage solution (see Electrode and bath solutions) with 0.5 mg ml−1 hyaluronidase (Sigma, St Louis, MO, USA). The hyaluronidase solution was replaced with cold, oxygenated storage solution, the retina was dissected out of the eyecup, and 200–250 μm slices were prepared from the isolated retina and maintained in oxygenated storage solution at room temperature.

Whole-cell recordings

Whole-cell patch recordings were made from BCs from retinal slices, as previously described (Eggers & Lukasiewicz, 2006a), using recording procedures and apparatus that also have been previously described (Lukasiewicz & Roeder, 1995). IPSCs were recorded from retinal BCs voltage clamped to 0 mV, the reversal potential for currents mediated by non-selective cation channels. Liquid junction potentials of 15 mV were corrected at the beginning of each recording. Electrodes were pulled from borosilicate glass (1B150F-4; World Precision Instruments, Sarasota, FL, USA) on a P97 Flaming/Brown puller (Sutter Instruments, Novato, CA, USA) and had resistances of < 5 MΩ. Patchit software (White Perch Software, Somerville, MA, USA) was used to generate voltage command outputs, acquire data and gate the drug perfusion valves. The data were digitized and stored on a personal computer using a Labmaster DMA data acquisition board (Scientific Solutions, Solon, OH, USA).

Solutions and drugs

The control solution used for dissection, storage and the extracellular recording solution for GABA-evoked responses contained (mm): 137 NaCl, 2.5 KCl, 1 MgCl2, 2.5 CaCl2, 28 glucose and 10 Hepes, was adjusted to pH 7.4 with NaOH and bubbled with O2. The extracellular recording solution used to examine spontaneous and light-evoked currents contained (mm): 125 NaCl, 2.5 KCl, 1 MgCl2, 1.25 NaH2PO4, 2 CaCl2, 20 glucose and 26 NaHCO3 and was bubbled with 95% O2–5% CO2. The intracellular solution contained (mm): 120 caesium gluconate, 1 CaCl2, 1 MgCl2, 10 Na-Hepes, 11 EGTA, 10 TEA-Cl and was adjusted to pH 7.2 with CsOH. Antagonists were applied to the slice chamber using a gravity-driven superfusion system. Unless otherwise indicated, all chemicals were obtained from Sigma.

Morphological identification of retinal cell classes

BCs were labelled with either Lucifer yellow (0.05%) or Sulforhodamine B (0.005%), dissolved in the intracellular solution. They were classified as either rod, ON cone or OFF cone BCs, based on their dendritic and axonal morphologies, and the stratification of their somas in the inner nuclear layer and their axon terminals within the ON and OFF sublaminae of the inner plexiform layer (Ghosh et al. 2004).

Light-evoked IPSC recordings

The methods to record light-evoked inhibitory post synaptic currents (L-IPSCs) have been previously described (Eggers & Lukasiewicz, 2006a). Mice were dark-adapted overnight and all dissection and recording procedures were performed under infrared illumination to preserve the light sensitivity of the preparations. Recordings were made in extracellular solution heated to 32°C, using thin stage and inline heaters (Cell Microcontrols, Norfolk, VA, USA). Light-evoked responses were filtered at 1 kHz with the four-pole Bessel filter on the Axopatch 200B (Axon Instruments, Foster City, CA, USA) and sampled at 2 kHz. To isolate inhibitory receptor inputs, strychnine (500 nm) was used to block glycine receptors, bicuculline methobromide (50 μm) to block GABAA receptors and (1,2,5,6-tetrahydropyridine-4yl) methyphosphinic acid (TPMPA, 50 μm) to block GABAC receptors. L-IPSCs were evoked with a full-field flash using a light-emitting diode (LED, Agilent HLMP-3950, λpeak= 565 nm, Palo Alto, CA, USA) positioned near the microscope stage. Stimulus intensity (1.85 × 103 photons μm−2 s−1) and duration were controlled by current applied to the LED.

GABA-evoked current recordings

Once a whole-cell recording was established, the Lucifer yellow- or sulforhodamine-filled BC axon terminal was briefly visualized and a glass electrode containing 30 μm GABA, dissolved in the control recording solution, was positioned near the axon terminal. GABA currents were evoked by puffing GABA (5–15 lbf in−2) onto the BC axon terminal using a Picospritzer II (General Valve). Recordings of GABA-evoked currents were made at room temperature and were recorded at 500 Hz. For GABA-evoked recordings, the control recording solution contained strychnine (10 μm), CNQX (10 μm) and d-AP5 (50 μm) to eliminate synaptic influences. Bicuculline (500 μm) and TPMPA (50 μm) were used to block GABAA and GABAC receptors, respectively.

Spontaneous IPSC recordings

Recordings of spontaneous currents were made in extracellular solution heated to 32°C, in the absence of light stimuli, and were filtered at 1 kHz and sampled at 2–5 kHz. Strychnine (500 nm), bicuculline (50 μm) and TPMPA (50 μm) were used to isolate receptor types, similar to the light-evoked recordings. Kainate (10 μm), which activates AMPA/kainate receptors on amacrine cells, was used to depolarize amacrine cells and increase spontaneous GABA release which enabled the recording of spontaneous GABAC receptor-mediated currents (Frech & Backus, 2004; Eggers & Lukasiewicz, 2006b).

Data analysis and statistics

Tack (White Perch Software, Somerville, MA, USA) and Clampfit (Axon Instruments) software were used to create average response records and to measure the peak, time to peak (from the onset of the light stimulus), charge transfer (Q, pA ms or fC) and decay time (D37, defined below) of L-IPSCs and the Q and decay time of GABA-evoked responses. All light-evoked IPSCs and GABA-evoked responses shown and used in our analyses are an average of two responses from the same cell. Since the decay time could not be easily fitted with either a single or double exponential curve, we determined the decay time by computing the time at which the L-IPSC declined to 37% of its peak amplitude (D37). Student's t tests (two-tailed, unequal variance) were used to compare response characteristics from WT and GABACR null BCs. An ANOVA with a Scheffé's post hoc test was used to compare differences among currents from rod, ON cone and OFF cone BCs. Differences were considered significant when P≤ 0.05. All average data are reported as mean ± standard error of the mean (s.e.m.), and n refers to number of cells; N refers to number of sIPSCs. Ratios of averages are reported as ratio ± propagated s.e.m. Propagated errors are defined as:

graphic file with name tjp0582-0569-m1.jpg

where Ratio = X/Y

Spontaneous IPSCs (sIPSCs) were selected so that the rise and decay phases did not contain any overlapping events, and Mini Analysis software (Synaptosoft, Decatur, GA, USA) was used to measure the amplitude and calculate the τdecay, the time constant of an exponential function fit to the decay from the peak to baseline, of each individual sIPSC. The distributions of sIPSC amplitude and τdecay were compared using the Kolmogorov–Smirnov test (K–S). To compute the average sIPSC, events were aligned at 50% of their rise time and sIPSCs were averaged using Clampfit. As rise time of GABAC receptor-mediated sIPSCs was too slow to be effectively aligned using the MiniAnalysis program, they were aligned by hand using Clampfit.

Results

GABAergic L-IPSCs show distinct timing across BC classes

Previous work characterizing currents evoked by GABA applications suggests a differential distribution of GABAA and GABAC receptor-mediated inhibition across BC classes in rat and ferret (Euler & Wassle, 1998; Shields et al. 2000). These results predict that there may be differential receptor filtering of GABAergic light-evoked inhibitory postsynaptic currents (L-IPSCs) across the distinct parallel BC pathways. However, because the magnitude and shape of L-IPSCs depends not only on receptor type and distribution but also on their activation by neurotransmitter release, differences in receptor distribution may not correlate directly with differences in L-IPSCs. Since GABAC receptor-mediated responses have slow kinetics (Eggers & Lukasiewicz, 2006b), we expected that differences in GABAC receptor distributions would primarily shape the decay of L-IPSCs. Thus, we examined the decay of pharmacologically isolated GABAergic L-IPSCs across all classes of WT BCs (in the presence of strychnine) by determining the time at which the response decayed to 37% of its maximum (D37). The GABAergic L-IPSCs (Fig. 1A–C) of rod BCs decayed slowest, followed by ON cone and then OFF cone BCs (Fig. 1D), suggesting that distinct proportions of GABAA and GABAC receptors shape light-evoked inhibition across BC classes.

Figure 1. The kinetics of GABAergic light-evoked IPSCs (L-IPSCs) vary across WT bipolar cell (BC) class.

Figure 1

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L-IPSCs were recorded from BCs voltage clamped to 0 mV, the reversal potential for excitatory currents mediated by non-selective cation channels, and elicited with a 30 ms full field stimulus (dark grey bar). A–C, WT GABAergic L-IPSCs (recorded in the presence of strychnine) from rod, ON cone and OFF cone BCs, respectively. D, the decay of the L-IPSCs (D37) varied by BC class with rod BCs (330.6 ± 38.1 ms, n= 10) significantly slower than either ON cone BCs (D37, 203.0 ± 24.5 ms, n= 7) or OFF cone BC (D37, 92.8 ± 31.6 ms, n= 3) (ANOVA, P < 0.01, rod versus ON P < 0.05; rod versus OFF, P < 0.01). ON cone BCs also had a slower D37 than OFF cone BCs (P < 0.05). Scale bars, 5 pA and 200 ms.

Proportions of GABAA and GABAC receptor-mediated inhibition vary with BC class

Differences in GABA release onto synaptic GABAA and GABAC receptors could also contribute to the decay of GABAergic L-IPSCs. To investigate how receptor properties and distributions shaped GABAergic responses in mouse retina, we by-passed release by directly activating presynaptic GABA receptors on morphologically identified WT BCs with focal GABA application onto the axon terminals (Shields et al. 2000). GABA-evoked currents were isolated by blocking glycine and ionotropic glutamate receptors, as noted in the Methods. GABAC and GABAA receptor-specific response components were separated with the antagonists bicuculline or TPMPA, respectively. The proportion of the total GABA-evoked current mediated by either GABAA or GABAC receptors differed across BC class (Fig. 2Aa–Ca). To quantify their contributions to the total current, we computed the ratio of the charge transfer (Q) for each isolated receptor (Q-GABAC or Q-GABAA) to the total charge transfer obtained in control solution (Fig. 2D). In rod BCs (Fig. 2Aa), GABAC receptors mediated most of the GABA-evoked total current (0.87 ± 0.05, n= 7), with a small contribution from GABAA receptors (0.17 ± 0.07, n= 12; GABACversus GABAAP < 0.0001, Fig. 2D). GABA-evoked currents in ON cone BC (Fig. 2Ba) were also dominated by GABAC receptors (GABAC 0.60 ± 0.03, n= 15; GABAA 0.29 ± 0.16, n= 3; GABACversus GABAA; P < 0.005), although the GABAC receptor-mediated proportion was significantly smaller than in rod BCs (ANOVA P < 0.001, Scheffé's post hoc test; P < 0.05; Fig. 2D). In OFF cone BCs (Fig. 2Ca), the GABAC receptor contribution was smallest of all BC classes (GABAC= 0.45 ± 0.06, n= 7, rod versus OFF BC P < 0.001) and was similar to the contribution by GABAA receptors (GABAA= 0.38 ± 0.09, n= 7; GABACversus GABAA; P= 0.3). These findings are in general agreement with earlier studies in other species (Euler & Wassle, 1998; Shields et al. 2000), and they allow us to compare the properties of GABA-evoked and light-evoked IPSCs in the mouse.

Figure 2. GABAA and GABAC contributions to GABA-evoked (30 μm) currents also vary across BC class.

Figure 2

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GABAA or GABAC receptor-mediated currents were isolated and measured using TPMPA or bicuculline, respectively, in WT rod (Aa), ON cone (Ba) and OFF cone (Ca) BCs. GABA-evoked currents from GABACR null rod (Ab), ON cone (Bb) and OFF (Cb) cone BCs compared with GABA-evoked currents from WT BCs of the same class. (The dark grey bar below each trace indicates the duration of the GABA puff that elicited the current.) D, fractional GABA-evoked current mediated by GABAA and GABAC receptors in WT BCs was calculated by normalizing GABAA and GABAC charge transfer (Q) to total Q. In WT rod and ON cone BCs, GABAC receptors (black bars) mediated significantly more of the total response (Q) than GABAA receptors (grey bars; P < 0.001 and 0.005, respectively). In WT OFF cone BCs the proportion of GABAA and GABAC contributions was similar (P= 0.3). For GABACR null BCs, the average Q of the GABA-evoked current was normalized to the average WT response (white bars), which gives an estimate of the contribution of GABAA receptors to the WT GABA-evoked current. In each BC class, this value is similar to the measured contribution of GABAA receptors (grey bars) to the WT current. In this histogram, the black error bars for GABAC (black) and GABAA (grey) represent the s.e.m. For Null/WT (white), the grey error bars represent the propagated error from the averages. E, in all BC classes, the GABAC receptor-mediated currents had slower decay times than the GABAA receptor-mediated currents (P < 0.05), indicated by the ratios of the average GABAC to GABAA decay (D37) values being greater than 1. Scale bars: A, B and Ca, 20 pA; Cb, 10 pA, and 500 ms in all.

To assess the contribution of GABAC and GABAA receptors to GABA-evoked current kinetics, we compared the decay times of the individual receptor-mediated currents in the same WT BCs. This approach minimizes any kinetic differences arising from variations in the distance between the puffer pipette and the axon terminals. The decays of WT GABAC receptor-mediated currents were longer than GABAA receptor-mediated currents in all BC classes, indicating that GABAC receptors determine the decay kinetics (Fig. 2E, P < 0.05). This is similar to observations made previously in mouse rod BCs (McCall et al. 2002), ferret BCs (Shields et al. 2000) and heterologous expression systems (Amin & Weiss, 1994). Furthermore, the correspondence in the decay ratios across BC classes suggests that the kinetic properties and, possibly, the receptor subunit composition of both GABAC and GABAA receptors are similar across BC classes.

To confirm distinct ratios of GABAA and GABAC receptors across BC class, we compared GABA-evoked currents from WT BCs and BCs from mice lacking GABACρ1 receptor subunits, which as a consequence lack retinal GABAC receptors (GABACR null) (McCall et al. 2002). We observed a large difference in rod BC GABA-evoked currents between WT and GABACR null mice (Fig. 2Ab). However, this difference diminished when we compared GABA-evoked currents in ON (Fig. 2Bb) and OFF cone (Fig. 2Cb) BCs. When we computed the ratio of average GABA-evoked charge transfer (Q) in GABACR null (GABAA only) to WT (GABAA+ GABAC) mice, we found that for each BC class (Fig. 2D), the GABACR null: WT ratio (white bars) was comparable to the WT GABAA: WT total ratio (black bars). This result is consistent with our previous observations that the GABAC-mediated current is eliminated in retinal BCs and that there is no compensatory up-regulation of GABAA receptors in GABACR null rod BCs (McCall et al. 2002; Eggers & Lukasiewicz, 2006a; Sagdullaev et al. 2006). These results now extend our previous conclusions, as we show no compensatory up-regulation in any BC class.

Spontaneous GABAA and GABAC receptor-mediated IPSCs are observed in all BC classes

The GABA-evoked responses from WT and GABACR null BCs suggest that GABAA and GABAC receptor contributions vary across mouse BC classes. However, applied GABA activates receptors at both extrasynaptic and synaptic locations and not all of these receptors may be activated during synaptic transmission. To isolate and characterize synaptically activated GABAA and GABAC receptors across BC classes in a way that is largely independent of transmitter release and clearance, we recorded pharmacologically isolated GABAA and GABAC receptor-mediated spontaneous IPSCs (sIPSCs). Differences in sIPSC decay should reflect differences in receptor subunit composition, while differences in sIPSC amplitudes should reflect differences in synaptic receptor cluster size. The decay times (τdecay) of GABAA receptor-mediated sIPSCs were similar across all three BC classes (Fig. 3Aa). In contrast, their amplitudes differed: GABAA receptor-mediated sIPSCs in OFF cone BC were significantly larger than in rod BCs (Fig. 3Ab) (Table 1, K–S, P < 0.05).

Figure 3. Analyses of spontaneous IPSCs (sIPCS) indicate that both GABAA and GABAC receptors are synaptically activated in all BC classes.

Figure 3

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A, normalized histograms showing distributions of τdecay (left) and amplitude (right) for GABAA receptor-mediated sIPSCs recorded from rod (top, N = 972), ON cone (middle, N = 137) and OFF (bottom, N = 317) cone BCs (inset: average sIPSCs). Symbols (♦) denote the average values ±s.e.m.Aa, there were no significant differences among the τdecay values for rod, ON cone and OFF cone BC GABAA sIPSCs (K–S, P= 0.3). Ab, GABAA sIPSCs from OFF cone BCs had significantly larger peak values (K–S, P < 0.05) than those from rod BCs. B, normalized histograms showing distributions of τdecay and amplitude for GABAC receptor-mediated sIPSCs from rod (N = 157), ON cone (N = 173) and OFF cone BCs (N = 72) (inset: average sIPSCs), in the presence of kainate (10 μm). Conventions are the same as in A. Ba, the sIPSCs from OFF cone BCs had significantly shorter τdecay than ON cone and rod BC sIPSCs (K–S, P < 0.001). Bb, the GABAC sIPSCs from OFF cone BCs had significantly smaller amplitudes than ON cone and rod BC GABAC sIPSCs (K–S, P < 0.01). Scale bars: A, 2 pA and 5 ms; B, 2 pA and 50 ms.

Table 1.

Parameters for sIPSCs

BC cell GABAC amp (pA) GABACτdecay (ms) N GABAA amp (pA) GABAAτdecay (ms) N Glycine amp (pA) Glycine τdecay (ms) N
OFF 4.7 ± 0.2 28.6 ± 2.6 72 7.1 ± 0.4 2.0 ± 0.5 317 21.9 ± 0.7 3.7 ± 0.5 1088
ON 5.2 ± 0.4 36.3 ± 4.1 173 6.2 ± 0.4 2.1 ± 0.5 137 NA NA NA
Rod 5.7 ± 0.2 34.1 ± 2.1 157 6.2 ± 0.4 2.0 ± 0.5 972 10.1 ± 0.4 3.6 ± 0.5 193
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GABAC receptor-mediated sIPSCs are difficult to record, but can be observed if spontaneous GABA release is increased by including kainate in the bath (10 μm) (Frech & Backus, 2004; Eggers & Lukasiewicz, 2006b). Across all BC classes, the decay of GABAC receptor-mediated sIPSCs was significantly longer than GABAA receptor-mediated sIPSCs (Table 1, K–S, P < 0.001). We also observed a small but significant difference in the decay of GABAC receptor-mediated sIPSCs between OFF cone BCs and ON cone or rod BCs (Fig. 3Ba; Table 1, K–S, P < 0.001 for both comparisons). Consistent with our observations of GABA-evoked currents, the amplitudes of GABAC receptor-mediated sIPSCs from OFF cone BCs were smaller than those from either ON cone or rod BCs (Fig. 3Bb, K–S, P < 0.01). Additionally, the amplitudes of GABAA and GABAC receptor-mediated sIPSCs were similar in rod BCs, while the amplitudes of GABAA receptor-mediated sIPSCs in both ON and OFF cone BCs were significantly larger than their respective GABAC sIPSCs (Table 1, K–S, P < 0.001).

These results show that GABAA and GABAC receptors can be activated synaptically in all BC classes. The similarities in decay times suggest that the subunit compositions of GABAA and GABAC receptors may be similar across BC classes. However, differences in sIPSC amplitudes suggest that GABAA receptor synaptic clusters should be the largest on OFF cone BCs, while GABAC receptor synaptic clusters should be largest on rod BC. These findings suggest that synaptic receptor properties play a role in shaping the L-IPSC. Rod BC responses are predominantly shaped by GABAC receptors, while GABAA receptors primarily shape OFF cone BC responses. In ON cone BC responses, GABAA and GABAC receptors play a more balanced role.

Pharmacologically isolated GABAA and GABAC receptor-mediated L-IPSCs vary with BC class

The relatively small differences found in sIPSCs across BC class were inconsistent with the much larger differences in light-evoked currents, suggesting that synapse distribution and GABA release may be more important than receptor cluster size in shaping GABAergic L-IPSCs. To characterize the light-evoked activation of GABA receptors, we recorded and compared pharmacologically isolated GABAA and GABAC receptor-mediated L-IPSCs across the three BC classes (Fig. 4A–C). When pharmacologically isolating GABAC receptors with bicuculline, we probably blocked the influence of serial inhibitory circuits on the GABAA and GABAC receptor-mediated inputs to BCs. Thus, these responses should reflect the maximal GABAC receptor-mediated input, as we previously described for rod BCs (Eggers & Lukasiewicz, 2006a).

Figure 4. GABAA and GABAC receptors differentially contribute to L-IPSCs across BC classes.

Figure 4

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Representative L-IPSCs (dark grey bar light stimulus duration 30 ms) mediated by GABAC and GABAA receptors from rod BCs (A), ON cone BCs (B) and OFF cone BCs (C). Scale bars, 5 pA and 200 ms. D, the histogram plots average Q of GABAC receptor-mediated L-IPSCs normalized to the Q of GABAA L-IPSCs for each BC class. GABAC receptor-mediated L-IPSCs were significantly larger than GABAA L-IPSCs in rod BCs (GABAA, n= 12; GABAC, n= 14, P < 0.01) but there was no significant difference in GABAC and GABAA L-IPSC Q for ON (GABAA, n= 13; GABAC, n= 5) or OFF cone BCs (GABAA, n= 7; GABAC, n= 5). E, the histogram plots the average L-IPSC decay (D37) of GABAC receptor-mediated normalized to the average GABAA L-IPSC D37 for each BC class. GABAC receptor-mediated currents were significantly longer than GABAA current in every class (rod, P < 0.001; ON, P < 0.01; OFF, P < 0.05). Error bars in D and E represent propagated standard errors of the average GABAA and GABAC values.

Rod BCs received the most light-evoked input via GABAC receptors with a smaller contribution via GABAA receptors (Fig. 4A and D, Table 2, P < 0.01) (Eggers & Lukasiewicz, 2006b). Both GABAA and GABAC receptors mediated L-IPSCs of similar magnitude in ON (Table 3, P= 0.5) and OFF cone BCs (Table 4, P= 0.9), so the relative contribution of GABAC receptor-mediated input in these two classes was smaller than in rod BCs (Fig. 4D). Across all BC classes, GABAC receptor-mediated L-IPSCs had slower decay times, as we observed for GABA-evoked currents and sIPSCs, as well as slower times to peak than GABAA receptor-mediated L-IPSCs (Fig. 4E, Tables 2–4, P < 0.05). However, we found no significant differences across the BC classes in GABAA L-IPSC decay times or times to peak (ANOVAs: decay, GABAA, P > 0.2; time to peak, GABAA, P > 0.2) or in GABAC receptor-mediated L-IPSC decay times or times to peak (ANOVAs: decay, GABAC, P > 0.2; time to peak, GABAC, P > 0.5). Taken together, these results suggest that the kinetics of the GABAA and GABAC receptor-mediated L-IPSCs were similar across BC classes, but the relative GABAA and GABAC receptor contributions vary. While the similarity between the decay (D37) of the total GABAergic (Fig. 1) and GABAA receptor-mediated L-IPSCs in OFF cone BCs (Table 4) suggests a primary role for GABAA receptors, this idea is not consistent with our observation of a significant GABAC receptor-mediated contribution to sIPSCs (Fig. 3) and L-IPSCs (Fig. 4) in OFF cone BCs. One potential explanation for this discrepancy is that GABAA receptor-mediated serial inhibition, among GABAergic amacrine cells, differentially suppresses GABAC receptor-mediated inputs (Zhang et al. 1997; Roska et al. 1998; Eggers & Lukasiewicz, 2006a).

Table 2.

Parameters for L-IPSCs from Rod BCs

L-IPSC type D37 (ms) Q (fC) Peak (pA) Time to peak (ms) n
GABAA 134.1 ± 35.5 954 ± 371 7.1 ± 1.2 158.9 ± 12.5 12
GABAC 472.4 ± 82.7 3950 ± 682 10.4 ± 1.4 225.7 ± 17.9 14
Glycine 279.1 ± 28.9 1635 ± 421 10.6 ± 1.6 154.0 ± 17.5 13
WT control 275.3 ± 19.8 3215 ± 368 13.5 ± 1.3 168.2 ± 9.2 43
GABACR null control 119.6 ± 14.3 1775 ± 559 13.1 ± 2.2 156.7 ± 8.9 15
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Table 3.

Parameters for L-IPSCs from ON cone BCs

L-IPSC type D37 (ms) Q (fC) Peak (pA) Time to peak (ms) n
GABAA  87.8 ± 19.4  1972 ± 1077 13.7 ± 2.6 136.3 ± 5.8 13
GABAC 242.5 ± 52.0 2877 ± 462 14.0 ± 1.8 207 ± 21.3 5
WT control 148.9 ± 10.7 2172 ± 400 16.3 ± 3.2 133.4 ± 6.7 17
GABACR null control 101.0 ± 16.5 1385 ± 533 19.6 ± 4.2 132.2 ± 8.4 7
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Table 4.

Parameters for L-IPSCs from OFF cone BCs

L-IPSC type D37 (ms) Q (fC) Peak (pA) Time to peak n
GABAA 163.5 ± 8.6 3081 ± 846 10.0 ± 3.6 157.9 ± 22.4 7
GABAC 397.4 ± 79.6 3225 ± 261 14.5 ± 4.0 260.7 ± 39.6 5
Glycine 280.0 ± 30.2 12204 ± 3448 65.1 ± 19.5 136.8 ± 16.5 7
WT control 253.8 ± 27.1 22770 ± 5474 108.0 ± 19.9 157.3 ± 30.3 13
GABACR null control 238.9 ± 32.0 16821 ± 7885  45.8 ± 19.7 164.1 ± 37.8 6
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Serial connections mediated by GABAA receptors suppress GABAC receptor-mediated L-IPSCs

To determine whether GABAA receptor-mediated serial inhibition is present across BC classes, we recorded GABAergic L-IPSCs and compared them to L-IPSCs recorded in the presence of bicuculline, when serial GABAergic signalling between amacrine cells was eliminated. Strychnine, which does not disrupt these serial connections (Eggers & Lukasiewicz, 2006a), was always present to isolate the GABAergic L-IPSCs. The addition of bicuculline increased the L-IPSC charge transfer (Q) across all the BC classes (Fig. 5A–D, ANOVA, P= 0.4). This suggests that GABAC receptor-mediated inputs to all BC classes were similarly suppressed by GABAA receptor-mediated serial inhibition.

Figure 5. Serial inhibition mediated by GABAA receptors decreases GABAC receptor-mediated L-IPSCs in all WT BC classes.

Figure 5

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A–C, representative GABAergic L-IPSCs recorded from WT rod (A, n= 9), ON cone (B, n= 7) and OFF cone (C, n= 6) BCs evoked by a light stimulus (1000 ms duration; dark grey bar) in the presence of strychnine (black trace) and when GABAA-mediated inhibition is blocked (strychnine + bicuculline, isolated GABAC receptor-mediated L-IPSCs). D, the histogram plots the average L-IPSC Q in the presence of strychnine + bicuculline normalized to the Q only in the presence of strychnine (dashed line). All WT BC classes showed a significant increase when GABAA input was blocked and there was no significant difference in proportional increase across BC class (ANOVA, P= 0.4). Scale bars: A, 5 pA; B and C, 10 pA, and 200 ms in all.

GABAC receptors shape L-IPSCs to rod and ON cone BCs but not OFF cone BCs

Since GABAC receptor-mediated inputs can be controlled by serial inhibition, our measures of the pharmacologically isolated GABAC receptor-mediated L-IPSCs may be overestimates of the GABAC receptor-mediated input to the total L-IPSCs. To estimate the GABAC receptor contribution when serial inhibition was present, we compared GABAergic L-IPSCs across BC classes from WT and GABACR null mice. The slow decay kinetics of WT GABAC receptor-mediated L-IPSCs (Fig. 4) predicts that L-IPSCs from GABACR null BCs will decay faster than WT BCs in any BC class with a significant GABAC receptor-mediated input. Consistent with this hypothesis, we found that the decay of L-IPSCs from GABACR null rod (Fig. 6A and D; P < 0.0001, Table 2) (Eggers & Lukasiewicz, 2006b) and ON cone BCs was significantly briefer than WT (Fig. 6B and D; P < 0.05, Table 3). In OFF cone BCs, by contrast, the decay of L-IPSCs from WT and GABACR null were similar (Fig. 6C and D; P= 0.7, Table 4), suggesting little or no GABAC receptor-mediated contribution when serial inhibition was present. Together our results from GABA-evoked currents and L-IPSCs show that there is a gradient of GABAC receptor-mediated input across BC classes and that this input proportionately prolongs the timecourse of GABAergic L-IPSCs.

Figure 6. GABAC receptors shape L-IPSCs in ON cone and rod BCs, but not OFF cone BCs.

Figure 6

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A–C, representative L-IPSCs (30 ms light stimulus duration, grey bar) from rod (A; adapted from Eggers & Lukasiewicz, 2006b), ON cone (B) and OFF cone (C) BCs in WT (black) and GABACR null (grey) mice. D, the histogram plots the average L-IPSCs decay (D37) from GABACR null BCs normalized to WT. GABACR null L-IPSCs in rod BCs (WT, n= 43; null, n= 15, P < 0.0001) and ON cone BCs (WT, n= 17; null, n= 4, P < 0.05) were significantly briefer than WT. There was no significant difference in L-IPSC decays from GABACR null and WT OFF cone BCs (WT, n= 13; null, n= 6, P= 0.7). Scale bars: A and B, 5 pA; C, 30 pA, and 200 ms in all. Error bars in D represent propagated standard errors of the average null to WT values.

Glycinergic inhibition is the dominant component of inhibition in OFF cone BCs

As many BCs also receive glycinergic inhibition (Eggers & Lukasiewicz, 2006b; Ivanova et al. 2006), we compared the properties of pharmacologically isolated glycinergic sIPSCs in rod and OFF BCs. Glycine receptor-mediated sIPSCs were never observed in ON cone BCs, consistent with previous observations (Ivanova et al. 2006). Figure 7A and B shows the distributions of decay (τdecay) and amplitude of glycinergic sIPSCs for OFF cone and rod BCs, as well as average glycinergic sIPSCs (inset). Rod and OFF cone BCs had similar decays, but the amplitude of glycine sIPSCs was significantly larger in OFF cone BCs (Table 1, K–S, P < 0.0001), suggesting that subunit composition was similar, but receptor cluster size differed in these two BC classes. The decay of glycine receptor-mediated sIPSCs also was significantly briefer than GABAC receptor-mediated sIPSCs (K–S, P < 0.001) and significantly longer than GABAA receptor-mediated sIPSCs (K–S, P < 0.001) in both rod and OFF cone BCs.

Figure 7. Glycine receptor-mediated inputs dominate L-IPSCs in OFF cone BCs.

Figure 7

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Glycine receptor-mediated sIPSCs were recorded from rod (black) OFF cone (dark grey), but not ON cone BCs. A, normalized histograms, with averages (♦) showing distributions of τdecay for glycine sIPSCs. There was no significant difference between the τdecay for OFF cone (n= 1088) and rod BC (n= 193) sIPSCs (K–S, P= 0.8). B, normalized histograms, showing distributions of peak amplitudes of glycine sIPSCs from rod and OFF cone BCs. The peak amplitude of glycine sIPSCs from OFF cone BCs were significantly larger than rod BCs (K–S, P < 0.0001). Inset, average glycine receptor-mediated sIPSCs. C, representative glycine receptor-mediated L-IPSCs (30 ms light stimulus, dark grey bar) recorded in both OFF cone (n= 7) and rod (n= 13) BCs. No light-evoked glycine receptor-mediated response was ever recorded in ON cone BCs (n= 5). D, the histogram plots glycine receptor-mediated L-IPSCs (Q) from rod (black) and OFF (grey) cone BCs. OFF cone L-IPSCs were significantly larger than rod and ON cone BCs (P < 0.05, the bar for ON cone BCs represents the baseline current). Rod BCs also had significantly more glycinergic input than ON cone BCs (P < 0.01). Scale bars: B, 2 pA and 5 ms; C, 10 pA and 200 ms.

To determine if these differential contributions in glycinergic sIPSCs contributed to light-evoked inhibition, we recorded and characterized glycinergic L-IPSCs. As we observed with glycinergic sIPSCs, we found no light-evoked glycinergic input to ON cone bipolar cells. L-IPSC responses (Q) (Fig. 7C) in OFF cone BCs were significantly larger than in rod BCs (Fig. 7D, P < 0.05, Tables 2–4). Furthermore, glycinergic L-IPSCs were significantly larger than GABAA or GABAC receptor-mediated inputs in OFF cone BCs (Table 4, P < 0.05). Although the kinetics of glycine receptor-mediated sIPSCs are significantly faster than GABAC receptor-mediated sIPSCs, their L-IPSCs have similar decay kinetics (P > 0.05, Tables 2 and 4), probably attributable to prolonged light-evoked glycine release (Eggers & Lukasiewicz, 2006b). These results suggest that glycine receptor-mediated inputs dominate L-IPSCs in OFF cone BCs in dark-adapted conditions.

The decay of combined glycinergic and GABAergic L-IPSCs varies with BC class

We have shown that the relative contributions of isolated glycine, GABAA and GABAC receptor-mediated inputs vary across BC class. To examine how the combination of these receptor-mediated inputs shape the total L-IPSCs, we compared their decay times (D37) across different WT BC classes (Fig. 8A–C). The decay of L-IPSCs in rod BCs was significantly slower than ON cone BCs (ANOVA. P < 0.001; Scheffé's post hoc test, rod versus ON cone BC; P < 0.001, Fig. 8D, Tables 2 and 3), consistent with our results showing that GABAC receptor-mediated input was greater in rod than ON cone BCs. Despite the relative lack of GABAC receptor-mediated input to OFF cone BCs, the decay (D37) of rod and OFF cone BCs was similar (Scheffé's post hoc test, P= 0.8, Fig. 8D, Table 4). The slow decay of L-IPSCs in OFF cone BCs was attributable to their large glycinergic input, as their total L-IPSCs were significantly longer than their GABAergic L-IPSCs (Figs 8D and 1D, respectively; P < 0.01). These results suggest that under dark-adapted conditions, GABAC receptor-mediated inputs are important for determining the timecourse of rod and ON cone BC L-IPSCs, but glycine receptor-mediated inputs predominate in OFF cone BCs.

Figure 8. The decay of combined glycinergic and GABAergic L-IPSCs varies with WT BC class.

Figure 8

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A–C, representative total L-IPSCs (glycinergic + GABAergic) from rod (A), ON cone (B) and OFF cone (C) BCs evoked by a light stimulus (30 ms light stimulus, dark grey bar). D, the histogram plots the average decay (D37) from each BC class. L-IPSCs from rod BCs were significantly slower than ON cone BCs (rod, n= 43; ON cone, n= 17, ANOVA, P < 0.001, rod versus ON Scheffé's post hoc, *P < 0.001), but similar to OFF cone BCs (n= 13, P= 0.86). The decay of L-IPSCs from OFF cone BCs also was significantly slower than ON cone BCs (**P < 0.05). Scale bars: A and B, 5 pA; C, 30 pA, and 200 ms in all.

Discussion

Using pharmacological tools and a mouse lacking retinal GABAC receptors, we have found that both GABAergic and glycinergic presynaptic inhibition differ with BC class in dark-adapted conditions. Distinct contributions of slow GABAC receptor-mediated inputs determined the decay time of GABAergic L-IPSCs in different BC classes. Rod BCs, with large GABAC receptor-mediated contributions, exhibited the slowest GABAergic L-IPSC decays and OFF cone BCs with small GABAC receptor-mediated contributions exhibited the fastest decays. Glycinergic inputs also differentially contributed to inhibition across BC classes. Glycinergic inputs are absent in ON cone BC responses and most prominent in the responses of OFF cone BCs, where they mediated a slow inhibition, similar to that of GABAC receptors in other BC classes.

Functional significance of the variance in presynaptic inhibition across BC classes

Excitatory responses to photoreceptor input have distinct kinetics across BC classes (Awatramani & Slaughter, 2000; Li & DeVries, 2006). Rod-mediated responses have slower rise and decay kinetics than cone-mediated responses (Schnapf & Copenhagen, 1982; Cadetti et al. 2005). Li & Devries (2006) show that when cones are electrically stimulated, by-passing phototransduction, ON cone BC responses have slower kinetics than OFF cone BC responses, suggesting that distinct postsynaptic glutamate receptors (Slaughter & Miller, 1981, 1983) shape ON and OFF bipolar cell responses. Consistent with this idea, ON and OFF cone BC light-evoked responses in the turtle retina showed distinct kinetics (Ashmore & Copenhagen, 1980). Experiments in the mammalian retina are still needed to demonstrate physiological differences of light-evoked excitatory responses in ON and OFF cone bipolar cells. Our data suggest that these distinct kinetics of the excitatory responses across BC class are well matched by the distinct kinetics of inhibition mediated by varying contributions of GABAC receptors across BC class. Although OFF cone BCs received little GABAC receptor-mediated input, their L-IPSCs still decay slowly in dark-adapted conditions because they are dominated by slowly decaying glycinergic inhibition.

We do not believe that this apparent difference is a mismatch between excitatory inputs and presynaptic inhibition in OFF cone BCs. Under the dark-adapted conditions used in our experiments, glycinergic inputs to OFF cone BCs arise primarily from AII amacrine cells, which are activated by signalling through the rod pathway (Strettoi et al. 1990). This OFF BC glycinergic input suppresses glutamate release and functions as the rod-mediated ‘OFF’ signal to OFF ganglion cells. Thus, the slow decay of glycinergic presynaptic inhibition in OFF cone BCs is actually well-matched to the slow timecourse of rod pathway excitation. In our previous experiments, we estimated the timecourse of glycine release, using a deconvolution analysis of rod BC L-IPSCs (Eggers & Lukasiewicz, 2006b), and showed that the slow decay kinetics of glycinergic L-IPSCs arise from prolonged release of glycine onto BCs. The similar kinetics of glycinergic L-IPSCs and sIPSCs in rod and OFF cone BCs (Tables 1, 2 and 4) suggests a similar, prolonged glycine release from AII amacrine cells to OFF cone BCs.

In contrast, under cone-mediated, light-adapted conditions AII amacrine cells receive only minor excitatory inputs (Protti et al. 2005; Trexler et al. 2005), resulting in little glycinergic input to OFF cone BCs. In the absence of this significant glycinergic input, our data predict that GABA-mediated inhibition most probably shapes L-IPSCs. Thus, our isolated GABAergic L-IPSCs approximate light-adapted responses and show a faster decay that reflects a large GABAA and a small GABAC receptor contribution. Under these conditions, cone-mediated responses of OFF cone BCs are again well matched to their presynaptic inhibition. Furthermore, this implies that the timecourse of presynaptic inhibition in OFF cone BCs depends on whether rod or cone signals are processed: slow glycinergic inputs matched to the slow rod input and fast GABA inputs matched to the brisk cone input.

Our findings may also explain the functional differences that we have observed between ON and OFF ganglion cell responses elicited via stimulation of cone pathways (Sagdullaev et al. 2006). When GABAC receptor-mediated input is eliminated, signalling in ON, but not in OFF ganglion cells is enhanced, suggesting that GABAC receptors only modulate ON BC output. However, immunocytochemical evidence indicates that GABAC receptors are expressed in both the ON and OFF sublaminae of the IPL (Interplexiform layer) (Enz et al. 1996; Shields et al. 2000; McCall et al. 2002), challenging the notion of differential GABAC receptor-mediated presynaptic inhibition. Our results demonstrate that GABAC receptor-mediated inhibition occurs in both ON and OFF cone BCs (Figs 3–5), but its magnitude differs between the two pathways (Figs 2, 3 and 6). Comparisons of GABA- and light-evoked currents from WT and GABACR null BCs indicate that GABAC receptors contributed significantly to ON cone BC responses, but their contribution to OFF cone BC responses was minor, at best. GABA-evoked currents showed a larger complement of GABAC receptors on OFF cone BCs than indicated by our L-IPSC results. This difference might be attributable to extrasynaptic GABAC receptors on OFF cone BCs, or to synaptic inputs that were limited by serial inhibitory circuits between amacrine cells (Zhang et al. 1997; Roska et al. 1998; Eggers & Lukasiewicz, 2006a).

Additionally, in the present study we have described the differences only between the major classes of BCs: rod, ON and OFF cone BCs. The ON and OFF ganglion cell pathway differences in GABAC receptor-mediated inhibition observed in all ON and OFF ganglion cell subtypes (Sagdullaev et al. 2006), suggests that similar differences occur in all ON and OFF cone bipolar cell types. We morphologically classified our BCs according to the scheme described by Ghosh et al. (2004) and found that GABAC receptor contributions fell into three categories, corresponding to rod, ON and OFF cone BCs. Although our conclusions are consistent with the major BC classifications, our sample sizes for different subtypes were insufficient to make conclusions about significant differences between subtypes. Thus, future studies may show differences within BC classes.

Temporally tuning inhibition with distinct inhibitory receptors

Similar to our observations of differential activation of GABAC receptors in the retina, temporal filtering through the expression of distinct inhibitory receptors also occurs in other areas of the CNS. In spinal cord and brainstem, the timecourse of IPSCs is temporally tuned by the relative activation of glycine and GABAA receptors with distinct kinetics (Jonas et al. 1998; O'Brien & Berger, 1999). In the hippocampus, the timecourses of inhibition and excitation mirror each other, and are controlled by distinct combinations of GABAA receptor subunits in different populations of interneurons (Hajos & Mody, 1997). Temporal tuning of inhibition is also important during development in many areas of the CNS. The decrease in the decay time of GABAergic IPSCs, attributable to changes in GABA receptor subunit composition (Brickley et al. 1996; Dunning et al. 1999; Okada et al. 2000) parallels the decrease in the timecourse of excitation (Takahashi, 2005).

Our findings demonstrate that distinct forms of presynaptic inhibition in parallel retinal BC pathways are attributable to different complements of GABA and glycine receptors. The timecourse of presynaptic inhibitory inputs generally matches excitatory inputs to distinct BCs reported in the literature, which are temporally filtered by different glutamate receptors types (Ashmore & Copenhagen, 1980; Schnapf & Copenhagen, 1982; Cadetti et al. 2005; Li & DeVries, 2006). Balancing the timecourses of excitation and presynaptic inhibition across parallel bipolar cell channels ensures that separate signals are transmitted effectively to their ganglion cell targets. This may be a strategy used both in the retina and elsewhere in the CNS to coordinate the inputs and outputs of neurons involved in parallel information processing.

Acknowledgments

This work was supported by NIH grants T32 EY13360 and F32 EY15629 (E.D.E.); EY014701 (M.A.Mc.); EY08922 (P.D.L.), The M. Bauer Foundation (P.D.L.); EY02687 (Washington University, Department Ophthalmology) and Research to Prevent Blindness (Washington University and University of Louisville, Departments of Ophthalmology). We thank members of the Lukasiewicz and McCall laboratories and Dr Rachel Wong, for helpful discussion and comments on this manuscript and James Debrecht for technical assistance.

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