1. Presynaptic Inhibition Modulates Spillover, Creating Distinct Dynamic Response Ranges of Sensory Output 
Botir T. Sagdullaev, Maureen A. McCall, Peter D. Lukasiewicz 
Neuron 
Volume 50, Issue 6, Pages (June 2006)
DOI: /j.neuron
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
Presynaptic Inhibition Modulates Spontaneous and Visually Evoked Spiking In Vivo
Representative raster plots (upper traces) and peristimulus time histograms (PSTHs, middle) illustrating spontaneous and light-evoked firing in wt (Aa) and GABACR null (Null) (Ab) sustained and transient ON-center ganglion cells (GCs); and wt (Ba) and Null (Bb) sustained and transient OFF GCs. The lower traces in (A) and (B) indicate the onset of a 2 s optimally sized stimulus, a bright spot (50 cd/m2) for ON GCs and a dark spot (10 cd/m2) for OFF GCs presented on an adapting background (23 cd/m2) and centered on the cell's receptive field center. The inset in (Aa) represents the in vivo recording paradigm (see Experimental Procedures). Responses were collected (50 ms bin width) during the stimulus presentation and for several seconds before and after the stimulus when the screen luminance returned to adapting level. The interstimulus interval was 4 s. Each PSTH represents the average of 8 individual responses shown above in the raster plots. Spontaneous and light-evoked firing rates were significantly increased only in ON GCs in Null mice compared to wt mice (C). ON GCs (C), spontaneous firing rate in: wt, ± 1.42, n = 50; versus Null, ± 1.47, n = 69; p < Light-evoked firing rate in: wt, ± 1.59, n = 47; versus Null, ± 1.31, n = 63; p = 0.02). OFF GCs (D), spontaneous firing rate in: wt, 6.52 ± 0.85, n = 44; versus Null, 7.95 ± 0.96, n = 26; p = Light-evoked firing rate in: wt, ± 4.24, n = 39; versus Null, ± 5.61, n = 26; p = Scale bars: horizontal, 1 s; vertical, 50 spikes/s. Error bars, ±SEM; ∗p < 0.05, ∗∗p < 0.01.
Neuron  , DOI: ( /j.neuron )
Copyright © 2006 Elsevier Inc. Terms and Conditions

3. Figure 2
Presynaptic Inhibition Extends the Dynamic Range of Responses to Light Increments
Stimulus paradigm for measuring light-evoked intensity-response functions (IRFs) in ON (Aa) and OFF GCs (Ba) in vivo. For ON GCs, responses were recorded to increments in light intensity of an optimal spot stimulus above a maintained light-adapting level (∼30 cd/m2). For OFF GCs, responses were recorded to decrements in light intensity of an optimal spot stimulus below the same maintained light-adapting level.
(Ab and Bb) Representative light-evoked responses (spikes) recorded extracellularly from ON (Ab) and OFF (Bb) GCs. The values to the right of each trace indicate the intensity of the light stimulus in cd/m2. Traces at the bottom of each set of responses in (Ab) and (Bb) indicate onset and offset of the stimuli. Duration of the light stimulus was 4 s and the interstimulus interval was 10 s for low-intensity stimulus steps and 60 s for high-intensity stimulus steps.
(Ac) Averaged and normalized intensity-response profiles of ON GCs in wild-type (wt) (black circles) and Null (gray circles) mice. Eliminating GABACR-mediated inhibition significantly decreased (p < 0.01) the dynamic range of ON GCs.
(Bc) Averaged and normalized intensity-response profiles of OFF GCs in wt (black triangles) and Null GCs (gray triangles). For comparison, the intensity-response profile of ON GCs in wt from (Ac) was replotted (dotted curve indicated with arrow). The intensity-response profiles for ON and OFF GCs were obtained by plotting the mean firing rate during the first 1 s of the response against log relative light intensity.
(C) Bar graph showing the dynamic ranges (5%–95% max response, see Experimental Procedures) of light-evoked responses in ON and OFF GCs in wt and Null mice in vivo.
(D) Bar graph showing the dynamic ranges of electrically evoked EPSCs (eEPSCs) in ON and OFF GCs in wt and Null mice. Inset in (D) represents the retinal circuitry and paradigm used for recording electrically evoked responses in vitro. eEPSCs were recorded from GCs in wt and Null mice in a light-adapted retina slice preparation. Slices were bathed in control solution 2 and GCs were voltage clamped at −35 mV to relieve NMDAR-mediated current from Mg2+ block. Bipolar cell (BC) inputs were activated by focal electrical stimuli (0.03–1000 μA; 2 ms) delivered by an extracellular electrode placed in the OPL. ON GCs have a wider dynamic range compared to OFF GCs in wt retina. Eliminating GABACR-mediated inhibition reduces the dynamic range of ON GCs.
Error bars, ±SEM; ∗p < 0.05, ∗∗p < 0.01.
Neuron  , DOI: ( /j.neuron )
Copyright © 2006 Elsevier Inc. Terms and Conditions

4. Figure 3
Elimination of GABACRs Enhances the NMDAR, but Not AMPAR, Component of eEPSCs in ON GCs
eEPSCs were recorded from GCs in wt and Null mice in a retinal slice preparation as described in Figure 2D. Bipolar cell inputs were activated by electrical stimuli (3–5 μA; 2 ms) delivered by an extracellular electrode placed in the OPL. eEPSCs from GCs in all wt (black traces) and Null mice (gray traces) exhibited NMDAR and AMPAR components (isolated after NMDARs were blocked with 50 mM D-AP5). eEPSCs from Null ON GCs had slower decays and larger charge transfers than wt GCs ([Aa]–[Ac]; p < 0.01), while eEPSCs were similar in both Null and wt OFF GCs ([Ba]–[Bc]; p > 0.5). The AMPAR component of the eEPSCs did not differ between wt and Null ON or OFF GCs (Ab and Bb). Each trace is an average of 2–4 responses. In this and all subsequent figures, response trace baselines were adjusted and superimposed for a better comparison.
Error bars, ±SEM; ∗p < 0.05, ∗∗p < 0.01.
Neuron  , DOI: ( /j.neuron )
Copyright © 2006 Elsevier Inc. Terms and Conditions

5. Figure 4
Pharmacological Blockade of GABACRs in wt Retina Selectively Enhances the NMDAR-Mediated Component of eEPSCs
eEPSCs were recorded from wt GCs held at −35 mV. Blockade of GABACRs with TPMPA (50 μM) significantly increased the eEPSCs in ON ([Aa] and [C]), but not OFF GCs ([B] and [C]). (Ab) Blockade of NMDARs with D-AP5 (50 μM) prevented TPMPA-induced increase in charge transfer of the eEPSC (QeEPSC). (Ac) Blockade of AMPARs with either CNQX (5 μM, data not shown) or GYKI (40 μM) did not alter the TPMPA-induced increase in QeEPSC.
Error bars, ±SEM; ∗p < 0.05, ∗∗p < 0.01.
Neuron  , DOI: ( /j.neuron )
Copyright © 2006 Elsevier Inc. Terms and Conditions

6. Figure 5
Presynaptic Inhibition Limits Spillover Activation of Perisynaptic NMDARs in ON wt GCs
(A) Averaged sEPSCs were recorded from ON GCs held at −75 mV and superfused with Mg2+-free solution in the presence of bicuculline and strychnine. Blockade of GABACRs by TPMPA increased the decay time (D37) and charge transfer of sEPSCs (QsEPSCs) ([Aa] and [Ac], black trace and bars). Blockade of NMDARs by D-AP5 reversed the TPMPA-induced enhancement ([Aa] and [Ac], gray trace and bars). (Ab) When NMDARs were initially blocked by D-AP5, TPMPA did not enhance the sEPSCs. All activity was blocked by subsequent application of GYKI (40 μM). In this and the following figure, the numbers in superscript and parenthesis indicate the order in which pharmacological agents were delivered.
(Ba) sEPSCs in Null ON GCs exhibited NMDAR-mediated current, which was absent in wt cells (see [Ab]).
(Bb) Decay times (D37) and charge transfers (Q) of sEPSCs in Null ON GCs (black bars) were greater than in wt GCs (dotted line, p < 0.05). D-AP5 decreased both QsEPSCs and D37 (gray bars).
(C) Frequency histograms show that eliminating GABACR-mediated inhibition either genetically (Null) or pharmacologically (wt + TPMPA) increased the number of events with larger QsEPSCs compared with wt mice.
Error bars, ±SEM; ∗p < 0.05, ∗∗p < 0.01.
Neuron  , DOI: ( /j.neuron )
Copyright © 2006 Elsevier Inc. Terms and Conditions

7. Figure 6
Multivesicular Release Occurs in the Absence of Presynaptic Inhibition when Pr Is High
(A) Inhibition modulates AMPAR-mediated sEPSCs when their saturation is reduced by a low-affinity antagonist. Mean sEPSCs from representative morphologically identified wt ON GCs in control conditions (D-AP5, bicuculline and strychnine, 0 Mg2+, Vh = −75 mV; 84–143 individual events were averaged for each condition). The low-affinity AMPAR antagonist γ-DGG (0.5–1.0 mM) reduced the sEPSCs' amplitude. The subsequent addition of TPMPA increased the sEPSCs' amplitude. Reducing the probability of release (Pr) by lowering extracellular Ca2+ reversed the effect of TPMPA. All sEPSCs traces in (Aa) are from the same cell.
(Ab) Summary graph showing that AMPAR-mediated sEPSC amplitudes were significantly reduced by γ-DGG (n = 15 cells, p < 0.01, paired t test). Block of presynaptic inhibition with TPMPA increased the amplitude of the sEPSC (n = 14, p < 0.01, paired t test). This increase was reversed after Pr was reduced by lowering extracellular Ca2+ (n = 7, p < 0.01, paired t test).
(B) In contrast, when presynaptic inhibition was preserved, lowering extracellular Ca2+ did not affect the sEPSCs' amplitude (Bb) (n = 10, p = 0.24, paired t test). In ([Ba], left), traces 3 and 4 from (Ba) are redrawn for comparison to representative sEPSCs in the presence of presynaptic inhibition ([Ba], right; each trace is an average of 270 and 337 individual events). In (Ab) and (Bb), gray lines are responses of individual cells and the black lines represent their mean values (±SEM) (see also Figure S4).
Neuron  , DOI: ( /j.neuron )
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8. Figure 7
Evidence that NMDARs Are Synaptic on OFF wt GCs and Activated during Spontaneous Glutamate Release
(Aa) sEPSCs recorded from morphologically identified ON (black) and OFF (gray) wt GCs. The traces for OFF and ON GCs represent an average of 302 and 235 events, respectively.
(Ab) D-AP5 speeds the decay of the sEPSC in OFF GCs (gray, the same cell as in Aa, 199 events), but does not affect sEPSCs in ON GCs (black, the same cell as in Aa, 208 events). In control conditions (bicuculline and strychnine, 0 Mg2+, Vh = −75 mV), the decay time, D37 (Ac), and the charge transfer, QsEPSC (Ad), of the OFF GCs' sEPSCs were larger than those recorded from ON GCs (p < 0.01). In the presence of D-AP5, sEPSCs from OFF GCs were similar to those recorded from ON GCs (p > 0.15).
(Ba) sEPSCs from ON GCs were exclusively mediated by AMPARs and blocked by GYKI
(Bb) In contrast, sEPSCs from OFF GCs were mediated by AMPA and NMDA receptors and were only partially blocked by GYKI The subsequent addition of D-AP5 eliminated the remaining sEPSCs.
(C) sEPSCs in OFF GCs are uniquantal and their amplitude is not affected by lowering the Pr (n = 6, p = 0.18, paired t test). Experimental conditions are the same as in Figures 6A and 6B (D-AP5, bicuculline and strychnine, 0 Mg2+, Vh = −75 mV).
(D) Effects of the glutamate uptake antagonist TBOA (5–10 μM) on sEPSCs in ON and OFF wt GCs. sEPSCs in TPMPA and after subsequent addition of TBOA are superimposed. The traces represent an average of 395 (TPMPA) and 356 (TPMPA + TBOA) events for ON GCs and 176 (TPMPA) and 351 (TPMPA + TBOA) events for OFF GCs. Summary bar graph shows the experimental paradigm and the effect of TBOA on sEPSC charge transfer (QsEPSCs) in seven ON and six OFF GCs. All events for an individual cell under a given condition were averaged and then normalized to their corresponding values in the presence of TPMPA (dashed line). Recording conditions were the same as in Figure 5A.
Error bars, ±SEM; ∗p < 0.05, ∗∗p < 0.01.
Neuron  , DOI: ( /j.neuron )
Copyright © 2006 Elsevier Inc. Terms and Conditions

9. Figure 8
Schematic Diagram of the Asymmetric Modulation of Glutamate Release from Bipolar Cells by Presynaptic Inhibition and Spillover Activation of Postsynaptic Glutamate Receptors on GCs
(Aa) The inhibitory GABAergic feedback from amacrine cells (ACs) via GABACRs limits glutamate release from ON bipolar cells and controls spillover activation of NMDARs that are located perisynaptically on ON GCs' dendrites. GABACR-mediated negative feedback confines synaptic transmission and extends the dynamic response range of ON GCs.
(Ab) In the absence of GABACR-mediated inhibition, the probability of glutamate release is enhanced and the modulation of the excitatory transmission is disrupted.
(B) In the OFF pathway, the activation of synaptically localized AMPARs and NMDARs on OFF GC dendrites is not limited significantly by GABACR-mediated feedback to OFF BCs. Because the excitatory inputs to OFF GCs are not significantly modulated by presynaptic inhibition, their output gain is high. For simplicity, only the inhibitory feedback component of a reciprocal synapse between a BC and an AC is shown.
Neuron  , DOI: ( /j.neuron )
Copyright © 2006 Elsevier Inc. Terms and Conditions