1. Segregated Glycine-Glutamate Co-transmission from vGluT3 Amacrine Cells to Contrast-Suppressed and Contrast-Enhanced Retinal Circuits 
Seunghoon Lee, Yi Zhang, Minggang Chen, Z. Jimmy Zhou 
Neuron 
Volume 90, Issue 1, Pages (April 2016)
DOI: /j.neuron
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Figure 1
Differential Co-release of Glycine and Glutamate from GACs to Specific GC Types
(A) (Top) Maximum z projection of a two-photon image stack taken from a recorded rBSGC (red, ON; cyan, OFF dendrites) in a vGluT3-Cre/ChR2-YFP (green) retina. (Bottom) Cross-sectional reconstruction of dendritic arbors, marked by green dashed lines in the upper panel, is shown.
(B) The z projection (top) and cross-sectional reconstruction (bottom) of an OFF αGC are shown.
(C) Blue light-evoked responses of an rBSGC in the presence of L-AP4 (20 μM), ACET (20 μM), and HEX (300 μM) (cocktail) are shown.
(D) Blue light-evoked responses of an OFF αGC to blue light flashes in the cocktail are shown.
(E and F) CNQX (40 μM) + CPP (20 μM) did not block the outward response in an rBSGC at 0 mV (E) nor did strychnine (STRY, 1 μM) block the inward response in an OFF αGC at −70 mV (F).
(G) STRY (bottom), but not SR95531 (SR, 50 μM, top), completely abolished the outward responses of an rBSGC at 0 mV in the cocktail + CNQX + CPP.
(H) CPP had little effect on the inward currents in an OFF αGC in the cocktail, but CPP + CNQX completely blocked the responses.
(I and J) Summaries of pharmacological effects on blue light-evoked outward currents in rBSGCs at 0 mV (I) and inward currents in OFF αGCs at −70 mV (J) are shown.
(K and L) Comparisons between blue light-evoked peak inward glutamatergic (at −70 mV) and peak outward glycinergic (at 0 mV) currents in rBSGCs (K) and OFF αGCs (L) in the cocktail are shown. Drug concentrations are the same as first indicated. Numbers in parentheses, cell tested; error bars, SEMs.
See also Figure S1.
Neuron  , 27-34DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 2 Ca2+-Dependent Co-release of Glycine and Glutamate from GACs
(A and B) Blue light-evoked glycinergic response in an rBSGC (A) and glutamatergic response in an OFF αGC (B) remained intact after 15- to 30-min perfusion of 18β-GA (25 μM), in the presence of a control cocktail of L-AP4 + ACET + HEX.
(C) Dual recording from a pair of GAC and rBSGC in the presence of the control cocktail, showing voltage-gated currents of GAC (top) in response to depolarizing steps (100 ms long, preceded by a 200-ms-long pre-step from −85 to −100 mV), outward postsynaptic response at 0 mV (middle), but no response at −70 mV (bottom) in the rBSGC. The same rBSGC also responded to blue light with an outward current at 0 mV but with no current at −70 mV (data not shown).
(D) Dual recording from a pair of GAC and OFF αGC, showing inward postsynaptic responses at −70 mV but no response at 0 mV from the OFF αGC. Blue light evoked inward currents at −70 mV (but no current at 0 mV) in the same OFF αGC (data not shown).
(E and F) (Top) The z projection of Alexa 594-filled GAC-rBSGC pair (E, same pair as in C) and GAC-OFF αGC pair (F, same pair as in D) from vGluT3-Cre/ChR2-YFP (green) retina. Arrowheads indicate GAC (pink), rBSGC (yellow), and OFF αGC (yellow). (Bottom) Enlarged view shows single optical sections of marked areas (1 and 2, white boxes) in upper panels, noting putative synaptic sites (white dashed circles) between GAC and rBSGC (E, OFF and ON sublamina) and between GAC and OFF αGC (F, both in OFF sublamina).
(G–J) CdCl2 (300 μM, G and H) and 0 mMEq [Ca2+]o (I and J) completely blocked the responses of rBSGC (at 0 mV) and OFF αGC (at −70 mV) to blue light flashes in the presence of the control cocktail. Drug concentrations (in μM) were as follows: L-AP4 (20), ACET (20), and HEX (300).
Neuron  , 27-34DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

4. Figure 3 Suppressed-by-Contrast Light Response Properties of rBSGCs
(A) Responses of an rBSGC to flashes of small light (left, on dark background) and dark (right, on bright background) spots (100 μm in radius) under on-cell loose-patch recording, showing transient suppression of tonic background spikes at both light onset and offset (upper). (Middle and bottom) Raster and rate plots, respectively, of the spikes in upper trace are shown.
(B) (Top) Responses of another rBSGC to a moving light bar (size, 600 × 200 μm; speed, 300 μm/s), showing spike suppression by both the leading edge (LE) and the trailing edge (TE) of the moving bar. Gray broken lines indicate time when LE and TE first enter ON and OFF dendritic field. Triangles, onset and offset of the moving bar stimulation. (Middle and bottom) Raster and rate plots, respectively, of the spikes in upper trace are shown.
(C) Responses of an rBSGC to a light spot (75 μm in radius) of various flash durations, showing that the duration of spike suppression at either light onset or offset was largely independent of the flash duration. Note that the reappearing spikes following the transient ON suppression sometimes had a higher spike rate than that of tonic background spikes (third and fourth traces).
(D) Example responses of ten different rBSGCs (all of which received blue light-evoked glycinergic input, data not shown) to a light spot (75 μm in radius) show variabilities in tonic background spike rate, duration of spike suppression at light onset and offset, and spike rate during recovery from suppression.
(E) (Top) Morphology of an rBSGC (marked 1 in D), showing a relatively small soma (17-μm diameter) and dendritic field size (<200-μm diameter), as well as a substantial number (7) of recurrent dendrites diving from the OFF (right) to the ON (left) IPL at positions indicated by black circles. Similar morphology also was exhibited by eight other rBSGCs in (D). (Bottom) Morphology of another rBSGC (marked 2 in D) shows relatively larger soma (22-μm diameter) and dendritic field sizes (∼300-μm diameter) and fewer (2) recurrent dendrites diving from the OFF to the ON arbor.
Neuron  , 27-34DOI: ( /j.neuron )
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5. Figure 4
Role of Glycine Release from GACs in the Suppressed-by-Contrast Trigger Feature of UDs
(A) Responses of a UD to flashes of light annuli of various radii (r, defined as the mean of inner and outer radii). All annuli were 50 μm thick except for the smallest one (25 μm thick). Green symbols, size of light annuli relative to that of recorded UD (gray, ∼100 μm in radius).
(B) Peak excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic currents (IPSCs) as a function of annulus radius. Drawings below the graph show dendritic sizes of UDs (gray, 100 μm in radius) and GACs (black, 50 μm in radius). (Inset) Normalized peak EPSCs and IPSCs against annulus radius are shown. Annulus radii for which the normalized EPSCs (ON) and IPSCs (ON and OFF) are significantly different are indicated (∗p < 0.05, paired Student’s t test). Error bars, SEMs.
(C and D) Examples (C) and summary (D) of STRY (1 μM) and SR (50 μM) effects on IPSC responses to center light spots (50–100 μm in radius) are shown.
(E) STRY (1 μM) diminished the spike suppression caused by a center light spot.
(F) Voltage traces of a UD under current clamp show hyperpolarization and suppression of maintained spikes at the onset and offset of a center light spot (100 μm in radius) (upper left); induction of maintained spikes by a depolarizing current step (20 pA) in a cocktail of (in μM) L-AP4 (20), ACET (20), HEX (300), and CNQX (40) (upper right); hyperpolarization and suppression of the induced spikes in response to blue light activation of GACs (lower left), and blockade of the blue light effect by STRY (1 μM) (lower right).
(G) Model of the circuitry and function of segregated glycine-glutamate co-transmission by GAC, showing a glycinergic drive to the suppressed-by-contrast circuit of UDs and a glutamatergic drive to the enhanced-by-contrast circuits of OFF αGC, ON and ON-OFF DSGCs, and a subpopulation of W3 cells.
See also Figure S2.
Neuron  , 27-34DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions