Temporally diverse excitation generates direction selective responses in on and off type retinal starburst amacrine cells

Report Temporally Diverse Excitation Generates DirectionSelective Responses in ON- and OFF-Type Retinal Starburst Amacrine Cells Graphical Abstract Authors James W Fransen, Bart G Borghuis Correspondence bart.borghuis@louisville.edu In Brief Fransen and Borghuis use whole-cell electrophysiology to demonstrate that ON- and OFF-type starburst amacrine cells, key players in retinal direction selectivity, receive temporally diverse excitation across their dendritic arbors Integration of this input in model simulations generates direction selective responses as an emergent receptive field property Highlights d Direct recordings of the motion-evoked responses of OFF starburst amacrine cells d ON and OFF starburst amacrine cells receive spatiotemporally patterned excitation d Spatiotemporally patterned excitation generates directionselective responses d Direction selectivity is an excitation-driven emergent receptive field property Fransen & Borghuis, 2017, Cell Reports 18, 1356–1365 February 7, 2017 ª 2017 The Author(s) http://dx.doi.org/10.1016/j.celrep.2017.01.026 Cell Reports Report Temporally Diverse Excitation Generates Direction-Selective Responses in ON- and OFF-Type Retinal Starburst Amacrine Cells James W Fransen1 and Bart G Borghuis1,2,* 1Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, KY 40202, USA Contact *Correspondence: bart.borghuis@louisville.edu http://dx.doi.org/10.1016/j.celrep.2017.01.026 2Lead SUMMARY The complexity of sensory receptive fields increases from one synaptic stage to the next In many cases, increased complexity is achieved through spatiotemporal interactions between convergent excitatory and inhibitory inputs Here, we present evidence that direction selectivity (DS), a complex emergent receptive field property of retinal starburst amacrine cells (SACs), is generated by spatiotemporal interactions between functionally diverse excitatory inputs Electrophysiological whole-cell recordings from ON and OFF SACs show distinct temporal differences in excitation following proximal compared with distal stimulation of their receptive fields Distal excitation is both faster and more transient, ruling out passive filtering by the dendrites and indicating a task-specific specialization Model simulations demonstrate that this specific organization of excitation generates robust DS responses in SACs, consistent with elementary motion detector models These results indicate that selective integration of spatiotemporally patterned excitation is a computational mechanism for motion detection in the mammalian retina INTRODUCTION Direction-selective ganglion cells (DSGCs) in the mammalian retina were discovered more than 50 years ago (Barlow and Hill, 1963) Despite an apparently compact circuit architecture (DSGCs are located just two [excitation] or three [inhibition] synapses from the photoreceptor input), our understanding of the neural mechanisms underlying direction selectivity (DS) remains incomplete DS originates in the dendrites of starburst amacrine cells (SACs) (Euler et al., 2002; Yoshida et al., 2001) and is transmitted to DSGCs through inhibitory synapses onto the DSGC dendrites (Briggman et al., 2011) Although several mechanisms have been shown to contribute to direction selective responses in the SAC dendrites, none has proven essential Thus, the fundamental computation of visual motion direction remains unresolved SACs comprise ON- and OFF-center subtypes with distinct anatomy and sensitivity to bright and dark moving stimuli, respectively (Figure 1A; Famiglietti, 1983; Peters and Masland, 1996; Taylor and Waăssle, 1995) ON SAC somas are located in the ganglion cell layer They are readily accessible for electrophysiological whole-cell recording, and their response properties have been studied extensively in rabbit (Dmitriev et al., 2012; Euler et al., 2002; Fried et al., 2002; Gavrikov et al., 2006; Hausselt et al., 2007; Lee et al., 2010; Lee and Zhou, 2006; Taylor and Waăssle, 1995; Zhou and Lee, 2008) and mouse (Ozaita et al., 2004; Pei et al., 2015; Vlasits et al., 2014, 2016; Wei et al., 2011) ON SACs preferentially respond to radial motion away from their soma (centrifugal) versus toward their soma (centripetal) (Euler et al., 2002; Hausselt et al., 2007; Lee and Zhou, 2006; Oesch and Taylor, 2010) This direction-dependent response asymmetry forms the basis for direction selective release of GABA from synaptic sites located in their distal dendrites Few studies have targeted OFF SACs for whole-cell recording (Vlasits et al., 2014) due to the less accessible location of their somas in the retina’s inner nuclear layer (Famiglietti, 1983) While one study showed motion-evoked Ca2+ responses in OFF SACs (Ding et al., 2016), no electrophysiological recordings of motion-evoked responses have been reported, and how OFF and ON SAC response properties compare is not known Several mechanisms have been demonstrated to contribute to ON SAC DS, and several models exist (Taylor and Smith, 2012) Reciprocal SAC-SAC GABAergic inhibition enhances DS (Lee and Zhou, 2006; Zhou and Lee, 2008), but SAC DS persists with GABA receptors blocked (Hausselt et al., 2007; Oesch and Taylor, 2010) and in the absence of surround stimulation required to evoke GABAergic surround inhibition (Hausselt et al., 2007); thus, inhibition is not strictly required An excitation-driven, dendrite autonomous model in which excitatory input interacts with cell-intrinsic properties recapitulates ON SAC DS (Hausselt et al., 2007) This model critically depends on a dendritic voltage gradient that is maintained by low membrane resistance at the soma combined with tonic excitation caused by tonic glutamate release from presynaptic ON bipolar cells While ON bipolar cells presynaptic to ON SACs tonically release glutamate (Borghuis et al., 2013), ON SAC DS in rabbit persist in the absence of tonic excitation following 1356 Cell Reports 18, 1356–1365, February 7, 2017 ª 2017 The Author(s) This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) A Figure OFF and ON SAC DS Shows Broad Temporal Tuning and Persists in the Absence of GABAergic Inhibition B C D E F pharmacological perturbation (Oesch and Taylor, 2010) Moreover, OFF-type bipolar cells presynaptic to OFF SACs not release glutamate tonically (Borghuis et al., 2013), indicating that OFF SACs may use a mechanism for DS that is different from that proposed for ON SACs (Hausselt et al., 2007) Recently, an alternative mechanism for DS, ‘‘space-time wiring,’’ was proposed based on connectomic reconstruction of the OFF and ON SAC presynaptic circuits (Greene et al., 2016; Kim et al., 2014) Both OFF and ON SACs showed cell-type-specific spatial organization of presynaptic bipolar cell contacts across the dendritic arbor In OFF SACs, type cone bipolar cells preferentially contact proximal dendrites, whereas type 3a cone bipolar cells preferentially contact intermediate or distal dendrites (further (A) Diagram showing the spatial arrangement of synaptic connections between OFF and ON starburst amacrine cells (SACs) and a direction-selective ganglion cell (DSGC) (B) Left: two-photon fluorescence images of tdTomato-expressing OFF SACs (magenta) in a whole-mount ChAT-Cre::Ai9 transgenic mouse retina before (top) and after whole-cell recording (bottom) Dye-fill (green) identifies the recorded cell (arrow) Right: illustration of the radial motion stimulus; a schematic of SAC morphology (green) was added for reference (C) Electrophysiological whole-cell recordings of the membrane voltage, excitatory, and inhibitory current responses of OFF and ON SACs during outward (red) and inward (black) motion stimulation (250 mm/s) (D) Top: average slope of the response onset for outward (red) and inward motion (black) for OFF (solid symbols) and ON SACs (open symbols) Hz modulation; sine wave slope was added for reference (dashed, gray; *p % 0.031) Bottom: average response amplitude (peak-trough) for all recorded SACs (n = 11 ON, n = OFF; *p % 0.013) Error bars indicate ±1 SEM (E) Example traces of the membrane voltage response during outward (red) and inward motion stimulation (black) at different stimulus velocities (x axis scaled to one period) Responses to outward and inward motion were normalized to have equal amplitude to emphasize the difference in response slope Dashed lines indicate linear fits to quantify slope of the response onset (F) Quantification of responses to outward and inward motion stimulation across cells (includes data shown in E; amplitude and slope were calculated prior to normalization) Shaded area represents ±1 SEM See also Figure S2 referred to as ‘‘distal’’; Vlasits et al., 2016) In ON SACs, type bipolar cells predominantly synapse proximally, and each of the three type bipolar cells synapse distally A subsequent connectomic study with superior methods (Ding et al., 2016) extended these results by demonstrating excitatory input from one additional OFF bipolar cell type (type 1) onto OFF SACs but agreed with the earlier claim that predominant bipolar cell type input differs between proximal versus distal regions of the SAC dendritic arbor The temporal kinetics of bipolar cell calcium responses (Baden et al., 2013) and glutamate release (Borghuis et al., 2013) at the anatomical levels where proximal and distal-connecting bipolar cell types stratify their axonal arbors suggests that they express sluggish and sustained and fast and transient responses to light stimulation, respectively Elementary motion detector models (Barlow and Levick, 1965; Hassenstein and Reichardt, 1956) predict that this difference in proximal versus distal excitation Cell Reports 18, 1356–1365, February 7, 2017 1357 is sufficient for generating DS in ON and OFF SACs However, the space-time wiring model for DS has remained controversial, and one electrophysiological study of mouse ON SACs found no support for it (Stincic et al., 2016) A direct test in OFF SACs is lacking To address this, we developed methods for efficient whole-cell recording of both ON and OFF SACs in the intact (whole-mount) mouse retina in vitro and applied these methods to study their visually evoked excitatory response properties RESULTS Radial Motion Stimuli Evoke DS Excitatory Responses in Both OFF and ON SACs We first established that mouse OFF SACs, like ON SACs, generate robust DS responses We made loose-patch spike recordings from postsynaptic, fluorescently labeled ON-OFF DSGCs (ooDSGCs; Figures S1A and S1B) and isolated the OFF SAC contribution to DS by eliminating light-evoked responses in the ON SAC presynaptic pathway (L-AP4, 20 mM) ON-pathway block eliminated ooDSGC responses to light increments, as expected, but left intact DS responses to light decrements (Figure S1B) This is consistent with recordings in rabbit (Kittila and Massey, 1995) and indicates robust DS responses in OFF SACs For direct measurements of motion-evoked responses in ON and OFF SACs, we obtained whole-cell recordings of fluorescently labeled SACs in whole-mount retinas of transgenic ChAT-Cre::Ai9 mice (Figure 1B) Recorded in current-clamp mode, the membrane voltage response of both cell types was direction selective, characterized by an increased response amplitude during outward compared with inward radial motion (Figure 1C) Responses to outward and inward radial motion also showed direction-selective harmonic distortion (skew; Figure 1C), consistent with harmonic frequency components (Figures S2A and S2B), as previously reported for ON SACs (Hausselt et al., 2007) Slope of response onset and peak-to-trough amplitude directly impact the activation of voltage-gated ion channels that drive DS synaptic release, particularly in the presence of leak currents Thus, we use these two physiologically relevant parameters, rather than Fourier amplitude and phase, to quantify the motion-evoked responses of ON and OFF SACs Next, we tested if the DS membrane voltage response reflected DS synaptic input, either excitatory or inhibitory, by recording synaptic currents during voltage clamp near the reversal potential for chloride (ECl, 67 mV) and cations (Ecation, mV), respectively We found significant DS in the slope and amplitude of excitatory postsynaptic potentials (EPSPs; Figures 1C and 1D; paired t test ON SACs, slope: p < 0.0001, amplitude: p = 0.001, n = 9; OFF SACs, slope: p = 0.009, amplitude: p = 0.035, n = 8) Inhibitory postsynaptic potentials (IPSPs; Figures 1C and 1D) were generally larger in OFF SACs than in ON SACs, but in neither cell type was the slope or amplitude of inhibition significantly different for outward versus inward motion (Figure 1D; ON SACs, slope: p = 0.58, amplitude: p = 0.21, n = 8; OFF SACs, slope: p = 0.84, amplitude: p = 0.84, n = 5) These data show that direction-selective excitation contributes to both ON and OFF SAC DS 1358 Cell Reports 18, 1356–1365, February 7, 2017 OFF and ON SACs Express Broad Velocity Tuning ooDSGCs and ON SACs show broad DS velocity tuning (He and Levick, 2000; Sivyer et al., 2010) To test if OFF and ON SACs exhibit similarly broad velocity tuning, we compared membrane voltage responses of OFF and ON SACs during motion stimulation across an 1 decade velocity range (62.5–500 mm/s) OFF and ON SAC responses were distinctly DS, both in slope and amplitude of the response at all but the lowest velocities (Figures 1E, 1F, and S2D), demonstrating broad velocity tuning of the mechanism underlying direction-selective responses in both cell types ON SAC DS is enhanced by reciprocal GABAa receptor-mediated (Zhou and Fain, 1995) inhibition between neighboring ON SACs (Lee and Zhou, 2006) but persists without it (Hausselt et al., 2007; Oesch and Taylor, 2010) To test if excitation alone is sufficient for DS in OFF SACs, we compared responses to inward versus outward radial motion under control conditions and with GABAa receptors blocked (gabazine; SR95531, 10 mM) Gabazine eliminated stimulus-evoked inhibitory currents and caused tonic depolarization as expected, but it did not eliminate directionselective differences in slope and amplitude of OFF and ON SAC responses to inward versus outward motion (Figures S2E and S2F) Thus, neither OFF nor ON SAC DS requires GABAergic inhibition Because glycinergic inhibition does not contribute significantly to ON and OFF SAC DS responses, as evidenced by near-complete loss of inhibition following GABAa receptor block, we conclude that DS is a SAC emergent receptive field property that can be generated through excitatory synaptic input alone SAC Excitation Is Faster and More Transient following Distal Compared with Proximal Receptive Field Stimulation If proximal and distal SAC dendrites receive excitatory input from temporally distinct bipolar cell populations, as indicated by connectomic reconstruction (Ding et al., 2016; Greene et al., 2016; Kim et al., 2014), then the time course of excitation at different distances from the SAC soma should differ To test this, we presented circular white noise (concentric rings of increasing diameter and fixed width, centered on the recorded cell’s soma) and recorded the excitatory response during binary white-noise luminance-contrast modulation of each ring (38 Hz; 100% binary contrast; Figure 2A) Reverse correlation of the excitatory current response recorded at the soma and the luminance history of each ring gave a linear approximation (impulse response, or ‘‘filter’’) of the time course of excitation at each ring’s eccentricity (Figure 2B) Simulations showed that a linear-nonlinear (LN) model (Carandini et al., 2005; Chichilnisky, 2001) constructed with the measured linear filters and a single static nonlinearity after spatial summation captured most of the response variance (Figures S3A–S3C) We found only modest differences between the static nonlinearities for each eccentricity when computed separately, indicating that the linear filters were representative of the time course of excitation at their respective eccentricities (Figures S3D and S3E) Since the static nonlinearity scales the linear response without influencing the shape of the temporal filter, it is not considered further here Excitatory receptive field centers extended 100 mm from the soma (Figure 2C), consistent with previous reports (Ding et al., 2016; Greene et al., 2016; Kim et al., 2014; Vlasits et al., 2016) A Figure SAC Excitatory Responses Are Faster and More Transient following Distal Compared with Proximal Receptive Field Stimulation Stimulus - ring #5 white 300 μm 15 μm EPSC, Vhold = -69 mV −100 −200 0.5 s B EPSC impulse response OFF SAC C ON SAC Eccentricity (μm) Vhold = -69 mV - 15 30 - 45 100 50 OFF SAC 150 100 50 ON SAC −50 45 - 60 60 - 75 75 - 90 100 150 120 110 100 90 80 90 - 105 100 f.u 50 Eccentricity (μm) D Time−to−peak (ms) Response amplitude (filter units) 15 - 30 Peak amplitude (filter units) 50 μm Current (pA) black E 50 100 Eccentricity (μm) (A) Left: example frame of the radial white noise stimulus All rings are equal width (15 mm) The stimulus shown represents one time point, with some rings black and others white according to each ring’s unique binary white noise stimulus sequence Schematic SAC morphology is shown for scale (magenta) Top right: example luminance time course for one stimulus ring Bottom right: excitatory current response of an OFF SAC recorded during radial white noise stimulation (B) Impulse responses (filters) of the excitatory synaptic input evoked by white noise stimulation at each eccentricity (Vhold = 69 mV) Traces represent averages of OFF SACs and 18 ON SACs ±1 SEM shown in gray Vertical dashed line illustrates longer time to peak of proximal OFF SAC filters Arrows indicate time to peak of proximal (solid arrows) and distal input (open arrows) Arrowheads indicate monophasic response profile of proximal (solid) versus biphasic response profile of distal input (open) Gray lines represent ±1 SEM (C) Amplitude of excitatory filters of OFF and ON SACs (D) Time to peak of excitatory filters of OFF and ON SACs Dashed lines represent linear fits Arrows indicate time to peak of proximal (solid arrows) and distal input (open arrows) (E) Biphasic index (BI), defined as the relative amplitude of the excitatory peak and trough (BI = At / At + Ap) of excitatory filters for OFF and ON SACs Arrowheads indicate monophasic response profile of proximal (solid) versus biphasic response profile of distal input (open) In (C)–(E), the shaded area represents ±1 SEM See also Figures S3 and S4 Biphasic index 105 - 120 120 - 135 135 - 150 250 Time (ms) 0.5 250 Time (ms) The most eccentric filters (e.g., 135–150 mm) revealed an inhibitory surround consistent with presynaptic inhibition of the bipolar cell axon terminals Most significant in the context of our study, which aimed to test for spatiotemporal differences in SAC excitation, time to peak of SAC excitatory filters showed a distinct temporal gradient with eccentricity in OFF SACs; this was also observed in ON SACs but was less pronounced (OFF SAC, 26 ± 1.1 ms/100 mm; ON SAC, 8.0 ± 1.2 ms/100 mm; Figure 2D) Independent measurements using conventional annulus stimuli gave similar results (Figures S4A and S4B) Here, too, differences in proximal and distal excitation were less pronounced in ON SACs than in OFF SACs In both ON and OFF SACs, filters representing distal excitation were more biphasic than filters representing proximal 0 excitation (25 mm versus 85 mm: biphasic index OFF SAC, 0.05 ± 0.05 versus 0.40 ± 0.05 [n = 9, p < 0.0001]; ON 50 100 SAC, 0.16 ± 0.04 versus 0.55 ± 0.06 Eccentricity (μm) [n = 17, p < 0.0001, paired t test]) (Figure 2E) This is consistent with the apparent transient and sustained response kinetics of bipolar cell glutamate release in different synaptic layers (Borghuis et al., 2013) Model Simulations Demonstrate Sufficiency of an Excitatory Mechanism for SAC DS To test if the observed spatiotemporal pattern of excitation in ON and OFF SACs is sufficient for DS, we constructed a linear model based on the cells’ average recorded filters (Figures 2B and 3A) Convolving these model ON and OFF SACs with a radial motion stimulus gave DS responses (Figures 3A and 3B) similar those recorded in the SACs (Figures 1C and 1D) An alternative model with spatially uniform temporal kinetics did not give DS responses (amplitude asymmetry in OFF SACs: 0.9% of control, ON SACs: 3.4% of control; Figure 3C) Thus, the observed DS Cell Reports 18, 1356–1365, February 7, 2017 1359 A B D C E G F H Figure Spatiotemporal Excitatory Interactions Generate an Outward-Motion-Preferring Response Asymmetry in OFF and ON SACs (A) Top: model spatiotemporal receptive field constructed from measured filters (Figure 2B) at their respective eccentricities (blue through red; subset of filters shown on right) Bottom left: space-time plot of the simulated receptive field (y = 0; blue, increasing excitatory current; red, decreasing excitatory current) Bottom right: space-time plot of the stimulus (y = 0) (B) Convolving the excitatory receptive field model with the motion stimulus generated asymmetric responses with increased amplitude and faster onset for outward compared with inward motion (C) Top: assigning the same filter (ring #5) to all eccentricities generated symmetrical motion-evoked responses Reversing filter sequence (surround becomes center, and vice versa) reversed the asymmetry of motion responses, causing a preference for inward motion (D) Simulated responses following specific manipulations of the model, as indicated (see text for details) (E) Summary of the amplitude and slope of simulated responses to outward (red circles) and inward (black circles) radial motion in a model OFF (left) and ON SAC (right) for the configurations shown in (B)–(D) (F) Direction selectivity indices (DSIs) calculated from the responses shown in (D) (G) Elementary motion detector model A correlator integrates excitatory input from two receptive fields (RF1 and RF2) separated by distance Dx In the original model (Hassenstein and Reichardt, 1956), low-pass filtering by RF1 generates a delay Dt in the transmitted signal that results in direction selectivity at the level of the correlator The detector shown would prefer motion in the RF1/RF2 direction, and responds maximally to stimulus velocity v = Dx /Dt (H) Illustration of hypothetical responses of RF1 and RF2 and their sum at the level of the correlator Asterisk indicates a motion response through synaptic release when the response threshold is crossed (red dashed line) In this example, the integrator uses summation; other integration modes (e.g., multiplication) may enhance detector performance Right: variation on the elementary motion detector model shown on the left, with motion detection based on integration of (legend continued on next page) 1360 Cell Reports 18, 1356–1365, February 7, 2017 (Figure 3B) resulted from interactions between spatially separated excitatory inputs with different temporal kinetics Indeed, reversing filter order (assigning center filters to the surround and surround filters to the center) changed the response from outward preferring to inward preferring (Figure 3C), demonstrating that the specific spatial organization of temporally diverse excitatory input onto the SAC dendrites generates outward-preferring DS Next, we performed three specific model tests to determine how different aspects of the SAC spatiotemporal receptive field contribute to DS To test the contribution of the inhibitory surround, we assigned filter values of zero to the outermost three rings (105–150 mm radius) This manipulation resulted in responses that were nearly identical to control conditions (amplitude asymmetry: 94.7% of control in OFF SACs and 87.3% of control in ON SACs; Figures 3D–3F) Thus, receptive field surround interactions from presynaptic inhibition onto the bipolar cell contribute negligibly to SAC DS To test the contribution of temporal interactions between proximal excitation (which has longer time-to-peak and sustained temporal kinetics) and distal excitation (which has shorter time-to-peak and transient temporal kinetics; Figures 2D, 2E, and 3A), we assigned filter values of zero to the centermost four rings, identified by their monophasic kinetics Losing center input reduced asymmetry in the motion-evoked response amplitude by 50.5% in OFF SACs and 76.8% in ON SACs (Figures 3D– 3F), demonstrating that central excitation contributes strongly to DS To test the contribution of response transience of distal excitation to DS, we eliminated transience by setting all positive filter values to zero This manipulation rendered all filters monophasic (i.e., sustained), while preserving differences in time to peak of proximal versus distal input The rationale is that temporal interactions between sustained and transient input may drive correlation detection even in the absence of a response latency difference (Figures 3G and 3H) Loss of transient excitation reduced asymmetry of the response amplitude by 50.1% in OFF SACs and 83.0% in ON SACs (Figures 3D–3F), demonstrating that distal transient excitation contributed strongly to DS Collectively, these model simulations show that spatiotemporal interactions of measured excitatory responses in both OFF and ON SACs are sufficient for generating DS with a magnitude that exceeds a commonly used threshold for direction selectivity (direction selectivity index [DSI] > 0.3; Figures 3E and 3F) Our data show that the SAC excitatory response to distal versus proximal visual stimulation differs In both ON and OFF SACs, proximal responses are sustained whereas distal responses are transient In addition, in OFF SACs, proximal responses are significantly more delayed than distal responses One interpretation is that the response properties of the presynaptic bipolar cells differ, which is consistent with connectomic reconstructions that show that contacting bipolar cell types segregate across the SAC arbors (Ding et al., 2016; Greene et al., 2016; Kim et al., 2014) However, our measurements cannot rule out that the observed differences in excitation arise postsynaptically, e.g., through differences in glutamate receptor composition of proximal versus distal synapses Selective expression of NMDA receptors in particular, which pass cations in a membrane voltage-dependent manner, could influence the time course of the postsynaptic excitatory response during spatial stimulation To test if NMDA receptors differentially shape the excitatory response at proximal versus distal synapses on the SAC dendrites, we recorded visually evoked SAC responses in the presence and absence of the NMDA receptor selective pharmacological blocker D-AP5 Amplitude and slope of the membrane voltage response remained direction-tuned with NMDA receptors blocked (Figure 4A), demonstrating that NMDA receptors are not required for ON and OFF SAC DS Current recordings further showed that the time course of excitation during circular white noise stimulation remained monophasic (sustained) proximal and biphasic (transient) distal in both ON and OFF SACs (Figures 4B–4D), demonstrating that these properties are NMDA-receptor independent NMDA-receptor block had a negligible effect on the temporal response in ON SACs but significantly shortened the duration of the sustained, proximal excitation in OFF SACs (Figure 4B) This faster shutoff of excitation with NMDA receptors blocked pushed the time to peak of the excitatory response forward in time, thus reducing the temporal difference between proximal and distal time to peak (Figure 4C) NMDA receptor block did not change the biphasic index for excitation at proximal or distal locations (Figure 4D) Bath-applied D-AP5 blocks NMDA receptors throughout the retina Thus, its effect on proximal OFF SAC excitation may be presynaptic (e.g., through specific NMDA-receptor-dependent inhibitory circuitry impinging on the proximal-connecting bipolar cell types) and/or postsynaptic (through preferential NMDA-receptor expression in proximal synapses) We resolved this ambiguity from current voltage (I-V) curves obtained by recording the current response to proximal visual stimulation at different holding potentials (Figure 4E) If synapses on the proximal OFF SAC dendrites express NMDA receptors, then subtraction of the I-V curves recorded in the absence and presence of D-AP5 should reveal a significant J-shaped residual current, characteristic of the NMDA receptor response (Dingledine et al., 1999) Indeed, OFF SACs showed a J-shaped difference current in control minus D-AP5 conditions, indicating that OFF SACs express NMDA receptors at proximal synapses (Figure 4F) ON SACs, on the other hand, did not show a J-shaped residual current during proximal stimulation, consistent with the observation that D-AP5 does not affect the time course of excitation in these cells (Figures 4B and 4C) D-AP5 broadly increased the current amplitude in ON SACs, indicating that NMDA receptors control the presynaptic bipolar cell response Thus, NMDA receptors influence the OFF SAC response by slowing its proximal excitatory spatially offset transient and sustained RF inputs Because the transient input may combine with the sustained input to cross threshold at a range of time points following onset of the sustained response, this detector would exhibit broad temporal tuning (see Discussion for details) To better illustrate the key features of the proposed model, in this schematic, the magnitude of differences in temporal response kinetic (transient versus sustained) has been increased relative to what was measured Cell Reports 18, 1356–1365, February 7, 2017 1361 A B C E D F Figure NMDA Receptors Slow Proximal Excitation in OFF SACs (A) Membrane voltage response (whole-cell current clamp) of OFF and ON SACs during inward (black) and outward (blue) radial motion in the absence (top) and presence (bottom) of the NMDA receptor blocker D-AP5 (50 mM) Panel shows single-cell examples representative of the recorded population (OFF n = 5; ON n = 6) Response slope and amplitude did not change significantly following NMDA receptor block (all p > 0.10) (B) Impulse responses (filters) of the excitatory synaptic input evoked by proximal, distal, and surround circular white noise stimulation (Vhold = 69 mV) in the absence (black) and presence (red) of D-AP5 (50 mM) Traces represent averages of OFF SACs and ON SACs ±1 SEM shown in gray (C) Response time-to-peak of the excitatory filters measured with circular white noise under control conditions (black) and with NMDA receptors blocked (red; data partially shown in B) Asterisks indicate significant differences (p < 0.05) (D) Biphasic index of the excitatory filters measured with circular white noise under control conditions (black) and with NMDA receptors blocked (red; data partially shown in B) Biphasic indexes were always greater at distal compared with proximal locations and did not change significantly following NMDA receptor block (n.s.) Error bars represent ±1 SEM (E) Current response of an OFF SAC at different holding potentials (bottom) during visual stimulation of the proximal receptive field (schematic in inset, top left; time course shown below traces) (legend continued on next page) 1362 Cell Reports 18, 1356–1365, February 7, 2017 input, but they not mediate the sustained versus transient difference in temporal kinetics of excitation at proximal versus distal synaptic sites DISCUSSION Targeted whole-cell recordings from ON and OFF SACs show that excitation in ON and OFF SAC distal versus proximal dendrites is temporally diverse; excitation following distal stimulation is both faster and more transient than that following proximal stimulation Model simulations show that this specific functional architecture generates direction-selective responses with a preference for outward motion—a hallmark property of ON and OFF SACs Because this mechanism acts at the level of the excitatory input, it appears to generate the first instance of direction selectivity in the SAC We conclude that selective integration of transient and sustained excitation is a fundamental mechanism for SAC direction selectivity Pharmacological perturbation showed that NMDA receptors prolong the proximal excitatory response and increase the temporal delay of proximal, but not distal, excitation in OFF SACs Thus, selective NMDA receptor expression is a cell-intrinsic mechanism for diversifying proximal versus distal temporal responses in OFF SACs Since temporal delay of excitation impacts DS, this mechanism likely contributes to these cells’ velocity tuning Importantly, excitation in ON and OFF SACs remained sustained proximal and transient distal following NMDA receptor block A parsimonious explanation, consistent with connectomic data (Ding et al., 2016; Greene et al., 2016; Kim et al., 2014) is that this difference in excitation reflects synaptic input from functionally distinct bipolar cell types across the dendritic arbor The circuit model supported by our results bears a striking resemblance to the visual motion detection circuits in Drosophila (Behnia et al., 2014; Leonhardt et al., 2016; Serbe et al., 2016; Tuthill and Borghuis, 2016) A similar synaptic architecture, characterized by dendritic domains with functionally distinct synaptic input, has been reported for mouse hippocampal pyramidal cells, where it serves as a mechanism for coincidence detection in synaptic plasticity and learning (Stuart and Spruston, 2015) Our work provides functional evidence for an excitation-based mechanism for SAC DS but does not rule out additional contributions from previously described mechanisms, including dendrite autonomous computation in ON SACs (Hausselt et al., 2007) and reciprocal SAC-SAC inhibition (Lee and Zhou, 2006) Our results apparently contradict those of a recent study that found no difference in the time course of excitation in ON SACs measured with flashed annuli (Stincic et al., 2016) One possible explanation is that the concentric white noise stimulus used in our study provides a more sensitive measure of the time-course of excitation during dynamic visual stimulation; another is that flashed annuli may evoke nonlinear postsynaptic response properties not activated by spatially dense white noise, which stimulates at a net-zero mean level across the SAC dendritic arbor Consistent with this explanation, ON SACs in our study showed substantially less temporal diversity in proximal versus distal excitatory conductance when stimulated with annuli compared with circular white noise (Figure S4B) We used linear systems analysis to compute the time course of excitation at different eccentricities The filter characteristics are linear approximations of the full excitatory response, which likely includes non-linearities The use of spatiotemporal white noise stimuli helped resolve response dynamics with high temporal resolution while minimizing nonlinearities from contrast and luminance gain control Contributions from additional non-linear mechanisms such as spatiotemporal interactions (motion detection) were excluded by definition due to the analysis method While spatiotemporally correlated stimuli (e.g., texture motion) would evoke the specific nonlinear response properties that drive DS synaptic release, our method was optimized for quantitative assessment of the time course of local excitation to test for predicted temporal differences in the excitatory response selective input from functionally diverse bipolar cell pathways impinging on proximal and distal dendrites (Greene et al., 2016; Kim et al., 2014) The magnitude of the asymmetry of ON and OFF SAC responses to outward compared with inward motion is small (Figure 1) compared with the robust directional asymmetry of GABA release evident in postsynaptic DSGCs (e.g., Park et al., 2014) In the current working model, the relatively small, initial asymmetry in the SAC membrane voltage is amplified nonlinearly by various mechanisms, including voltage-gated channels and reciprocal inhibition, and thresholded to generate strongly DS synaptic release DS of ooDSGC responses extends over a wide range of stimulus velocities This broad temporal tuning is present already at the level of the GABAergic inhibitory input from SACs shown here (Figure 1F) and in rabbit (Sivyer et al., 2010) Broad temporal tuning is inconsistent with a strictly spatiotemporal delay-based mechanism for motion detection, which would give narrow temporal tuning with peak sensitivity defined by the ratio of spatial extent of the excitatory input (Dx) and relative delay between proximal and distal input (Dt; Figures 3G and 3H) Our data show that two independent components of OFF and ON SAC excitatory responses contribute to coincidence detection underlying DS (Figure 3H): (1) a relative delay (Dt) between proximal and distal excitation, which was substantial in OFF SACs (26 ms/100 mm) but minor in ON SACs (8 ms/100 mm; Figure 2D); and (2) a difference in temporal kinetics (transient versus sustained) between proximal and distal excitation, which was observed in both OFF and ON SACs (Figures 2B and 2E) Because the transient response can trigger SAC synaptic release as long as the sustained response persists (Figure 3H), transient-sustained integration provides a mechanistic basis for the well-established but incompletely understood broad temporal tuning of ooDSGCs (Sivyer et al., 2010) (F) Current-voltage relation of the response to proximal visual stimulation Left: amplitude of the initial, transient response component (‘‘T’’ in E, average of latter three stimulus cycles) Right: amplitude of the sustained response component (‘‘S’’ in E) Magenta curve shows the difference between the current response under control (black) and D-AP5 conditions (red) Asterisks indicate significant differences (p < 0.05) The significant J-shaped difference curve of the sustained response component in OFF SACs (top right, arrowhead) indicates that in OFF SACs, NMDA receptors contribute to the excitatory current at proximal synapses In (B), (C), and (F), the shaded area represents ±1 SEM Cell Reports 18, 1356–1365, February 7, 2017 1363 EXPERIMENTAL PROCEDURES Animals All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Louisville and were in compliance with National Institutes of Health guidelines Mice of either sex, maintained on C57BL/6J backgrounds, were studied between 1.5 and months of age Data were obtained from transgenic ChAT-IRES-Cre mice (Jackson Laboratory stock #006410) crossed with the Ai9 tdTomato ROSA26 reporter line (Jackson Laboratory stock #007905) ooDSGC recordings of Figure S1 used an Thy1iGluSnFR line (Looger lab, Janelia Research Campus) with low-level expression of iGluSnFR in various cell types, including ooDSCGs Electrophysiological Recording Whole-cell electrophysiological recordings were obtained from the ventral, whole-mount mouse retina in vitro as described previously (Borghuis et al., 2013) See Supplemental Experimental Procedures for details Visual Stimulation Visual stimuli were generated with an Apple G4 computer and custom C-language software Stimuli were displayed using a DLP video projector (HP AX325AA; Hewlett-Packard), with the image projected onto the photoreceptor layer using the microscope condenser Stimuli comprised inward and outward drifting radial square waves (Figure 1B), flashed annuli, and concentric binary white noise (Figure 2A) Data Analysis and Model Simulations Data are presented as mean ± SEM unless stated otherwise Statistical significance was assessed using Student’s t test as indicated Model analysis was performed with numerical simulations using custom algorithms in MATLAB (MathWorks) SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and four figures and can be found with this article online at http://dx.doi.org/ 10.1016/j.celrep.2017.01.026 AUTHOR CONTRIBUTIONS J.W.F and B.G.B conceived of the study, designed the experiments, and collected and analyzed the data B.G.B developed the model analysis and simulations B.G.B wrote the manuscript Both authors read and approved the final version of the manuscript ACKNOWLEDGMENTS We thank Dr J Demb for helpful comments on the model analysis and simulations This work was supported in part by grants from the E Matilda Ziegler Foundation for the Blind and the University of Louisville School of Medicine Received: April 25, 2016 Revised: December 5, 2016 Accepted: January 11, 2017 Published: February 7, 2017 Behnia, R., Clark, D.A., Carter, A.G., Clandinin, T.R., and Desplan, C (2014) Processing properties of ON and OFF pathways for Drosophila motion detection Nature 512, 427–430 Borghuis, B.G., Marvin, J.S., Looger, L.L., and Demb, J.B (2013) Two-photon imaging of nonlinear glutamate release dynamics at bipolar cell synapses in the mouse retina J Neurosci 33, 10972–10985 Briggman, K.L., Helmstaedter, M., and Denk, W (2011) Wiring specificity in the direction-selectivity circuit of the retina Nature 471, 183–188 Carandini, M., Demb, J.B., Mante, V., Tolhurst, D.J., Dan, Y., Olshausen, B.A., Gallant, J.L., and Rust, N.C (2005) Do we know what the early visual system does? 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DISCUSSION Targeted whole-cell recordings from ON and OFF SACs show that excitation in ON and OFF SAC distal versus proximal dendrites is temporally diverse; excitation following distal stimulation... temporal responses in OFF SACs Since temporal delay of excitation impacts DS, this mechanism likely contributes to these cells? ?? velocity tuning Importantly, excitation in ON and OFF SACs remained