Article

A Central Role for Mixed Acetylcholine/GABA

Transmission in Direction Coding in the Retina
Highlights Authors
d ACh initiates responses to motion in natural scenes/low- Santhosh Sethuramanujam,
contrast stimuli Amanda J. McLaughlin,
Geoffery deRosenroll, Alex Hoggarth,
d Non-linear ACh-NMDA interactions amplify low-contrast David J. Schwab,
responses Gautam B. Awatramani
d Spatiotemporally distinct profiles of GABA and ACh underlie
low-contrast DS
Correspondence
gautam@uvic.ca
d Optogenetic stimulation of the SAC network in isolation
drives direction selectivity In Brief
Sethuramanujam et al. demonstrate that
the network of retinal GABAergic/
cholinergic starburst amacrine cells can
differentially transmit excitatory and
inhibitory signals and stimulate
directional responses in downstream
ganglion cells. These results demonstrate
for the first time a physiological role for
mixed transmission in the directional-
selective retinal circuit.

Sethuramanujam et al., 2016, Neuron 90, 1243–1256

June 15, 2016 ª 2016 Elsevier Inc.
http://dx.doi.org/10.1016/j.neuron.2016.04.041
Neuron
Article
A Central Role for Mixed Acetylcholine/GABA
Transmission in Direction Coding in the Retina
Santhosh Sethuramanujam,1 Amanda J. McLaughlin,1 Geoffery deRosenroll,1 Alex Hoggarth,1 David J. Schwab,2
and Gautam B. Awatramani1,*
1Department of Biology, University of Victoria, Victoria, BC V8W 3N5, Canada
2Department of Physics and Astronomy, Northwestern University, Evanston, IL 60208, USA
*Correspondence: gautam@uvic.ca
http://dx.doi.org/10.1016/j.neuron.2016.04.041
SUMMARY
A surprisingly large number of neurons throughout
the brain are endowed with the ability to co-release
both a fast excitatory and inhibitory transmitter.
The computational benefits of dual transmitter
release, however, remain poorly understood. Here,
we address the role of co-transmission of acetylcho-
line (ACh) and GABA from starburst amacrine cells
(SACs) to direction-selective ganglion cells (DSGCs).
Using a combination of pharmacology, optoge-
netics, and linear regression methods, we estimated
the spatiotemporal profiles of GABA, ACh, and gluta-
mate receptor-mediated synaptic activity in DSGCs
evoked by motion. We found that ACh initiates re-
sponses to motion in natural scenes or under low-
contrast conditions. In contrast, classical glutama-
tergic pathways play a secondary role, amplifying
cholinergic responses via NMDA receptor activation.
Furthermore, under these conditions, the network
of SACs differentially transmits ACh and GABA to
DSGCs in a directional manner. Thus, mixed trans-
mission plays a central role in shaping directional
responses of DSGCs.
INTRODUCTION
In the mammalian retina, direction-selective (DS) responses are
first observed in the radiating dendrites of GABAergic/cholin-
ergic starburst amacrine cells (SACs; Euler et al., 2002; Yonehara
et al., 2013). SAC dendrites transmit information to postsynaptic
direction-selective ganglion cells (DSGCs) and endow them with
the ability to code direction (Vlasits et al., 2014; Yoshida et al.,
2001). A specific asymmetric dendro-dendritic wiring connecting
SACs to DSGCs allows SACs to mediate a ‘‘null’’ GABAergic in-
hibition (Briggman et al., 2011; reviewed by Borst and Euler,
2011; Demb, 2007; Vaney et al., 2012). DSGCs are thought to
compute direction by integrating asymmetric SAC inhibition
with a non-directional bipolar cell glutamatergic excitation
(Yonehara et al., 2013; Park et al., 2014; Figure 1A). In addition,
SACs also release acetylcholine (ACh) (Massey and Neal,
1979; Massey and Redburn, 1983), but the role of cholinergic
excitation is not well understood.
Elegant optical stimulation and paired-recording techniques
have revealed that nicotinic ACh receptor (nAChR)-mediated
inputs to DSGCs are large and reliable and exhibit millisecond-
scale kinetics (Lee et al., 2010; Yonehara et al., 2011). Pharma-
cological studies investigating the role of ACh in mediating the
DSGC’s light response, however, have provided inconsistent re-
sults. Some studies suggest that ACh may contribute up to 50%
of the DSGC’s light response (Ariel and Daw, 1982; Grzywacz
et al., 1998; Kittila and Massey, 1997; Lipin et al., 2015; Weng
et al., 2005), while others indicate a weak or negligible contribu-
tion of these receptors (Fried et al., 2005; Lee et al., 2010; Park
et al., 2014). Regardless of the extent of nAChR contribution,
blocking these receptors does not appear to compromise the
ability of DSGCs to compute direction. To better understand
the role of nAChRs in mediating directional selectivity of DSGCs,
here we used pharmacology and a complementary linear regres-
sion method (Manookin et al., 2010; Taylor and Vaney, 2002) to
examine the contribution of nAChRs during physiologically intact
conditions. Regression analysis was based on the distinct bio-
physical signatures of the individual synaptic receptors medi-
ating the DSGC’s light response and provided for the first
time a simultaneous estimate of the spatiotemporal profiles of
GABA, ACh, and glutamate signaling at dendrites of DSGCs.
Interestingly, we found that during natural/low-contrast stimu-
lation, SACs serve as the primary source for both excitation and
inhibition to DSGCs. Under these conditions, glutamatergic exci-
tation played only a secondary role in amplifying the response to
cholinergic input, acting mainly on NMDA receptors (NMDARs)
and not AMPA receptors (AMPARs). In response to moving stim-
uli, SAC GABA and ACh were differentially transmitted to DSGCs
in a manner that drives directional spiking responses (Figure 1B).
Using optogenetics, we directly demonstrated that the SAC
network alone can trigger directional responses in DSGCs in
the absence of glutamate input. Together, these results lead
us to posit a novel intermediary role for the SAC network in
conveying directional information to ganglion cells, relying pre-
dominantly on mixed cholinergic/GABAergic transmission.
RESULTS
Directional Information to ON-OFF DSGCs Is Routed
through the SAC Network
The first piece of evidence that SACs (rather than bipolar cells)
initiate activity in DSGCs came from examining the effects of
blocking nAChRs on DSGC responses to a natural movie
Neuron 90, 1243–1256, June 15, 2016 ª 2016 Elsevier Inc. 1243

(E) The polar plot illustrates the peak ON spike rate evoked in eight directions (three trials) in control (black) and in the presence of an nAChR antagonist (red). The
DSI is indicated by the solid radial line. Example spike trains for both preferred (0
) and null (180
) motion in control and in the presence of nAChR antagonist are
illustrated around the polar plot.
(F) Similar to (E) but using low-contrast stimuli (C50).
(G) Plot of the average peak ON spike rate for responses evoked in the preferred direction under conditions shown in (E) and (F) (n = 8; mean ± SEM is plotted;
*p < 0. 001). For extended analysis of DS properties and quantification of the OFF spike rates, see Figure S2.
(Figures 1C and 1D). Natural movies were obtained using a CCD
camera mounted on a mouse’s head while it freely roamed in its
cage (Froudarakis et al., 2014). Spike recordings were made
from ON-OFF DSGCs in an isolated whole-mount retina, onto
which this movie was projected. DSGCs responded to certain
moving features in the movie (Figure 1C), and rotating the
movie revealed that indeed these responses were DS (Figure S1,
available online). Interestingly, the application of an nAChR
antagonist (100 mM hexamethonium; HEX; or 50 mM curare)
strongly and reversibly suppressed DSGC responses, indicating
a critical role for cholinergic excitation in generating responses
to motion in the movie (Figures 1C and 1D). This strong suppres-
sion of DSGC responses contrasts with the weaker effect of
nAChR antagonists reported in the literature (Ariel and Daw,
1244 Neuron 90, 1243–1256, June 15, 2016
Figure 1. Cholinergic Inputs Play a Central
Role in Initiating Responses in DSGCs
during Natural/Low-Contrast Stimulation
Conditions
(A) A schematic of the standard model for direction
selectivity in which DSGCs compute direction
by comparing glutamatergic excitation mediated
by bipolar cells (Glu; red) with GABAergic inhibition
from null-side SACs (null SAC shown in green).
Arrow indicates the preferred direction of the
DSGC.
(B) Diagram of the proposed model in which the
SAC network generates direction selectivity. SAC
dendrites are optimally stimulated by centrifugal
motion (soma-to-dendrite; Euler et al., 2002)
and therefore preferred-side SACs (blue) drive
preferred-direction cholinergic excitation, and null-
side SACs (green) provide null-direction GABAergic
inhibition (and also ACh). As only null-side SACs
make strong synaptic connections with DSGCs
(Briggman et al., 2011), ACh transmission may
occur through paracrine mechanisms (dotted blue
arrows), while GABAergic inhibition acts via con-
ventional synaptic mechanisms (solid green arrow).
The secondary contribution of bipolar cells medi-
ated by NMDARs has not been shown for simplicity.
(C) Spike raster plot in response to the repeated
presentation of a 5 s natural movie clip (100 trials,
presented at 0.1 Hz; a uniform gray background
was presented during the intervals). The spiking
in response to natural stimuli were DS (see Fig-
ure S1). The gray trace (top) shows the changes in
light intensity over the DSGC receptive field. Trials
in the presence of an nAChR antagonist (100 mM
HEX) are indicated in red. The instantaneous
spike rates (estimated through convolution with a
Gaussian kernel, s = 25 ms) averaged over seven
trials are shown below for control (black) and in
nAChR antagonists (red).
(D) The average total number of spikes generated/
trial under different conditions (n = 7; mean ± SEM is
plotted; *p < 0. 001) demonstrates that the potent
block by nAChR antagonists is completely reversible.
1982; Fried et al., 2005; Grzywacz et al., 1998; Kittila and Mas-
sey, 1997; Lee et al., 2010; Lipin et al., 2015; Park et al., 2014;
Weng et al., 2005), possibly due to the differences in the visual
stimuli used. One major difference was that the equivalent
contrast of the movie used here (as estimated by Tadmor and
Tolhurst, 2000) is significantly lower than that of high-contrast
moving bars, spots, or gratings that have been typically used
in previous studies. Hence, we next tested the role of nAChRs
in mediating responses to moving spot stimuli of varying
contrast.
Indeed, saturating ‘‘high’’ contrast spots (C100) moving on
a featureless background evoked stronger spiking responses
in DSGCs compared to natural stimulation (the maximum peak
firing rate was 206 ± 14 Hz, n = 8, compared to 95 ± 10 Hz,

n = 7 under natural stimulation conditions). Consistent with
the previous literature, we found that the application of
nAChR antagonists reduced the DSGC peak spike rate to
the leading edge of full contrast moving spots (250 mm in diam-
eter, 1 mm/s; Figures 1E and 1G) but did not compromise its
ability to code direction (Figures 1E and S2). Similar effects
were observed on the trailing edge response (OFF response),
and measuring spike rate or total spike number did not signifi-
cantly impact our results (Figure S2).
When ‘‘low’’ contrast spots that produced 50% of the maximal
response (C50; 110 ± 12 Hz, n = 8) were used, application of
nAChR antagonists completely blocked the spiking response
(Figures 1F and 1G). This suggested that cholinergic inputs play
a central role in mediating DSGC responses in this regime.
Thus, it appears that simple low-contrast stimuli are similar to
natural scenes in their sensitivity to cholinergic antagonists. For
simplicity, in the rest of this study we utilized low-contrast moving
stimuli to probe the properties of the DS circuit. Importantly, low-
contrast stimuli drive a reliable DS response in DSGCs, indicating
that cholinergic/GABAergic inputs are balanced in such a way
that DS responses are maintained, despite being weakly stimu-
lated (Figures 1F and S2).
The Role of nAChRs, AMPARs, and NMDARs in Contrast
Encoding
To better understand the organization of excitatory inputs to
DSGCs, we used voltage-clamp methods to assess the roles
of AMPARs, nAChRs (Figure 2), and NMDARs (Figure 3). Surpris-
ingly, we found that these receptors were not activated in parallel
when stimulus contrast was varied, giving rise to several unex-
pected consequences, as described below.
First, we recorded contrast response functions for AMPAR/
nAChR-mediated excitatory postsynaptic currents (EPSCs) in
DSGCs (Figures 2A and 2B; VHOLD 60 mV, near ECl;
D-AP5 was included in the bath to block NMDARs), which
were fit using the Naka-Rushton equation (Naka and Rushton,
1966; Figure 2B). Applying an nAChR antagonist substantially
Figure 2. AMPAR- and nAChR-Mediated
Synaptic Inputs to DSGCs Are Activated at
Distinct Contrasts
(A) EPSCs measured in a voltage-clamped DSGC
mediated by AMPARs and nAChRs in control
conditions (top) and in the presence of an nAChR
antagonist (AMPAR only; middle). The contribution
of nAChRs (bottom) was estimated by taking the
difference (control  nAChR antagonists).
(B) The peak EPSC amplitude plotted as a function
of contrast for the different conditions shown in
(A) (n = 7 DSGCs; mean ± SEM is plotted) was fit
by the Naka-Rushton equation (see Experimental
Procedures). The vertical dashed line indicates
the contrast required to evoke a half-maximal
response (C50) measured in control conditions.
Responses were normalized to the peak amplitude
measured in control.
(C) A plot of the fractional nAChR (blue)- and
AMPAR-mediated currents (red) as a function of
contrast.
decreased responses to stimuli below C50, but only reduced
maximal responses by 49% ± 9% (n = 7, p < 0.005; Figure 2B).
In addition, the C50 was significantly larger in the presence of an-
tagonists (C50 = 24% ± 3% in control; C50 = 41% ± 3% in the
presence of nAChR antagonist; n = 7; p < 0.005; Figure 2B). This
suggested that the direct AMPAR-mediated inputs from bipolar
cells contribute less to responses evoked by low- compared to
high-contrast stimuli. Conversely, when the contrast response
function of the cholinergic input to DSGCs was estimated by
subtracting the responses measured in nAChR antagonists
from those measured under control conditions (Figure 2A), it
became apparent that cholinergic inputs dominate in the low-
contrast regime, accounting for >80% of the total excitatory in-
puts for contrast levels below C50 (Figure 2C).
Previous studies have shown that voltage-dependent
NMDARs play a major role in mediating the DSGC’s light re-
sponses (Kittila and Massey, 1997; Lee et al., 2010; Poleg-Pol-
sky and Diamond, 2016), raising the possibility that glutamate
signals were mediated by these receptors in the low-contrast
regime. To test this hypothesis, synaptic responses evoked
by low-contrast stimuli (C50) were measured at different holding
potentials in the presence of an nAChR antagonist (Figure 3A).
Indeed, synaptic responses measured under these conditions
were highly non-linear, as depicted in the current-voltage (I-V)
plot (Figure 3C), indicating the involvement of NMDARs. Subse-
quent addition of an NMDAR antagonist decreased the current
amplitudes and linearized the I-V, shifting the reversal potential
close to 40 mV (Figures 3B and 3D). This indicated that
the residual component was made up of weak GABAA recep-
tor (GABAR)- and AMPAR-mediated conductances (GABAR
2.1 nS; AMPAR 0.6 nS). In contrast, the I-V of the currents
blocked by D-AP5 was ‘‘J’’ shaped, characteristic of NMDARs.
The maximal conductance (12.5 nS at +40 mV; Figure 3E) was
large compared to other ganglion cell types (Buldyrev et al.,
2012; Manookin et al., 2010). Despite the large NMDA conduc-
tance, the absence of any spiking activity in the same condi-
tions in which NMDAR currents were measured indicates that
Neuron 90, 1243–1256, June 15, 2016 1245

NMDARs on their own do not drive spikes, i.e., they are
‘‘silent.’’
In the context of nAChR-mediated activity, however, we
found that NMDARs play an important role in amplifying low-
contrast responses (Figures 3F and 3G). At C50, responses
were reduced (20% to 80%; average suppression 55% ± 8%
of control, n = 8; Figure 3F), in contrast to the nAChRs, which
completely blocked responses at this contrast level (Figure 1G).
The remaining spikes observed in the presence of D-AP5 at
C50 were completely blocked by the additional application of
an nAChR antagonist, confirming that they were mediated by
ACh alone (data not shown). The finding that both nAChR
and NMDAR antagonists strongly affect the response to low-
contrast stimuli suggests a significant non-linear interaction
between these receptors. Furthermore, as nAChRs, but not
NMDARs, could independently drive spikes in DSGCs, it sug-
gests that SAC release of ACh initiates responses in DSGCs,
while bipolar cell glutamate augments these responses via
NMDARs. Thus, in DSGCs, nAChRs appear to usurp the role
of functionally absent AMPARs in recruiting NMDARs, consti-
tuting a novel mechanism whereby SACs can control excitation
to DSGCs. However, as the effects mediated by NMDARs
appear secondary to cholinergic excitation, they are unlikely
1246 Neuron 90, 1243–1256, June 15, 2016
Figure 3. NMDAR-Mediated Activity at Low
Contrast Relies on SAC ACh
(A and B) An example of a DSGC in which a half-
maximal contrast response is blocked by HEX
(inset). In the continued presence of HEX, voltage
clamping the same cell reveals the presence of
‘‘silent’’ synaptic conductances (A), which were
partially blocked by the NMDAR antagonist, D-AP5
(50 mM; B).
(C–E) The mean current measured in the boxed
region in (A) and (B) are plotted against holding
potential in (C) and (D), respectively. The I-V rela-
tionship of responses blocked by D-AP5 is shown
in (E), had the characteristics of NMDAR currents,
and could be fit by a sigmoidal function (gray
dashed line). The I-Vs were constructed from re-
sponses averaged over five cells (mean ± SEM is
plotted).
(F) Spiking responses of a DSGC to preferred and
null direction for stimuli eliciting half-maximal (C50)
and maximal (C100) responses, recorded in control
(black) and in the presence of the NMDAR antag-
onist (gray).
(G) Plots of the average peak spike rate measured
in DSGCs evoked by high- and low-contrast stimuli
in control or in the presence of D-AP5 (n = 8;
mean ± SEM is plotted; *p < 0.001).
to be critical for the generation of direc-
tion selectivity (Figure 8).
Relative Contribution of nACh,
GABA, and AMPARs to Preferred
Motion Revealed by Linear
Regression Analysis
So far, our pharmacological analysis sug-
gests that nAChRs mediate the dominant
excitatory inputs at low contrast and are therefore necessary to
generate directional responses observed under these condi-
tions. However, the potential for indirect presynaptic network
(Dmitrieva et al., 2007; Elgueta et al., 2015; Strang et al., 2015)
and/or off-target effects of nAChR antagonists make it difficult
to unambiguously interpret these results. To estimate the relative
contribution of AMPARs, GABARs, and nAChRs to the DSGC’s
synaptic response without application of antagonists for these
receptors (which could disturb the dynamics of the circuit), we
developed a linear regression model. The only antagonist
included, to simplify this experiment, was the well-characterized
NMDAR antagonist D-AP5, which is not critical for DS computa-
tion (Figure 8). The linear regression analysis relies on the distinct
‘‘basis functions’’ of individual receptors, which is described by
their I-V relationships (Figure 4). The composite synaptic light-
evoked currents measured over a range of holding potentials
were modeled as a linear weighted sum of the individual receptor
components (nAChRs, GABARs, and AMPARs).
To measure the voltage-dependent properties of synaptic
nAChRs and GABARs in DSGCs, we used an optogenetic strat-
egy to stimulate SACs and measured monosynaptic responses
in DSGCs in pharmacological isolation. In transgenic mice in
which ChR2 is selectively expressed in SACs (Chat-Cre::Ai32;

SAC-ChR2; Figure S3), cholinergic synaptic currents were
measured in a cocktail of glutamate agonists/antagonists that
is commonly used to block photoreceptor-driven responses
(50 mM D-AP5 + 50 mM L-AP4 + 10 mM CNQX), as well as
the added presence of a GABAA receptor antagonist (10 mM
SR-95531). nAChR-mediated currents were inwardly rectifying
(Haghighi and Cooper, 1998) and were well described by the
Woodhull equation (Figures 4A and 4B; note that spermine was
included in the intracellular solution to maintain rectification; see
Experimental Procedures). Similarly, SAC-ChR2 was used to
stimulate GABAR-mediated synaptic responses in a cocktail
that blocked photoreceptor input as well as nAChRs (100 mM
HEX). Isolated GABAR-mediated currents were found to be
slightly outwardly rectifying (Figures 4C and 4D), and their I-V rela-
tionship was fit by a single exponential equation, as previously
described (Manookin et al., 2010). To measure the AMPAR basis
function, photoreceptor-driven responses were measured in
DSGCs in the presence of NMDAR, GABAR, and nAChR antago-
nists. In contrast to GABAR- and nAChR-mediated responses,
Figure 4. Multiple Types of Synaptic Re-
ceptors Expressed by DSGCs Can Be
Distinguished by Their Voltage-Dependent
Characteristics
(A) A transgenic mouse line with ChR2 expression
specific to SACs (Figure S3) was used to directly
stimulate nAChR currents measured at a series of
holding potentials (indicated on right). The stimulus
was a 100 ms intense blue flash. Photoreceptors
and GABARs were pharmacologically blocked in
this experiment.
(B) Normalized peak current responses were
plotted against holding potential (mean ± SEM is
plotted). The I-V was fit by the Woodhull equation
(solid blue line; see Experimental Procedures).
(C) Similar to (A), SACs expressing ChR2 were
stimulated in conditions that isolated GABAR-
mediated activity.
(D) The GABA I-V was fit by an exponential function
(solid green line).
(E) AMPAR currents were pharmacologically iso-
lated in the presence of a cocktail of NMDAR,
GABAR, and nAChR antagonists. These responses
were driven by photoreceptors.
(F) The AMPA I-V was well described by a linear fit
(solid red line).
AMPAR-mediated currents were linear
(Figures 4E and 4F). Thus, each of the syn-
aptic receptors functionally expressed
by DSGCs has a distinct biophysical
signature, making it feasible to distinguish
their relative contributions to light-evoked
responses under physiological conditions.
To estimate the relative contributions
of the different receptors to the total
response evoked by low-contrast mov-
ing stimuli, preferred direction responses
were measured in DSGCs over a range
of holding potentials (Figure 5A). To
ensure adequate voltage clamp, the superior coding population
of electrically coupled DSGCs (Trenholm et al., 2013a) was
excluded. The composite light-evoked synaptic I-V relationship
was modeled by a weighted linear sum of the basis functions
for AMPARs, GABARs, and nAChRs, which yielded an estimate
of a fractional conductance for each receptor type (Figure 5B).
Interestingly, examining the I-V relationship of the early phase
of the synaptic response revealed inwardly rectifying currents,
indicating they were largely mediated by nAChRs (compare Fig-
ure 5B, top, and Figure 4B). A dominant cholinergic component
in the early phase was consistently observed across six cells
(gACh = 87% ± 6%, gGABA = 5% ± 2%, and gAMPA = 8% ± 5%;
where gACh, gGABA, and gAMPA indicate the conductance for
each receptor at 60 mV; Figure 5C). Although the I-V relation-
ship appeared less rectified during the peak synaptic response,
it remained best estimated by a combination of nAChR and
GABAR components with minimal contribution of AMPARs (Fig-
ures 5B and 5C; gACh = 70% ± 3%, gGABA = 18% ± 3%, and
gAMPA = 12% ± 5%; n = 6).
Neuron 90, 1243–1256, June 15, 2016 1247
Figure 5. Cholinergic SAC Inputs Dominate DSGC Responses to Low-Contrast Preferred Motion under Near-Physiological Conditions
(A) Photoreceptor-mediated synaptic responses in a DSGC to low-contrast preferred motion shown at four different holding potentials (indicated on the right;
mean ± SEM of three trials is plotted in black and gray, respectively). Responses are measured in the presence of an NMDAR antagonist (50 mM AP5). The inset
shows the active circuit components under these conditions.
(B) The I-V relationship computed from the current averaged over the two boxed regions in (A) (open circles, top; closed circles, bottom). The average current was
modeled by a weighted linear sum of the basis functions of each receptor as estimated in Figure 4 (black solid line; see Experimental Procedures). The computed
conductances (gAch, gGABA, and gAMPA) at 60 mV are indicated in each graph. Responses were dominated by nAChRs (blue lines).
(C) The average I-V relationship of the early and peak responses (open circles, top; closed circles, bottom) measured in six cells (mean ± SEM). The average
estimated contributions of the ACh, GABA, and AMPARs are indicated in blue, green, and red, respectively.

To validate the linear regression analysis, we compared the
performance of three different models (ACh + AMPA + GABA,
ACh + GABA, or AMPA + GABA) under different experimental
conditions that systematically biased the contribution of the indi-
vidual receptor components. For low-contrast responses, the
errors associated with the fitting procedure (root mean square
error; RMS error) are similar for the three-component model
and the ACh + GABA model. The AMPA + GABA model, how-
ever, performed relatively poorly. Together, these results confirm
the lack of detectable AMPAR-mediated glutamatergic input
in this regime (Figures 6A–6E). On the other hand, responses
evoked by high-contrast stimuli were best fit with the three-
component model, consistent with the idea that nAChRs,
AMPARs, and GABARs contribute to the synaptic responses
(Figures 6F–6J). Both of the two-receptor component models
performed poorly in the high-contrast regime. Furthermore,
AMPARs and nAChRs appear to contribute equally to the synap-
tic response (Figure 6G), consistent with the pharmacology (Fig-
ure 2B). Finally, when we measured responses to high-contrast
stimuli in the presence of nAChR antagonists, we found
AMPA + GABA were sufficient to model the synaptic responses
(Figures 6K–6O). The ACh component usually observed in con-
trol conditions was completely abolished by nAChR antagonists
(n = 6; p < 0. 001, t test). Under these conditions, adding an ACh
component did not improve the fit, and the ACh + GABA model
provided a poor estimate of the response. Thus, we conclude
that linear regression provides a reliable estimate of the relative
contribution of different synaptic receptors underlying DSGC re-
sponses to preferred direction motion.
Spatiotemporal Profiles of ACh and GABA Underlying
Low-Contrast Direction Selectivity
Previous studies have demonstrated SAC-mediated inhibition,
but not excitation, to be spatially asymmetric (Lee et al., 2010;
Yonehara et al., 2011). However, these studies relied on artificial
optical or electrical methods to stimulate SACs, not capturing
their dynamic behavior in the context of the network. Further-
more, these experiments were performed in conditions in which
network activity was pharmacologically blocked, casting doubt
on the physiological relevance of ACh (Taylor and Smith,
2012). Thus, the temporal dynamics of ACh and GABA evoked
by moving stimuli remain to be investigated. As we found basis
functions for the nAChRs, GABARs, and AMPARs did not signif-
icantly change over the duration of the synaptic response (Fig-
ure S4), we could reiterate the linear regression procedure
throughout the entire light response at 1 ms intervals (Figure 7B).
This provided a direct estimate of the temporal dynamics of
nAChR-, GABAR-, and AMPAR-mediated synaptic responses
evoked by moving stimuli (Figure 7A; see Figure S5 for compar-
isons of data to model predictions).
As noted previously, the contribution of AMPARs under these
conditions was minimal (Figures 7B and 7C). Importantly, under

1248 Neuron 90, 1243–1256, June 15, 2016

Figure 6. Experimentally Varying nAChR or AMPAR Activity Verifies the Fidelity of Multicomponent Linear Regression Models
(A) A schematic of the circuit involved under low-contrast motion where direction selectivity is driven by ACh and GABA (the contributions of NMDARs are omitted
for clarity; all conductance measurements were performed in the presence of an NMDAR antagonist [50 mM AP5]).
(B–D) The I-V of the peak phase of preferred direction response (mean ± SEM is plotted) for low-contrast motion shown in Figure 5C (bottom) was fit to a three-
component linear model (ACh + AMPA + GABA, B) or to two receptor component models (ACh + GABA,C; or AMPA + GABA, D). The residuals between the data
and their respective fits are shown below, shaded in gray. Similar results were obtained when the error analysis was performed for the duration of the light
response (Figure S4).
(E) A comparison of the average root mean square (RMS) error across six cells is plotted for the three models. Note that the AMPA + GABA model performs poorly
compared to the other two models (*p < 0. 001).
(F–J) Similar to (A)–(E), except high-contrast stimuli are used to increase the contribution of AMPARs (see Figure 2B). Under these conditions (F), the three-
component model (G) performs significantly better than the two-component models (H and I), as shown in (J) (*p < 0. 01; n = 5).
(K–O) Similar to (F)–(J) but with nAChRs blocked pharmacologically, as depicted in (K). Under these conditions, the ACh + GABA model (M) performs relatively
poorly compared to the ACh + GABA + AMPA (L) or AMPA + GABA models (N), as summarized in (O) (n = 5; *p < 0. 001).

low-contrast stimulation conditions, ACh and GABA signals
differed in several respects during motion in the preferred or
null directions. First, during preferred motion, excitation arose
a few milliseconds before inhibition (40 ± 13 ms delay between
peak gACh and gGABA; n = 6 DSGCs; Figures 7B and 7F), which
corresponds to a spatial lag of 40 mm (given the stimulus veloc-
ity of 1 mm/s). Interestingly, as inhibition increased, cholinergic
excitation ceased, consistent with the idea that cholinergic
release was strongly controlled by GABAergic inhibition (Fried
et al., 2005). The temporal delay between excitation and inhibi-
tion results in a net excitation in the preferred direction in a similar
time window in which spiking was observed in DSGCs (Figures

Neuron 90, 1243–1256, June 15, 2016 1249

Figure 7. Differential Transmission of ACh and GABA Mediates Direction Selectivity in Ganglion Cells
(A) A schematic illustrating the circuit mechanism involved in conveying low-contrast direction information to DSGCs (photoreceptor and bipolar cells driving
SACs are not shown for clarity), as described in Figure 1B.
(B) Spiking responses in a DSGC evoked by low-contrast stimuli moving in the preferred (P) or null (N) directions (as indicated; top panel). The temporal evolution
of GABA (green traces)-, ACh (blue traces)-, and AMPAR-mediated conductances (red) estimated at 60 mV in the same DSGCs using linear regression methods
(see text for details). Subtracting the GABA from the ACh conductance (ACh  GABA, bottom traces) provides an indication of net excitation (yellow box) or
inhibition.
(C–G) A comparison of the synaptic response properties evoked by low-contrast stimuli across six DSGCs. Plots of the peak ACh and AMPA conductances
evoked by null versus preferred stimuli (C), null versus preferred peak GABA conductances (D), net excitatory (peak ACh  GABA) conductances evoked by null
versus preferred motion (E), time to peak of the GABA versus ACh conductances evoked by preferred motion (F), and time to peak of the GABA versus ACh
conductance evoked by null motion (G).

7B and 7E). Second, during null motion, GABAergic inhibition
was significantly stronger than that observed during preferred
motion (Taylor and Vaney, 2002; Figures 7B and 7D), while
cholinergic excitation was approximately equal in both direc-
tions (Figures 7B and 7C), indicating a differential transmission
of ACh and GABA signals to DSGCs. In the null direction, both
GABAergic inhibition and cholinergic excitation had a similar
time course (Figures 7B and 7G), but since the GABA conduc-
tance was significantly larger than the cholinergic conductance,
the net activity was inhibitory (Figure 7B; bottom trace). While
directional inhibition and spatiotemporal offset inhibition/excita-
tion have been previously demonstrated to be key factors in
shaping direction selectivity (Taylor and Vaney, 2002), what is
surprisingly revealed here is that these features can arise from
the SAC network alone under physiological conditions, without
direct bipolar cell input to DSGCs.
Optogenetic Stimulation of SACs Evokes DS Responses
In general, results from optogenetic experiments are difficult to
interpret because these stimulation techniques are not usually
designed to drive physiologically relevant spatiotemporal pat-
terns of activity. However, convincing evidence that SACs alone
could generate direction selectivity through mixed transmission
was obtained when we measured responses to moving stimuli
evoked by either photoreceptors or ChR2-expressing SACs in
the same DSGC (Figures 8A–8D). In these experiments, after
characterizing the tuning properties of the DSGCs under normal
stimulation conditions (Figure 8B), photoreceptor output was
blocked using a standard cocktail of glutamate receptor antag-
onists/agonist (Figure 8C). The stimulus intensity was then
increased by 5 log units in order to stimulate ChR2 (Figure 8D).
Remarkably, we found that stimulation of SAC-ChR2 rein-
stated the DS spiking responses after photoreceptor blockade
(Figures 8B–8D). Importantly, each cell’s preferred direction
measured in control conditions remained unchanged when
measured under SAC-ChR2 stimulation (Figures 8E and 8F),
and SAC-ChR2-mediated direction selectivity was observed in
DSGCs coding a variety of directions (Figure 8F), indicating
that this mechanism was not specific for a particular DSGC
subtype. The observed direction-selective index (DSI; see
Experimental Procedures) during SAC-ChR2 stimulation was
increased compared to control (Figure 8G), likely due to a

1250 Neuron 90, 1243–1256, June 15, 2016

Figure 8. The SAC Network Alone Can Drive Direction Selectivity in Ganglion Cells
(A) A circuit diagram indicating that all synapses were pharmacologically blocked except GABA and ACh SAC to DSGC synapses. In these experiments, SACs
were directly stimulated with ChR2.
(B–D) Directional responses in the SAC-ChR2 retina under control (B) or in the presence of a cocktail of agents that block photoreceptor synapses (C). In the
continued presence of blockers, directional responses are recovered in the DSGC when stimulus intensity was increased by 5 orders of magnitude to drive
SAC-ChR2 (D). Stimulus intensity is indicated as R*/s on the right.
(E) Polar plots of spiking measured in (B) and (D).
(F) The direction encoded by the optogenetically stimulated SAC network (SAC-ChR2) is plotted against the direction encoded during stimulation of intact
photoreceptors (PR; n = 14 DSGCs). The dashed line is a reference line with a slope of 1.
(G) DSI computed under the two stimulation conditions are plotted against each other (for the same set of DSGCs in F).
(H) SAC-ChR2-mediated direction-selective responses in DSGCs (top panel) were abolished in the presence of the GABAR blocker (10 mM SR-95331; n = 5;
middle panel). Responses were completely blocked by the subsequent application of nAChR antagonists (n = 5). Figure S6 shows the temporal kinetics of the
nAChR and GABAR conductances generating this DS response.
(I–K) The mean (± SEM) number of spikes/peak spike rate (I), relative direction (0 indicates the same direction as photoreceptor-drive responses, J), and DSI (K),
evoked in DSGCs by optogenetic stimulation of SACs with bright spots moving at different velocities in the preferred direction.

Neuron 90, 1243–1256, June 15, 2016 1251

threshold effect (the ‘‘iceberg effect’’; Carandini and Ferster,
2000) associated with the weaker responses observed in this
condition (control, 43 ± 6 spikes; SAC-ChR2, 10 ± 1 spikes;
n = 14, p < 0. 001). Varying the velocity of the stimulus showed
that the ChR2-stimulated responses were optimally evoked
at 1 mm/s (Figures 8I–8K), similar to photoreceptor-mediated
responses (Trenholm et al., 2013b). In contrast, ChR2 stimula-
tion did not drive responses to slower stimuli (0.4 mm/s), where
photoreceptor responses are apparent (Trenholm et al., 2013b).
However, whether these tuning properties reflect the intrinsic
properties of SACs or those of ChR2 is not clear.
Finally, blocking GABARs abolished the directionally selective
spiking, suggesting that cholinergic inputs on their own were not
DS. The subsequent addition (or addition alone; n = 5; data
not shown) of an nAChR antagonist blocked all spiking activity
(Figure 8H), confirming that there was no residual photoreceptor
activity and that DS responses were indeed driven by SACs. Mea-
surements of SAC-ChR2-evoked synaptic conductances indi-
cated that direction selectivity was driven by a combination of (1)
symmetrical cholinergic excitation, (2) directional GABAergic inhi-
bition, and (3) directional timing differences between excitation
and inhibition, all similar to physiological conditions (Figure S6).
A combination of results obtained from natural and artificial stimu-
lation of the SAC network highlights the importance of this network
in the generation of direction selectivity in ganglion cells.
In summary, results from pharmacology, linear regression, and
optogenetic experiments demonstrate that under low-contrast
conditions (1) cholinergic inputs robustly drive DSGC spiking
activity (with the help of NMDARs), and (2) ACh and GABA
are differentially transmitted to DSGCs, shaping their DS spiking
properties. Thus, mixed ACh/GABA transmission mediated by
the SAC network plays a more important role in conveying DS in-
formation than previously envisioned.
DISCUSSION
An Intermediary Stage for Processing Directional
Information
It is widely assumed that DSGCs compute motion by integrating
excitation mediated by bipolar cells and inhibition mediated by
SACs (Figure 1A), leaving a less well-defined role for ACh (Borst
and Euler, 2011; Demb, 2007; Taylor and Smith, 2012; Vaney
et al., 2012). Here we posit that fast cholinergic and GABAergic
inputs from the SAC network are key factors in computing DS
responses in ganglion cells, while excitatory bipolar cell inputs
play a secondary role in amplifying DS responses through
NMDAR activation.
The importance of ACh in the DS computation was revealed
using pharmacology, as well as an in-depth analysis of the
synaptic conductances mediating the DSGC’s light response.
Linear regression analysis supports the pharmacological results
and further demonstrates that GABA and ACh are differentially
transmitted to the DSGC. In addition, the re-instatement of direc-
tion selectivity in ganglion cells after photoreceptor blockade
in SAC-ChR2 retinas demonstrates the intrinsic capacity of the
SAC network to differentially transmit ACh and GABA signals
to DSGCs. While a variety of amacrine cells are known to excite
ganglion cells via direct chemical (Kim et al., 2015; Krishnasw-
1252 Neuron 90, 1243–1256, June 15, 2016
amy et al., 2015) or electrical synapses (Kolb and Famiglietti,
1974; Vaney, 1991; Xin and Bloomfield, 1997), the finding
that a single population of amacrine cells can provide feature-
specific information to a ganglion cell through direct excitation
and inhibition is unprecedented.
A Novel Synaptic Organization of the Excitatory
Synapses in the DS Circuit
The synaptic organization of excitatory synapses in the DS cir-
cuit appears to be specialized in a number of aspects, promoting
space- and time-dependent non-linear interactions. Most strik-
ingly, AMPAR-mediated synaptic activity in DSGCs is absent
at low contrast and only emerges at the higher-contrast levels.
This is unusual, as in most ganglion cells the AMPAR conduc-
tance is the dominant excitatory conductance (Diamond and
Copenhagen, 1993; Sagdullaev et al., 2006; Sethuramanujam
and Slaughter, 2015; Zhang and Diamond, 2009), and AMPARs
and NMDARs are usually activated in parallel when contrast
(Buldyrev et al., 2012; Diamond and Copenhagen, 1995; Manoo-
kin et al., 2010) or temporal frequency (Stafford et al., 2014)
is varied. Whether different types of bipolar cells differentially
use NMDARs and AMPARs, or whether these receptors are
expressed at the same synapses and the system relies on the
distinct affinities of these receptors (Patneau and Mayer, 1990)
to signal different levels of glutamate release evoked by varying
contrasts remains to be investigated.
In contrast to the expression of ‘‘silent’’ NMDARs described
here, previous studies have demonstrated that bipolar cells can
drive spiking in DSGCs solely using NMDARs (Cohen and Miller,
1995; Kittila and Massey, 1997; Weng et al., 2005). Similar
NMDAR-mediated spiking has also been observed in other types
of ganglion cells (Buldyrev et al., 2012; Diamond and Copenha-
gen, 1993). However, this apparent difference can be easily
reconciled by considering the specific pharmacological condi-
tions used to isolate NMDAR-mediated activity. Specifically, pre-
vious studies have used AMPAR antagonists to isolate NMDARs,
which also block feedforward inhibition. Without inhibition, intact
NMDAR-mediated activity is likely enhanced (Poleg-Polsky and
Diamond, 2016). In contrast, we assessed NMDAR synaptic
functions in the presence of nAChR antagonists, leaving feedfor-
ward inhibition intact. In these conditions, NMDAR-only spiking
was not observed despite its strong potential to mediate robust
excitation (gNMDA 10 nS at +40 mV). Thus, we conclude that
in the physiological context of low-contrast stimulation, NMDAR
inputs are silent, as widely observed in other parts of the brain
(Kerchner and Nicoll, 2008). Thus, our results suggest a funda-
mentally different role for NMDARs than previously envisioned
(Cohen and Miller, 1995; Kittila and Massey, 1997; Weng et al.,
2005), where DSGCs only respond to glutamate during coinci-
dent cholinergic activity initiated by SACs. This requirement
ensures that SAC inputs control the timing of the DSGC excitatory
responses, a key factor in determining direction selectivity (Taylor
and Vaney, 2002; Lee et al., 2010; Fried et al., 2005).
Differential Transmission of GABA and ACh Underlies
Direction Selectivity in Ganglion Cells
More than 30 years ago, Ariel and Daw (1982) envisioned that
cholinergic excitation and GABAergic inhibition drove direction
selectivity in ganglion cells, based on simple pharmacology.
Here, we evaluate for the first time the temporal dynamics of
GABAR- and nAChR-mediated synaptic responses evoked by
low-contrast moving stimuli and found GABA and ACh to be
differentially transmitted to the DSGC, supporting this model.
Specifically, in the preferred direction, SACs mediate cholinergic
excitation that preceded the onset of inhibition. SAC ACh also
gates the bipolar cell glutamate signals by recruiting NMDARs,
producing an extremely robust, well-timed excitation. In the
null direction, SACs co-release ACh and GABA on a similar time-
scale, generating a net GABAergic inhibition (note NMDARs are
not recruited under these conditions). The functional relevance
of the mixed transmission we’ve described can be easily under-
stood in the context of the present framework of the DS circuit
(Borst and Euler, 2011; Demb, 2007; Taylor and Smith, 2012; Va-
ney et al., 2012), described below.
Anatomical studies have shown that SACs with somas located
on the null side of the DSGC’s receptive field (null-side SACs,
where null side refers to the side from which null stimuli enter
the DSGC’s receptive field) make ten times the number of syn-
aptic contacts with the DSGC compared to preferred-side SAC
dendrites (Briggman et al., 2011). Direct electrical/optogenetic
stimulation of SACs in pharmacological isolation indicates a
similar asymmetrical spatial distribution of GABA inhibition (Fried
et al., 2002; Lee et al., 2010; Wei et al., 2011; Yonehara et al.,
2011). During motion, the intrinsic DS preferences of SAC
dendrites (Euler et al., 2002) and asymmetries of the wiring/
functional connectivity, along with network interactions (Kostadi-
nov and Sanes, 2015; Lee et al., 2010; Münch and Werblin,
2006), lead to the formation of an anisotropic postsynaptic
GABAergic inhibition. Our results are consistent with these
well-established synaptic mechanisms of the DS circuit. How-
ever, the finding that direction selectivity in ganglion cells can
be driven in the absence of bipolar cell inputs using optogenetics
brings into question the significance of more recent models in
which distinct bipolar cells with different kinetics are proposed
to play a key role in the DS computation (Greene et al., 2016;
Kim et al., 2014).
The spatial constraints arising from anatomical connectivity
that give rise to anisotropic GABAergic inhibition appear to be
relaxed for SAC excitation. ACh excitation appears isotropic
either when driven artificially (Ariel and Daw, 1982; Grzywacz
and Amthor, 1993; Kittila and Massey, 1997; Lee et al., 2010;
Yonehara et al., 2011) or when driven with moving stimuli as
we have shown here (Figure 7; but see Lee et al., 2010; Pei
et al., 2015). As suggested in previous studies (Briggman et al.,
2011), isotropic excitation could be an outcome of the paracrine
nature of ACh transmission. The dense plexus of SAC dendrites
releasing ACh (with each point containing overlapping dendrites
originating from 30–60 SACs; Keeley et al., 2007) and the diffuse
expression of acetylcholinesterase (Nichols and Koelle, 1968)
together promote paracrine transmission of ACh in the retina
(Ariel and Daw, 1982; Ford et al., 2012; Schmidt et al., 1987).
Paracrine transmission would allow DSGCs to pool cholinergic
signals arising from many dendrites orientated in different direc-
tions. In contrast to ACh, the clearance of GABA from the synap-
tic cleft relies on strong uptake mechanisms that confine its
action to the synapse. In this way, co-release of ACh and
GABA by SACs could lead to distinct spatiotemporal patterns
of activity at the level of the DSGC. Alternatively, a heteroge-
neous set of ACh and GABA SAC-DSGC synapses with distinct
symmetric and asymmetric wiring patterns could also explain
why ACh, but not GABA, is isotropic. Experimental verification
of the mechanisms underlying isotropic ACh signaling awaits
the development of a new generation of genetically encoded
optical sensors that can directly probe ACh dynamics at the level
of single synapses.
Technical Limitations of Voltage-Clamp Techniques
Applied to Mouse DSGCs
The original study characterizing synaptic conductances in
DSGCs in rabbit retina assumed that a majority of the inputs
were mediated by bipolar cells, based on the linear AMPAR-like
I-V relationship of the measured synaptic responses (Taylor and
Vaney, 2002). However, since NMDAR- and nAChR- mediated
currents have complementary voltage-dependent relationships,
together they would tend to linearize the total response and
make it difficult to tease apart the three receptor components
(ACh, NMDA, and AMPA) known to drive DGSCs. Here, in mouse
DSGCs, using a large range of holding potentials and by blocking
NMDARs, we simplify the analysis and clearly reveal the
separate contributions of linear AMPARs and rectifying nAChRs
in the composite synaptic response using regression analysis.
Our results suggest that at high contrasts, both SACs and
bipolar cells contribute equally to DGSC responses, while at
low contrast, cholinergic inputs dominate (Figure 6).
It should be noted that when comparing cholinergic conduc-
tances during null and preferred motion, it is possible that cholin-
ergic excitation in the null direction is underestimated due to
space-clamp errors caused by the large inhibitory conductance
evoked during null motion (Poleg-Polsky and Diamond, 2011),
and whether cholinergic excitation is directional (Figure 7C;
Lee et al., 2010; Pei et al., 2015) remains to be confirmed using
alternative methods (Park et al., 2014). Importantly, voltage-
clamp errors do not appear to strongly bias the estimation of
nAChR or AMPAR contributions to the low-contrast preferred
motion response, as the I-V relationships of the synaptic re-
sponses changed in a predictable manner using a variety
of experimental manipulations (Figure 6). Thus, the major
conclusion that cholinergic excitation is a prominent component
of DSGC responses under low-contrast conditions is well
substantiated.
Conclusions
Mixed neurons releasing both fast excitatory/inhibitory transmit-
ters are found throughout the CNS, including the lateral/medial
habenula (Root et al., 2014; Shabel et al., 2014), auditory brain-
stem (Gillespie et al., 2005), CA3 region of the hippocampus (Gil-
lespie et al., 2005; Gutiérrez et al., 2003), and basal forebrain
cholinergic system (Saunders et al., 2015), as well as in the retina
(Brecha et al., 1988; Vaney and Young, 1988). Modulating the
balance between inhibition and excitation can provide mixed
neurons with an effective means of controlling their net output
(Shabel et al., 2014). However, we have found that co-release
of mixed transmitters does not necessarily lead to a simple
cancellation. By virtue of the divergent transmitter release and
Neuron 90, 1243–1256, June 15, 2016 1253
clearance mechanisms, together with the distinct biophysical
properties of postsynaptic receptors, mixed neurons can finely
control the spatiotemporal profiles of neural activity. This may
represent a unique way to generate exquisitely timed inhibition
and excitation that is important for high-fidelity computations
(Cafaro and Rieke, 2010), even under conditions of high network
variability. In the case of DSGCs, a specific wiring of SACs to
DSGCs (Briggman et al., 2011) allows inhibition and excitation
to be separated in space and time despite GABA and ACh orig-
inating from the same source, providing a unique signaling
mechanism for conferring upon downstream ganglion cells the
ability to code direction using minimal hardware.
EXPERIMENTAL PROCEDURES
exponential equation accounting for its weak rectification (Manookin et al.,
2010),
fGABA = eaV + b;
where a = 0.0105 and b = 0.4602 are constants. Responses mediated by
AMPARs were linear, resulting in fAMPA = 1. The reported receptor conduc-
tances (gACh, gGABA, and gAMPA) were estimated near the resting membrane
potential 60 mV. Population data have been expressed as mean ± SEM.
Student’s t test was used to compare values under different conditions and
the differences were considered significant when p % 0.05.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures
and six figures and can be found with this article online at http://dx.doi.org/
10.1016/j.neuron.2016.04.041.

All procedures were performed in accordance with the Canadian Council on AUTHOR CONTRIBUTIONS
Animal Care and approved by the University of Victoria’s Animal Care Commit-
tee. Experiments were performed using adult Hb9eGFP (RRID: MGI_109160),
S.S. designed and performed most experiments, analyzed results, and
or ChatCre (RRID: MGI_5475195) crossed with reporter mice, Ai9 (RRID:
contributed to writing the paper. A.J.M. and A.H. collected data that contrib-
MGI_3809523) or Ai32 (RRID: MGI_5013789). Retinae were extracted from
uted to Figures 3F and 3G and Figures 1E and 1F, respectively. G.D. and
mouse eyes under dim red light and flat mounted on a piece of filter paper
D.J.S. helped design the conductance analysis/natural stimuli. G.B.A. de-
with a pre-cut window, through which visual stimuli were presented. Superior
signed experiments and wrote the paper.
coding GFP+ DSGCs were identified in the Hb9eGFP retina using two-photon
laser-scanning microscopy techniques (Trenholm et al., 2013a), while other
ACKNOWLEDGMENTS
types of GFP DSGCs were identified by their medium-sized elliptical somata
and by their DS responses. Light stimuli, produced using a digital light projec-
We thank Kerry Delaney, Bob Chow, and Craig Brown for their useful discus-
tor (Hitachi Cpx1, refresh rate 75 Hz), were focused onto the outer segments of
sions, and Laura Hanson and Rishi Vasandani for providing helpful technical
the photoreceptors using the sub-stage condenser. The background lumi-
support. This work was supported by operating grants from the Foundation
nance, measured with a calibrated spectrophotometer (Ocean Optics), was
Fighting Blindness (Canada), Brain Canada, and Canadian Institutes for Health
set to 10 photoisomerisations/s (R*/sec). Visual stimuli created in the Matlab
Research (CIHR MOP 142301) awarded to G.B.A.
environment (Psychtoolbox) were of positive contrasts, ranging between 15%
and 1,000% (Weber contrast). In some experiments, 50–100 ms light flashes
Received: November 2, 2015
using a blue LED (470 nm) were used to drive ChR2 (10 mW/cm2 or 108
Revised: March 9, 2016
R*/sec). All experiments were performed at 35
C–37
C.
Accepted: April 18, 2016
Light-evoked synaptic currents were recorded at membrane potentials be-
Published: May 26, 2016
tween 70 mV and +20 mV, stepped in 5–10 mV increments, and were fit to
the following equation using Matlab’s built-in Levenberg-Marquardt algorithm:
REFERENCES
IðVÞ = gACh  fACh ðVÞ  ðV  ECat Þ + gGABA  fGABA ðVÞ  ðV  ECl Þ + gAMPA
 fAMPA ðV  ECat Þ; Ariel, M., and Daw, N.W. (1982). Pharmacological analysis of directionally
sensitive rabbit retinal ganglion cells. J. Physiol. 324, 161–185.
where I is the instantaneous total current at the membrane voltage V,
Borst, A., and Euler, T. (2011). Seeing things in motion: models, circuits, and
Ecat the cationic reversal potential = 0 mV, and ECl the chloride reversal
mechanisms. Neuron 71, 974–994.
potential = 56mV. fACh, fGABA, and fAMPA are basis functions for these recep-
tors, normalized to 1 at 60 mV (described below). gACh, gAMPA, and gGABA Brecha, N., Johnson, D., Peichl, L., and Wässle, H. (1988). Cholinergic ama-
denote the conductance mediated by nAChR, AMPAR, and GABARs, respec- crine cells of the rabbit retina contain glutamate decarboxylase and gamma-
tively, estimated by the fitting procedure. The fitting was performed at a time aminobutyrate immunoreactivity. Proc. Natl. Acad. Sci. USA 85, 6187–6191.
interval of 1 ms (Figure 7) or over a 30–50 ms period (Figures 5 and 6). Briggman, K.L., Helmstaedter, M., and Denk, W. (2011). Wiring specificity in
The Woodhull equation, which well describes the voltage-dependent inward the direction-selectivity circuit of the retina. Nature 471, 183–188.
rectification of nAChRs (Haghighi and Cooper, 1998), was used to define its Buldyrev, I., Puthussery, T., and Taylor, W.R. (2012). Synaptic pathways that
basis function (fACh), shape the excitatory drive in an OFF retinal ganglion cell. J. Neurophysiol.
Gmax 107, 1795–1807.
fACh ðVÞ =  
Cafaro, J., and Rieke, F. (2010). Noise correlations improve response fidelity
1 + ½S
kd
and stimulus encoding. Nature 468, 964–967.

Carandini, M., and Ferster, D. (2000). Membrane potential and firing rate in cat
VzdF
kd = kdð0Þ  e RT ; primary visual cortex. J. Neurosci. 20, 470–484.
Cohen, E.D., and Miller, R.F. (1995). Quinoxalines block the mechanism of
where Gmax is the maximum conductance, [S] is the internal concentration of
directional selectivity in ganglion cells of the rabbit retina. Proc. Natl. Acad.
spermine (100 mM), kd is the dissociation constant at a given voltage V, kd (0)
Sci. USA 92, 1127–1131.
is the dissociation constant at 0 mV estimated as 10.66 mM, z is the valence
of spermine assumed to be 3.8 at physiological pH (Haghighi and Cooper, Demb, J.B. (2007). Cellular mechanisms for direction selectivity in the retina.
1998), d is the fraction of the membrane electrical field sensed by spermine Neuron 55, 179–186.
from the intracellular side and was estimated to be 48.4%, F is the Faraday Diamond, J.S., and Copenhagen, D.R. (1993). The contribution of NMDA and
constant, and R and T are the universal gas constant and absolute tempera- non-NMDA receptors to the light-evoked input-output characteristics of retinal
ture, respectively. The GABAR basis function (fGABA) was described by an ganglion cells. Neuron 11, 725–738.

1254 Neuron 90, 1243–1256, June 15, 2016

Diamond, J.S., and Copenhagen, D.R. (1995). The relationship between light-
evoked synaptic excitation and spiking behaviour of salamander retinal
ganglion cells. J. Physiol. 487, 711–725.
Dmitrieva, N.A., Strang, C.E., and Keyser, K.T. (2007). Expression of alpha 7
nicotinic acetylcholine receptors by bipolar, amacrine, and ganglion cells of
the rabbit retina. J. Histochem. Cytochem. 55, 461–476.
Elgueta, C., Vielma, A.H., Palacios, A.G., and Schmachtenberg, O. (2015).
Acetylcholine induces GABA release onto rod bipolar cells through hetero-
meric nicotinic receptors expressed in A17 amacrine cells. Front. Cell.
Neurosci. 9, 6.
Euler, T., Detwiler, P.B., and Denk, W. (2002). Directionally selective calcium
signals in dendrites of starburst amacrine cells. Nature 418, 845–852.
Ford, K.J., Félix, A.L., and Feller, M.B. (2012). Cellular mechanisms underlying
spatiotemporal features of cholinergic retinal waves. J. Neurosci. 32, 850–863.
Fried, S.I., Münch, T.A., and Werblin, F.S. (2002). Mechanisms and circuitry
underlying directional selectivity in the retina. Nature 420, 411–414.
Fried, S.I., Münch, T.A., and Werblin, F.S. (2005). Directional selectivity is
formed at multiple levels by laterally offset inhibition in the rabbit retina.
Neuron 46, 117–127.
Froudarakis, E., Berens, P., Ecker, A.S., Cotton, R.J., Sinz, F.H., Yatsenko, D.,
Saggau, P., Bethge, M., and Tolias, A.S. (2014). Population code in mouse V1
facilitates readout of natural scenes through increased sparseness. Nat.
Neurosci. 17, 851–857.
Gillespie, D.C., Kim, G., and Kandler, K. (2005). Inhibitory synapses in the
developing auditory system are glutamatergic. Nat. Neurosci. 8, 332–338.
Greene, M.J., Kim, J.S., and Seung, H.S.; EyeWirers (2016). Analogous
convergence of sustained and transient inputs in parallel on and off pathways
for retinal motion computation. Cell Rep. 14, 1892–1900.
Grzywacz, N.M., and Amthor, F.R. (1993). Facilitation in ON-OFF directionally
selective ganglion cells of the rabbit retina. J. Neurophysiol. 69, 2188–2199.
Grzywacz, N.M., Amthor, F.R., and Merwine, D.K. (1998). Necessity of acetyl-
choline for retinal directionally selective responses to drifting gratings in rabbit.
J. Physiol. 512, 575–581.
Gutiérrez, R., Romo-Parra, H., Maqueda, J., Vivar, C., Ramı̀rez, M., Morales,
M.A., and Lamas, M. (2003). Plasticity of the GABAergic phenotype of the ‘‘glu-
tamatergic’’ granule cells of the rat dentate gyrus. J. Neurosci. 23, 5594–5598.
Haghighi, A.P., and Cooper, E. (1998). Neuronal nicotinic acetylcholine recep-
tors are blocked by intracellular spermine in a voltage-dependent manner.
J. Neurosci. 18, 4050–4062.
Keeley, P.W., Whitney, I.E., Raven, M.A., and Reese, B.E. (2007). Dendritic
spread and functional coverage of starburst amacrine cells. J. Comp.
Neurol. 505, 539–546.
Kerchner, G.A., and Nicoll, R.A. (2008). Silent synapses and the emergence of
a postsynaptic mechanism for LTP. Nat. Rev. Neurosci. 9, 813–825.
Kim, J.S., Greene, M.J., Zlateski, A., Lee, K., Richardson, M., Turaga, S.C.,
Purcaro, M., Balkam, M., Robinson, A., Behabadi, B.F., et al.; EyeWirers
(2014). Space-time wiring specificity supports direction selectivity in the retina.
Nature 509, 331–336.
Kim, T., Soto, F., and Kerschensteiner, D. (2015). An excitatory amacrine cell
detects object motion and provides feature-selective input to ganglion cells
in the mouse retina. eLife 4, http://dx.doi.org/10.7554/eLife.08025.
Kittila, C.A., and Massey, S.C. (1997). Pharmacology of directionally selective
ganglion cells in the rabbit retina. J. Neurophysiol. 77, 675–689.
Kolb, H., and Famiglietti, E.V. (1974). Rod and cone pathways in the inner
plexiform layer of cat retina. Science 186, 47–49.
Kostadinov, D., and Sanes, J.R. (2015). Protocadherin-dependent dendritic
self-avoidance regulates neural connectivity and circuit function. eLife 4,
http://dx.doi.org/10.7554/eLife.08964.
Krishnaswamy, A., Yamagata, M., Duan, X., Hong, Y.K., and Sanes, J.R.
(2015). Sidekick 2 directs formation of a retinal circuit that detects differential
motion. Nature 524, 466–470.
Lee, S., Kim, K., and Zhou, Z.J. (2010). Role of ACh-GABA cotransmission in
detecting image motion and motion direction. Neuron 68, 1159–1172.
Lipin, M.Y., Taylor, W.R., and Smith, R.G. (2015). Inhibitory input to the direc-
tion-selective ganglion cell is saturated at low contrast. J. Neurophysiol. 114,
927–941.
Manookin, M.B., Weick, M., Stafford, B.K., and Demb, J.B. (2010). NMDA
receptor contributions to visual contrast coding. Neuron 67, 280–293.
Massey, S.C., and Neal, M.J. (1979). The light evoked release of acetylcholine
from the rabbit retina iN vivo and its inhibition by gamma-aminobutyric acid.
J. Neurochem. 32, 1327–1329.
Massey, S.C., and Redburn, D.A. (1983). The cholinergic amacrine cells of
rabbit retina receive on and off input: an analysis of [3H]-ACh release using
2-amino-4-phosphonobutyric acid (APB) and chloride free medium. Vision
Res. 23, 1615–1620.
Münch, T.A., and Werblin, F.S. (2006). Symmetric interactions within a homo-
geneous starburst cell network can lead to robust asymmetries in dendrites of
starburst amacrine cells. J. Neurophysiol. 96, 471–477.
Naka, K.I., and Rushton, W.A. (1966). S-potentials from luminosity units in the
retina of fish (Cyprinidae). J. Physiol. 185, 587–599.
Nichols, C.W., and Koelle, G.B. (1968). Comparison of the localization of
acetylcholinesterase and non-specific cholinesterase activities in mammalian
and avian retinas. J. Comp. Neurol. 133, 1–16.
Park, S.J., Kim, I.J., Looger, L.L., Demb, J.B., and Borghuis, B.G. (2014).
Excitatory synaptic inputs to mouse on-off direction-selective retinal ganglion
cells lack direction tuning. J. Neurosci. 34, 3976–3981.
Patneau, D.K., and Mayer, M.L. (1990). Structure-activity relationships for
amino acid transmitter candidates acting at N-methyl-D-aspartate and quis-
qualate receptors. J. Neurosci. 10, 2385–2399.
Pei, Z., Chen, Q., Koren, D., Giammarinaro, B., Acaron Ledesma, H., and Wei,
W. (2015). Conditional knock-out of vesicular GABA transporter gene from
starburst amacrine cells reveals the contributions of multiple synaptic mech-
anisms underlying direction selectivity in the retina. J. Neurosci. 35, 13219–
13232.
Poleg-Polsky, A., and Diamond, J.S. (2011). Imperfect space clamp permits
electrotonic interactions between inhibitory and excitatory synaptic conduc-
tances, distorting voltage clamp recordings. PLoS ONE 6, e19463.
Poleg-Polsky, A., and Diamond, J.S. (2016). NMDA receptors multiplicatively
scale visual signals and enhance directional motion discrimination in retinal
ganglion cells. Neuron 89, 1277–1290.
Root, D.H., Mejias-Aponte, C.A., Qi, J., and Morales, M. (2014). Role of gluta-
matergic projections from ventral tegmental area to lateral habenula in aver-
sive conditioning. J. Neurosci. 34, 13906–13910.
Sagdullaev, B.T., McCall, M.A., and Lukasiewicz, P.D. (2006). Presynaptic
inhibition modulates spillover, creating distinct dynamic response ranges of
sensory output. Neuron 50, 923–935.
Saunders, A., Granger, A.J., and Sabatini, B.L. (2015). Corelease of acetylcho-
line and GABA from cholinergic forebrain neurons. eLife 4, http://dx.doi.org/
10.7554/eLife.06412.
Schmidt, M., Humphrey, M.F., and Wässle, H. (1987). Action and localization of
acetylcholine in the cat retina. J. Neurophysiol. 58, 997–1015.
Sethuramanujam, S., and Slaughter, M.M. (2015). Properties of a glutamater-
gic synapse controlling information output from retinal bipolar cells. PLoS ONE
10, e0129133.
Shabel, S.J., Proulx, C.D., Piriz, J., and Malinow, R. (2014). Mood regulation.
GABA/glutamate co-release controls habenula output and is modified by an-
tidepressant treatment. Science 345, 1494–1498.
Stafford, B.K., Manookin, M.B., Singer, J.H., and Demb, J.B. (2014). NMDA
and AMPA receptors contribute similarly to temporal processing in mamma-
lian retinal ganglion cells. J. Physiol. 592, 4877–4889.
Strang, C.E., Long, Y., Gavrikov, K.E., Amthor, F.R., and Keyser, K.T. (2015).
Nicotinic and muscarinic acetylcholine receptors shape ganglion cell response
properties. J. Neurophysiol. 113, 203–217.
Neuron 90, 1243–1256, June 15, 2016 1255
Tadmor, Y., and Tolhurst, D.J. (2000). Calculating the contrasts that retinal
ganglion cells and LGN neurones encounter in natural scenes. Vision Res.
40, 3145–3157.
Taylor, W.R., and Smith, R.G. (2012). The role of starburst amacrine cells in
visual signal processing. Vis. Neurosci. 29, 73–81.
Taylor, W.R., and Vaney, D.I. (2002). Diverse synaptic mechanisms generate
direction selectivity in the rabbit retina. J. Neurosci. 22, 7712–7720.
Trenholm, S., McLaughlin, A.J., Schwab, D.J., and Awatramani, G.B. (2013a).
Dynamic tuning of electrical and chemical synaptic transmission in a network
of motion coding retinal neurons. J. Neurosci. 33, 14927–14938.
Trenholm, S., Schwab, D.J., Balasubramanian, V., and Awatramani, G.B.
(2013b). Lag normalization in an electrically coupled neural network. Nat.
Neurosci. 16, 154–156.
Vaney, D.I. (1991). Many diverse types of retinal neurons show tracer coupling
when injected with biocytin or Neurobiotin. Neurosci. Lett. 125, 187–190.
Vaney, D.I., and Young, H.M. (1988). GABA-like immunoreactivity in cholin-
ergic amacrine cells of the rabbit retina. Brain Res. 438, 369–373.
Vaney, D.I., Sivyer, B., and Taylor, W.R. (2012). Direction selectivity in
the retina: symmetry and asymmetry in structure and function. Nat. Rev.
Neurosci. 13, 194–208.
Vlasits, A.L., Bos, R., Morrie, R.D., Fortuny, C., Flannery, J.G., Feller, M.B., and
Rivlin-Etzion, M. (2014). Visual stimulation switches the polarity of excitatory
input to starburst amacrine cells. Neuron 83, 1172–1184.
1256 Neuron 90, 1243–1256, June 15, 2016
Wei, W., Hamby, A.M., Zhou, K., and Feller, M.B. (2011). Development
of asymmetric inhibition underlying direction selectivity in the retina. Nature
469, 402–406.
Weng, S., Sun, W., and He, S. (2005). Identification of ON-OFF direction-selec-
tive ganglion cells in the mouse retina. J. Physiol. 562, 915–923.
Xin, D., and Bloomfield, S.A. (1997). Tracer coupling pattern of amacrine and
ganglion cells in the rabbit retina. J. Comp. Neurol. 383, 512–528.
Yonehara, K., Balint, K., Noda, M., Nagel, G., Bamberg, E., and Roska, B.
(2011). Spatially asymmetric reorganization of inhibition establishes a mo-
tion-sensitive circuit. Nature 469, 407–410.
Yonehara, K., Farrow, K., Ghanem, A., Hillier, D., Balint, K., Teixeira, M.,
Juttner, J., Noda, M., Neve, R.L., Conzelmann, K.K., et al. (2013). The first
stage of cardinal direction selectivity is localized to the dendrites of retinal gan-
glion cells. Neuron 79, 1078–1085.
Yoshida, K., Watanabe, D., Ishikane, H., Tachibana, M., Pastan, I., and
Nakanishi, S. (2001). A key role of starburst amacrine cells in originating
retinal directional selectivity and optokinetic eye movement. Neuron 30,
771–780.
Zhang, J., and Diamond, J.S. (2009). Subunit- and pathway-specific localiza-
tion of NMDA receptors and scaffolding proteins at ganglion cell synapses in
rat retina. J. Neurosci. 29, 4274–4286.