LECTURE
Neurochemical organization of the ﬁrst visual synapse
Noga Vardi,1 Anuradha Dhingra,1 Lingli Zhang,1 Arkady Lyubarsky,2 Tian Li Wang1,3 andKatsuko Morigiwa1,4
Department of 1Neuroscience and 2Ophthalmology, University of Pennsylvania, PA, USA(Received for publication on July 2, 2002)

Abstract. The retina employs two main synaptic relays in which information converges to higher
order cells, and at the same time is modiﬁed by lateral inhibitory interneurons. At the ﬁrst synaptic
layer, rod and cone terminals contact second order neurons (horizontal and bipolar cells), and in turn,horizontal cells contact cones and bipolar cells. In this talk/review we describe the structures and theneurochemicals involved in transmitting the visual signal at this synaptic complex. (Keio J Med 51 (3):154–164, September 2002)
Key words: mGluR6, G-protein, knockout mouse, ERG, cone synapse

The retina employs two main synaptic relays, inwhich information passes forward via glutamatergic
synapses. This information is simultaneously modiﬁedby lateral inhibitory connections at each layer (Fig.
1A). The main function of the forward and lateralprocessing is to form the center-surround receptive ﬁeld
structure of the light responses of several parallel circuits (Fig. 1B). In the ﬁrst synaptic layer (termed ‘‘outerplexiform layer’’ or OPL), a rod terminal transmits information about single photons (scotopic range) to rod
bipolar cells. A cone terminal transmits information
about mesopic (twilight) and photopic (daylight) luminances to ten types of bipolar cell.1–3 The bipolar cells
are divided into two main classes: the OFF class, which
depolarizes to glutamate, and the ON class, which
hyperpolarizes to glutamate (Fig. 1B). Large cells,
which collect information from many surrounding cones(horizontal cells), modify this transmission to provide
surround information.4–6 In the second synaptic layer(termed ‘‘inner plexiform layer’’ or IPL), bipolar cells
provide information to ganglion cells, and this information is modiﬁed by about 21 types of amacrine cells.
In this review, we shall discuss the neurochemical basisof the diverse responses in the rod- and cone synaptic
complexes.

Results
Glutamate depolarizes OFF bipolar cells via ionotropic
glutamate receptors
A single cone transmits information to about
400–1,000 second-order dendrites via two specialized
structures: ﬂat (or basal) contact and synaptic triad(Fig. 1C).7 In primates, the basal contact consists of anelectron-dense cone membrane apposing an electrondense bipolar membrane; the cleft between them is ﬁlled with vertical striated material and the bipolar dendrite belongs to OFF bipolar cells. The synaptic triad
consists of an electron dense synaptic ribbon, which ispresynaptic to three processes: two ‘‘lateral elements’’of horizontal cell processes, and a ‘‘central element’’ ofan ON bipolar dendrite.
To determine which receptors are expressed by the
OFF bipolar cells, we and others examined the localization of several ionotropic glutamate receptor subunits (iGluR) in the cat.8–12 Others studied the localization of ionotropic subunits in primates.13–15 It was
found that OFF dendrites express most of the known
iGluR subunits including the AMPA (a-amino-3hydroxy-5-methyl-4-isoxazole propionic acid) subunits

Presented at the 1250th Meeting of the Keio Medical Society in Tokyo, March 6, 2002.
Present Afﬁliations: 3Howard Hughes Medical Institute and Johns Hopkins Oncology Center, The Johns Hopkins University, MD, 4Departmentof Neurobiology, Physiology and Behavior, University of California, CA, USA
Reprint requests to: Dr. Noga Vardi, Department of Neuroscience, University of Pennsylvania, Philadelphia, PA, 19104, e-mail:noga@retina.anatomy.upenn.edu
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in the ground squirrel showed that different types ofOFF bipolar cells express different receptors, some express the AMPA receptor, and some the kainate receptor.16 Examination of the immunostainings also
revealed that horizontal cells express both AMPA andkainate receptors, and that the receptor is concentrated
at the electron-dense horizontal cell membrane just beneath the synaptic ribbon (Fig. 2B). ON bipolar cells
generally do not express iGluR subunits, but in the cat,they did stain for the GluR2/3 and GluR4 subunits (Fig.
2C).9 None of the second order neurons expressed the
NMDA (N-methyl-D-aspartate) subunits.17–19
Glutamate hyperpolarizes ON bipolar cells via themetabotropic receptor mGluR6 coupled to the G-protein
Go1Fig. 1 The ﬁrst visual synapse is a complex designed to transmit
center surround information to parallel pathways. (A) A semi-thin(0.5 mm) epon radial section of a monkey retina stained with toluidine
blue. Retinal layers for this and the rest of ﬁgures are: OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL,
ganglion cell layer. Unless otherwise stated, all sections are radial. (B)A functional schematic of the cone circuits. The white circle represents the dimension of the bipolar receptive ﬁeld center; within thecenter, light response decays according to a Gaussian distribution:strong in the very center, weaker at the outskirts. The gray circle andthe corresponding Gaussian curve represent the receptive ﬁeld surround. The surround is wider and shallower. A cone pedicle (cp)contacts horizontal cells (H) and ON (white) and OFF (black) bipolarcells. Voltage responses to light for ON (V(On)) and OFF V(Off))cells are opposite. (C) An electron micrograph of a monkey cone
pedicle (cp). The pedicle contains specialized ribbons (r), which arepresynaptic to two lateral elements of horizontal cell processes (h)and a central element (ce) of an ON bipolar dendrite. In addition
multiple OFF bipolar dendrites (fb) contact the pedicle at its base in a
structure called ‘‘ﬂat contact’’.

GluR1, GluR2, GluR2/3, and GluR4, and the kainate
subunits GluR5, GluR6/7. Within the cell, receptor
was concentrated in association with the OFF dendrite
electron-dense membrane at the basal contact (Fig.
2A). These studies did not distinguish between different
types of bipolar cells. However, a physiological study

In contrast to OFF bipolar cells, in which glutamate
gates the receptor and opens cation channels, in ON
bipolar cells, glutamate closes cation channels. Abouta decade ago, Nawy & Jahr20 and Shiells & Falk21
discovered that this glutamate response depends on aG-protein. Thus, a search to identify the receptor concentrated on the recently cloned metabotropic glutamate receptors. One such receptor, mGluR6, was foundto be retina-speciﬁc, localized to dendritic tips of rod
bipolar cells, and its deletion eliminated the darkadapted electroretinogram (ERG) b-wave.22,23 Since
this wave originates from the ON responses of rod
bipolar cells, its elimination proved that light response
in ON bipolar cells requires mGluR6. Further studies
showed mGluR6 to be expressed not only in rod bipolar cells, but also in ON cone bipolar cells.8,24–26 To
examine expression in higher mammals, we partiallysequenced human mGluR6, and made an antibody thatrecognized this receptor in cat, rabbit and primates. In
the monkey, mGluR6 was localized exclusively todendrites of rod bipolar cells and ON cone bipolarcells (Fig. 3). Interestingly, the receptor was not concentrated under the synaptic ribbon close to the releasesite, but about 400 nm away at a region of the ON
bipolar dendrite that was in contact with the electrondense cone membrane (Fig. 3B, C).26
We next wished to identify the G-protein in the ON
cells. Immunostainings for about 7 alpha subunits(at1=2 ; aq ; as ; ai ; aolf , and ao ) revealed that only the ao
subunit was localized to dendritic tips of ON bipolarcells. Furthermore, this subunit was localized to dendrites of all types of ON bipolar cells; it was not localized to axon terminals of ON bipolar cells, or to OFF
bipolar cells (Fig. 4).27 Thus, Go seemed a good candidate to mediate the ON response. Subsequent tests
showed that the ao subunit is capable of interacting with
mGluR6 in vitro,28 and that antibodies to this subunit
reduce the light response.29 The ﬁnal proof that Go

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Fig. 2 Ionotropic glutamate receptor subunits (iGluR) are expressed by all types of second order neurons at their dendritic tips (electron
micrographs, immunostain in cat). (A) iGluR6/7 is present at a ﬂat contact formed by an OFF bipolar dendrite (fb). Note the exquisite localization to the membrane apposing the cone membrane. (B) iGluR4 is present in horizontal cell processes (h); it is highly localized to the electron-dense membrane just under the ribbon (r). (C) iGluR2/3 is present in a central element (ce), formed probably by an ON bipolar dendrite.

mediates the ON response came from testing the electroretinograms of mice deﬁcient in both splice variantsof the ao subunit.30 These mice lacked both the scotopic
and the photopic b-waves (Fig. 5). The scotopic b-wave
reﬂects the activity of rod bipolar cells, while the photopic b-wave reﬂects the activity of ON cone bipolarcells. The a-wave, which reﬂects the activity of photoreceptors, was normal. Thus, ON responses in all ON
bipolar cells require Go .
The next step was to determine which splice variantof Gao is localized to these cells, and whether all ON
bipolar cells express the same splice variant.31 We ﬁrst
determined expression by RT-PCR (reverse transcription followed by polymerase chain reaction) and Western blotting using antibodies speciﬁc for the ao1 or the
ao2 splice variants. Both splice variants were expressed.
Immunostaining for the ao1 splice variant revealed apattern similar to that obtained with an antibody thatrecognized both splice variants. Double staining for ao1
and protein kinase C (PKC) conﬁrmed that rod bipolarcells stain for ao1 (Fig. 6A). Double staining for ao(previously established to stain all types of ON bipolarcells) and ao1 showed colocalization in all bipolar cells(Fig. 6B). Thus, all types of ON bipolar cells express
ao1 .
Localization of the ao2 splice variant required anindirect approach because the antibody for ao2 was
not suitable for immunostaining. This was done using
two antibodies that recognize both splice variants andapplying them to mouse retina deﬁcient in ao1 . Thus
staining reﬂects expression of ao2 . Both antibodies gave
similar staining: bipolar cell somas, dendrites, and aband in stratum 1 of the IPL. However, this stain wasmuch weaker than the stain present in the wild type
mouse. Double labeling for ao2 (with anti-ao ) and PKC
showed that the two proteins colocalized, indicatingthat rod bipolar cells express the ao2 splice variant (Fig.

6C). In addition to rod bipolar cells, some bipolar cellsomas negative for PKC were positive for the ao2 splicevariant. This suggests that at least some cone bipolarcells also express ao2 .
To determine which splice variant is critical forthe light responses, we examined the ERG response ofmice lacking either ao1 or ao2 . Mice lacking the ao1
splice variant showed a reduced but signiﬁcant a-wave,
but completely lacked the b-wave. Mice lacking the ao2
splice variant had normal ERG a- and b-waves.31
Horizontal cells provide GABAergic signal to OFF andON bipolar cells
About a decade ago, the neurotransmitter in mammalian horizontal cells was controversial because it
was difﬁcult to consistently show immunostaining for
GABA (g-aminobutyric acid) and GAD (glutamic
acid decarboxylase) in these cells. Furthermore, initialstaining for GABAA receptors was negative in theouter plexiform layer.32 With the improvement of antibodies against GABA and optimizing ﬁxation, we wereable to show that all horizontal cells in cat and monkey
contain GABA. We next investigated the localizationof GAD. There are two isoform of GAD, one with amolecular weight of 65 kDa (GAD65 ), and one with amolecular weight of 67 kDa (GAD67 ).33 Horizontal
cells in cat and monkey stained for GAD; however,cat horizontal cells (both A- and B-types) stained for
GAD67 , and monkey horizontal cells (both H1 and H2)stained for GAD65 (Fig. 7A).34 Rabbit horizontal cells
also stained for GAD, but the pattern was more complex: both types of horizontal cells stained for GAD67 in
the tips of their processes, and type A also stained for
GAD65 . This staining depended on eccentricity, at thevisual streak somas and primary dendrites (but not theirtips) stained for GAD65 ; ventral to the visual streak,Keio J Med 2002; 51 (3): 154–164

Fig. 3 mGluR6 is expressed by all ON bipolar dendritic tips in apposition to the electron-dense cone membrane. (A) Light micrograph,monkey, visualized with diaminobenzidine (DAB) reaction product.
Punctate stain for mGluR6 is seen in two locations, in the upper partof the OPL, single puncta represent dendritic tips of rod bipolar cells(rb), and lower in the OPL puncta are organized in lines just under
the cone pedicle; these represent cone bipolar dendrites (cb). (B)Electron micrograph, monkey. Three triads show that staining is
restricted to the central elements (ce). No staining appears in ﬂat(OFF) bipolar cells (fb). Within the central element, mGluR6 concentrates on the bipolar membrane region that apposes an electrondense cone membrane (arrows). It is weak or undetectable just under
the ribbon (arrowhead), where the dendrite apposes a horizontal cell
membrane. (C) Electron micrograph, rat. mGluR6 is seen in one central element and in certain contacts resembling ﬂat contacts (arrows).
However, when these processes were traced over several sequential
ultra-thin sections, it became clear that they were central elements.

staining intensity decreased gradually and it was undetected at about 1.5 mm from the center of the streak.35
To further establish that horizontal cells are
GABAergic it was necessary to show that GABA
receptors are postsynaptic to horizontal cells. This wastested by examining the localization of the a1 and b 2=3
subunits of the GABAA receptor. Both of these subunits localized to the OPL just beneath the cones (Fig.
7B).36 Fine localization by electron microscopy showedthat the strongest stain was present on dendrites in apposition to horizontal cell processes, both on OFF and
ON dendrites (Fig. 7C, D).37 To our surprise, we wereunable to detect staining on cone or rod membranes.

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Fig. 4 Gao is expressed by all ON bipolar dendrites colocalized with
mGluR6 in the dendritic tips. Top, wild type (WT) and Gao null (KO)mouse retinas immunostained for Gao . Immunostain is strong in
bipolar somas and dendrites, and is also present in the IPL. Middle,
semi-thin epon section of monkey retina visualized with DAB reaction product Stain for Gao is apparent in bipolar somas and theirdendrites: both rod bipolar dendritic tips (rb) in the upper part of the
OPL and cone bipolar dendritic tips (cb) located just below cone
pedicles. Bottom, double staining for Gao and mGluR6. mGluR6(green) is present at the tip of every dendrite stained for Gao (red).

Possible function of horizontal cell input to bipolar cells
Bipolar cells respond to light with a center-surround
receptive ﬁeld structure. The center is formed by convergence of several cones (in cat area centralis, 4–7)38
onto a bipolar cell, and the response sign is determinedby glutamate action: glutamate depolarizes OFF bipolarcells and hyperpolarizes ON bipolar cells. Because light
reduces glutamate release, light on the OFF bipolar
receptive ﬁeld center hyperpolarizes, and on the ON
receptive ﬁeld center depolarizes. The surround isformed by input from horizontal cells; these cells collectinput from a large number of cones (in cat, @200)39

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Fig. 5 Rod- and cone-driven b-waves are absent from the electroretinogram of the null mouse. (Row A) Animals dark-adapted for 2
hours were stimulated with dim ﬂashes. Such ﬂashes elicited a roddriven, corneal-positive b-wave in the heterozygotes, but no positivegoing responses in the null mice. The estimated ﬂash intensities inphotoisomerizations per rod ðFÞ and the number of responses ðnÞ
averaged for each trace shown are as follows: for the P21 and P31
mice, F ¼ 20, n ¼ 11; for the P44 mouse, F ¼ 3, n ¼ 40. (Row B)
Dark-adapted animals were stimulated with an intense ﬂash (isomerizing @1% of the rhodopsin). This elicited in the heterozygote a negative a-wave (shaded), followed by a positive-going b-wave. In thenull mice the a-wave was normal, but the b-wave was absent. The
ﬂash intensities ðFÞ and the number of responses ðnÞ averaged were:
for the P21 and P31 mice, F ¼ 10 6 , n ¼ 2–4. For the P44 mouse
responses to three intensities are shown: F ¼ 20, n ¼ 20; F ¼ 500,
n ¼ 16; F ¼ 10 6 , n ¼ 2. (Row C) Mice were adapted to a brightbackground (540 nm, 20,000 R* rod1 s1 ) that completely suppressed
the cGMP-activated current of the rods. They were then stimulatedwith an intense white ﬂash that isomerizes about 1% of the M-conepigment and 0.1% of the UV-cone pigment in adult mice. The cone
driven a-wave was not visible in the P21 animals, but was pronouncedin P31 and P44 animals (both Gao þ= and Gao =). A typical conedriven b-wave (positive-going response with superimposed oscillations, peaking about 70–90 ms after the ﬂash) was observed in the
Gao þ= mice of all age groups, but was absent in the Gao = mice.
For P21 and P31 Gao þ= mice, n ¼ 10; for P21 Gao =, n ¼ 20; forP31 and P44 Gao =, n ¼ 40. The slow positive-going potential in
P21 mouse is probably an artifact due to movement of the lightly
anesthetized mouse. (D–G) ERG a- and b-waves’ peak amplitude isvariable due to variable contact with the electrode, and due to rapid
growth between 21 and 30 day postnatal. However, at all ages, therod- and cone-driven b-waves were missing from the Gao = mouse.

Fig. 6 ON bipolar dendrite express both splice variants of Gao (mouse). (Left) double staining for Gao1 (green) and PKC (red, a marker forrod bipolar cells). All PKC positive cells also stain for Gao1 (double arrowhead), but certain somas negative for PKC are also positive (arrow).
These are probably ON cone bipolar somas. Also, some processes, stained for Gao1 , are unstained for PKC; these are cone bipolar dendrites(short arrow). (Middle) double staining for Gao1 (green) and Gao (red, marks all ON bipolar somas). The two stains are 100% colocalized,
indicating all ON bipolar cells express Gao1 . (Right) double staining for PKC (red) and Gao (green) on a Gao1 -null mouse. ON this mouse,
staining for Gao labels cells expressing Gao2 . Gao2 is expressed by rod bipolar cells (double arrowheads) and certain ON cone bipolar cells(arrow).

thus averaging light luminance over a wide area. A
strong component of the surround is accomplished by
feedback inhibition to cones. By feeding back to thecones, horizontal cells create surround already at thelevel of photoreceptors, and this is then transmitted
to ON and OFF bipolar cells via glutamate. This feedback had been recognized more than 20 years ago inlower vertebrates,4,40 and conﬁrmed recently in mammals.41,42 Surprisingly, however, the molecular mechanism of the feedback (i.e., which transmitter is released
and which receptors responds to it) is still debated.43
Horizontal cells could also provide direct antagonistic surround to bipolar cells, via GABA receptors
located on their dendritic tips.37 However, this mode of

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Fig. 8 Retina expresses two types of chloride cotransporters: KCC2
(a chloride extruder), and NKCC (a chloride accumulator) (monkey).
Staining for KCC2 is present in OPL, bipolar somas (BP) in INL, in
IPL, and in ganglion cell somas (GC). Staining for NKCC is strong
and punctate in the upper part of the OPL, and somewhat diffusedlower in the OPL. Stain is very weak in the IPL, and strong in thenerve ﬁber layer (NFL). PR, photoreceptors.

hyperpolarizes, so for GABA to antagonize it, it should
depolarize. GABA could depolarize a cell if the chloride equilibrium potential (ECl ) is maintained higher
than the resting potential. Early reports measuring[Cl ] i in mudpuppy, indeed found that [Cl ] i in ON
bipolar cells was higher than in OFF bipolar cells.44 We
tested this conjecture in mammalian retinas by examining the expression pattern of cation-coupled chloride
cotransporters.
Retina expresses two types of chloride cotransporters:
KCC2 and NKCC
Fig. 7 Horizontal cells are GABAergic, probably releasing GABA
onto ON and OFF bipolar dendritic tips expressing GABAA receptors (monkey). (A) A semi-thin section stained for GAD65 . Stain ispresent in both types of horizontal cells (HC) and their processes in
the OPL, in amacrine cells (AC), and in the IPL. (B) A semi-thinsection stained for the a1 subunit of the GABAA receptor. Stain ispresent in the middle of OPL in bipolar dendrites (bd), and in amacrine cells and IPL. (C) Electron micrograph showing part of a cone
pedicle (cp) and its postsynaptic processes. Staining for GABAA (a1 )is present in OFF bipolar dendrites forming ﬂat contacts (fb), and in
ON bipolar cells forming the central element of the triad (ce). (D)Electron micrograph. GABAA staining on a bipolar membrane (arrow) apposing a horizontal cell process (h) is much stronger than on
the membrane facing another bipolar cell (arrowheads). Stain isvisualized with an un-intensiﬁed DAB reaction product.

input requires separate treatment for the ON and OFF
bipolar cells. In OFF bipolar cells, glutamate depolarizes and GABA could antagonize it by hyperpolarizing. In contrast, in ON bipolar cells, glutamate

Three families of cation-coupled chloride cotransporters are known: the K-Cl cotransporter (KCC) uses
Kþ gradient to extrude Cl , and the Na-K-Cl (NKCC)
and Na-Cl cotransporters (NCC) use Naþ gradient toaccumulate Cl . Neuronal cotransporters are mainly
KCC2 and NKCC1.45,46 In retina, immunostaining for
KCC2 gave strong staining both in OPL and in IPL(Fig. 8). In OPL, the pattern resembled ‘‘dashed lines’’,in IPL it was punctate throughout the layer. Immunostaining for NKCC gave strong staining only in the OPL
with puncta high in the OPL and somewhat diffusedstain lower in the OPL (Fig. 8).
Distribution of NKCC and KCC2 in adult retina correlates with known ECl
Horizontal cells express NKCC, the chloride accumulator (Fig. 9, left).47 This ﬁts robust evidence that

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ECl in horizontal cells is positive to E rest : (1) GABA
depolarizes horizontal cells in lower vertebrates and in
mammals44,48,49; (2) [Cl ] i measured with a chloridesensitive electrode predicts ECl at about 17 mV, i.e.,@10 mV positive to dark E rest .44,48
Bipolar axon terminals (of rod bipolar and OFF cone
bipolar cells) express KCC2, the chloride extruder (Fig.
9, right). This ﬁts evidence that their GABA feedback
from amacrine cells is inhibitory, i.e., hyperpolarizing,
which implies ECl negative to E rest ,50,51 (reviewed by
Freed).52
Ganglion cells express KCC2. Somas (Fig. 8) anddendrites, identiﬁed by EM (Fig. 9, bottom), were
stained in the plasma membrane.47 Also, co-staining rat
retina with anti-KCC2 and anti-thy1 (a marker for ganglion cell dendrites) showed strong colocalization.53
Direct measurement of [Cl ] i or ECl under conditions
that do not disturb [Cl ] i are rare because mostrecordings are performed whole cell, where Cl is free
to diffuse from the electrode into the cell. Nevertheless
it is clear that GABA hyperpolarizes ganglion cells:(1) [Cl ] i measured by a chloride-sensitive electrode,estimated ECl at 49 mV, about 20 mV negative todark E rest 44; (2) Blocking GABAA and glycine receptors increases excitation.54–57 Whether ganglion cells
also express NKCC is unclear.
OFF bipolar dendrites express KCC2 and ON bipolar
dendrites express NKCC
Staining in OFF bipolar dendrites was examined byelectron microscopy. When stained for KCC2, OFFbipolar cell dendrites stained at their tips (Fig. 10A),
but these remained unstained for NKCC (Fig. 10C).
Staining for ON bipolar cells was examined both bycolocalization with mGluR6 (Fig. 10B) and by electron
microscopy (Fig. 10C, D). Dendritic tips of rod bipolarcells and ON cone bipolar cells stained for NKCC, but
not for KCC2.
Discussion

Fig. 9 Staining for the chloride cotransporters agrees with known
ECl . (Left three) double labeling for NKCC and calbindin (marker forhorizontal cells), rabbit. Stainings are colocalized in horizontal cellsomas (HC) and their primary processes. Strong punctate stain for
NKCC in the upper OPL is not clocalized with horizontal cell processes (see ﬁgure 10). (Right three) double labeling for KCC2 andPKC (marker for rod bipolar cells), rabbit. Stained with PKC is anaxon of a rod bipolar cell (ax) and its terminal in stratum 5 of the IPL(at). KCC2 was localized in the axon terminal but not in the axon
itself. Scale bar, 5 mm. (Bottom) electron micrograph, monkey. Staining for KCC2 is associated with the plasma membrane of ganglion cell
dendrites (highlighted in pink, gc-den).

The neurochemical organization of the ﬁrst visual
synapse is unique in its complexity (summarized in Fig.
11), and it is designed to transmit center-surround antagonistic signal to about 10 parallel pathways.
Parallel pathways
There are about 10 parallel pathways in photopic
vision, about 5 in the OFF channel and 5 in the ON
channel. Within each channel, the parallel pathways
are thought to transmit different temporal frequen-Keio J Med 2002; 51 (3): 154–164

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Fig. 10 Dendritic tips of OFF bipolar cells express KCC2 and those of ON bipolar cells express NKCC (monkey). (A, C, D) Electron micrographs. (A) KCC2 is present in ﬂat contacts (fp) and not in central elements (ce); cp, cone pedicle; r, ribbon. (B) Double staining for NKCC andmGluR6. Staining is colocalized in large puncta representing rod bipolar dendritic tips (rb) and in smaller puncta representing dendritic tips of
ON cone bipolar cells (cb). Diffuse stain for NKCC below the puncta represents horizontal cells’ primary dendrites. (C) NKCC is expressed by
the central element and is absent from ﬂat bipolar dendritic tips. (D) NKCC is expressed by rod bipolar dendritic tips (rb); rt, rod terminal.

cies.3,58–61 The fundamental differences between the
OFF and the ON channels are now understood. The
OFF bipolar cells express ionotropic glutamate receptors; when bound to glutamate, these receptors open
and permit an inﬂux of cations. In contrast, the ON
pathways express mGluR6. When bound to glutamate,this receptor activates the G-protein Go1 , which closes a
non-speciﬁc cation channel. In the absence of glutamate
the channel is maintained at an open state, which
depends on intracellular [cGMP]. However, the identityof the channel, and how Go1 causes its closure are still
unknown.
Also unsolved is what molecular mechanisms underlie the differences between cell types. In the ON pathways, we have shown that all types of ON bipolarcells express mGluR6 and Go1 , thus the differential
responses have to arise either from their different distance from the release site,16 or by differential modulations of the cascade. In the OFF pathways, different
bipolar types may express different combinations of thefour iGluR subunits that form a receptor, but this has
not been resolved yet.

Bipolar cell center-surround receptive ﬁeld
It is clear from multiple recordings that feedback
from horizontal cells to cones contributes to bipolarcells surround by shifting the activation curve for Ca 2þ ,but how horizontal cells manage to shift this curve is
not clear. Some investigators believe that the inhibition
is GABAergic, but others attribute the feedback to anephatic effect, in which the shift is created by a current
through hemichannels located on the two appossing
horizontal cell membranes under the ribbon.43 To add
to the puzzle, we should note that even the morphology
and the location of the feedback synapse have not been
identiﬁed because the synaptic complex contains noconventional structures that might disclose the locationof the feedback synapse.
In addition to feedback, horizontal cells affect bipolar cell responses by directly synapsing onto them. It is
now accepted that the horizontal to bipolar synapse
is GABAergic, and that bipolar cells respond via
GABAA and GABAC receptors. We have shown thatOFF bipolar cells express KCC2 and we predict that

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Does the ON rod bipolar cell maintain a chloride
gradient?

Fig. 11 The ﬁrst visual cone synaptic complex: structure and neurochemistry. The cone pedicle (cp) releases glutamate onto horizontal
cell processes (h) and ON and OFF bipolar cell dendrites by an exocytotic process that occurs at the release site located just under thesynaptic ribbon (r) (to which synaptic vesicles are tethered). Endocytosis occurs at regions ﬂanking the release site (arrowhead). The conecontacts the second order neurons at three specialized regions, atthese cotacts, the cone membrane is electron-dense (thick lines). The
contact between cone and horizontal cell is at the invagination, justbeneath the ribbon; the horizontal cell membrane at this contact iselectron-dense; and it expresses ionotropic glutamate receptors(iGluRs). The contact between cone and OFF bipolar dendrite (ﬂatcontact) is at the cone base; the bipolar membrane is electron-dense
and it expresses iGluRs. The contact between cone and ON bipolar
dendrites is at the mouth of the invagination, the bipolar membrane isonly slightly dense; it expresses mGluR6, Go1 , and an unidentiﬁed
non-selective cation channel. Horizontal cell processes contact ON
bipolar cells at the triad, but they probably contact both ON and OFF
bipolar dendrites as they ascend to invaginate the cone. These contacts are largely unspecialized, but they are revealed by GABAA receptor that concentrates on bipolar membranes apposing horizontal
cells. Along with the GABAA receptor, ON bipolar dendrites express
NKCC and OFF bipolar dendrites express KCC2.

they maintain an ECl negative to rest. Under these
conditions, GABA would antagonize responses to glutamate and will contribute to surround response. ON
bipolar dendrites express NKCC, so they are predicted
to maintain an ECl positive to rest. If so, GABA could
contribute to surround response by depolarizing thecells. Recent measurement of ECl in bipolar cells conﬁrmed that ECl in rod bipolar cells was higher than thatin ON cone bipolar cells, which was higher than in OFF
cone bipolar cells.62 The difference between rod bipolar cells and OFF cells was signiﬁcant, but the difference
between ON and OFF cone bipolar cells was not.

Theoretical considerations require that the rod
bipolar’s dendrite maintains different ECl than its axon
terminal. As explained above, dendrites should depolarize to GABA, but axon terminals should hyperpolarize. This is because GABAergic amacrine cells
that feedback onto the rod bipolar axon terminal depolarize to light.63 Thus when a light stimulus depolarizes rod bipolar cells, it also depolarizes the A17
amacrine cell. GABA would then be released with adelay and hyperpolarize the rod bipolar cell. This prediction is supported by the expression pattern of thechloride cotransporters (NKCC in dendritic terminal,
KCC2 in axon terminal). ECl measurements by physiological methods are controversial, one study measured
the same ECl for GABA applied to dendrites and toaxons,62 and another study measured a difference.64
Whether or not it is theoretically possible to maintaina gradient within such a compact cell is not clear because it depends on the level of activity of the chloride
transporters and on the diffusion parameters within thecell. It should be mentioned that chloride gradients
within larger cells (hippocampal neurons) have beenreported.65
Acknowledgements: We thank Yi-Jun Shi, Sally Shrom, Tehilla BarYehuda, and Jian Li for excellent technical assistance. Supported by
grant EY11105.

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