Mammalian retinal horizontal cells are unconventional GABAergic neurons

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*Department of Neurobiology and Genetics, Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS UMR_7104,

Inserm U 964, Universite de Strasbourg, Illkirch, France

�Institut de la Vision, INSERM UMR_S968, CNRS UMR_7210, Universite Pierre et Marie Curie Paris 06, Paris, France

�Imaging Center, Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS UMR_7104, Inserm U 964,

Universite de Strasbourg, Illkirch, France

§Eurexpress Platform, Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS UMR_7104, Inserm U 964,

Universite de Strasbourg, Illkirch, France

¶Department of Gene Technology and Developmental Neurobiology, Institute of Experimental Medicine, Budapest, Hungary

**Centre d’Investigation Clinique 503, Inserm-Centre Hospitalier National d’Ophtalmologie des Quinze-Vingts, Paris, France

��Fondation Ophtalmologique Adolphe de Rothschild, Paris, France

Retinal horizontal cells (HCs) are long known to be involved inthemodulation of information transfer between photoreceptorsand bipolar cells (Baylor et al. 1971; reviewed in Kamermansand Spekreijse 1999; Piccolino 1995) but the mechanism by

Received August 4, 2010; revised manuscript received November 9,2010; accepted November 10, 2010.Address correspondence and reprint requests to Michel J. Roux,

Department of Neurobiology and Genetics, Institut de Genetique et deBiologie Moleculaire et Cellulaire, CNRS UMR 7104, Inserm U 964,Universite de Strasbourg, 1 rue Laurent Fries BP10142, 67404 IllkirchCedex, France. E-mail: [email protected]

1These authors contributed equally to this study.2The present address of Eric Wersinger is The Johns Hopkins UniversitySchool of Medicine, Department of Otolaryngology Head and NeckSurgery, 720 Rutland Ave. Ross 818, Baltimore, MD 21205, USA.Abbreviations used: aCSF, artificial CSF; AMPA, a-amino-3-hydroxy-

5-methylisoxazole-4-propionate; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; DAB, 3,3¢-diaminobenzidine; GAD, glutalic aciddecarboxylase; GFP, green fluorescent protein; HC, horizontal cell; INL,inner nuclear layer; IPL, inner plexiform layer; NGS, normal goat serum;OPL, outer plexiform layer; PBS, phosphate buffer saline; PFA,paraformaldehyde; PLP, pyridoxal 5¢-phosphate.

Abstract

Lateral interactions at the first retinal synapse have been ini-

tially proposed to involve GABA by transporter-mediated re-

lease from horizontal cells, onto GABAA receptors expressed

on cone photoreceptor terminals and/or bipolar cell dendrites.

However, in the mammalian retina, horizontal cells do not

seem to contain GABA systematically or to express mem-

brane GABA transporters. We here report that mouse retinal

horizontal cells express GAD65 and/or GAD67 mRNA, and

were weakly but consistently immunostained for GAD65/67.

While GABA was readily detected after intracardiac perfusion,

it was lost during classical preparation for histology or elec-

trophysiology. It could not be restored by incubation in a

GABA-containing medium, confirming the absence of mem-

brane GABA transporters in these cells. However, GABA was

synthesized de novo from glutamate or glutamine, upon

addition of pyridoxal 5¢-phosphate, a cofactor of GAD65/67.

Mouse horizontal cells are thus atypical GABAergic neurons,

with no functional GABA uptake, but a glutamate and/or glu-

tamine transport system allowing GABA synthesis, probably

depending physiologically from glutamate released by photo-

receptors. Our results suggest that the role of GABA in lateral

inhibition may have been underestimated, at least in mam-

mals, and that tissue pre-incubation with glutamine and pyri-

doxal 5¢-phosphate should yield a more precise estimate of

outer retinal processing.

Keywords: horizontal cell, inhibitory transmission, mouse,

neurotransmitter transporter, retina, vesicular release.

J. Neurochem. (2011) 116, 350–362.

JOURNAL OF NEUROCHEMISTRY | 2011 | 116 | 350–362 doi: 10.1111/j.1471-4159.2010.07114.x

350 Journal of Neurochemistry � 2010 International Society for Neurochemistry, J. Neurochem. (2011) 116, 350–362� 2010 The Authors

which they do so is still highly debated. It was initially thoughtto be mediated by GABA, as in lower vertebrates HCs havebeen demonstrated to release GABA [via reverse uptake andnot by a Ca2+-dependent, vesicular mechanism (Schwartz1987)] and that cones were responding to externally appliedGABA (Tachibana and Kaneko 1984). However, subsequentwork has put forward alternative hypothesis for the basis ofcenter-surround receptive fields, through a modulation of thesynaptic cleft pH (Hirasawa and Kaneko 2003; Cadetti andThoreson 2006; Davenport et al. 2008) or by an ephapticmechanism implicating hemigap junctions (Kamermans et al.2001; Pottek et al. 2003; Fahrenfort et al. 2004).

In mammalian retinas, expression of GABA receptors oncone terminals is controversial: GABAA and GABAC recep-tors were described in cultured pig cones (Picaud et al.1998b) and in mouse cones from degenerated rd1 retinas(Pattnaik et al. 2000), but responses to GABA were notdetected in cones from macaque (Verweij et al. 2003) or greysquirrel (S. DeVries, personal communication) retinas. Whilethis argues against a major role for HC feedback onto cones,data obtained in the last decade points toward an implicationof this inhibitory neurotransmitter in the mammalian outerretinal processing. HCs express the vesicular GABA andglycine amino-acid transporter (VIAAT or VGAT) in mouse,macaque and guinea pig retinas (Haverkamp et al. 2000;Cueva et al. 2002; Jellali et al. 2002; Guo et al. 2010) as wellas in cultured human HCs (Jellali et al. 2002). In addition,HCs were shown to express the synaptic proteins required forvesicular release in the rabbit (Hirano et al. 2005, 2007) andguinea pig (Lee and Brecha 2010) retinas – though there is nowell-defined pre-synaptic specialization in HC terminals, norpost-synaptic density in the contacted photoreceptors andbipolar cell dendrites. On the post-synaptic side, a role forGABA in the triadic synapse formed by photoreceptors,bipolar and horizontal cells, is suggested by the expression ofGABA receptor in bipolar cells dendrites (reviewed in Wassleet al. 1998). Moreover, the differential expression of the twoCl) co-transporters, NKCC and KCC2, in ON and OFFbipolar cell dendrites (Vardi et al. 2000), allowing GABA tobe depolarizing in the ON pathway (Varela et al. 2005a;Duebel et al. 2006) is compatible with HC influencing thecenter-surround receptive fields of bipolar cells through feed-forward GABAergic signaling.

However, heterogeneous results were published regardingthe GABAergic nature of mammalian HCs. Notably, innocturnal rodents, GABA and/or glutamic acid decarbox-ylase (GAD) were detected in HCs only during development(Schnitzer and Rusoff 1984; Agardh et al. 1986; Versaux-Botteri et al. 1989; Fletcher and Kalloniatis 1997; Yamasakiet al. 1999; Schubert et al. 2010). Furthermore, by contrastwith classic GABAergic neurons, mammalian HCs do notexpress membrane GABA transporters, not even the lowaffinity betaine/GABA transporter BGT-1 (Honda et al.1995; Johnson et al. 1996; Guo et al. 2009, 2010).

As green fluorescent protein (GFP) expression was drivenby the GAD65 promoter in a subset of adult HCs in atransgenic mouse line, we re-investigated the presence ofGABA in these cells. GAD65 and/or GAD67 were detectedboth by immunohistochemistry and in situ hybridization,however at a lower level than in amacrine cells. GABA waspresent in HCs after fixation by intracardiac perfusion,especially when vesicular release was blocked by addition ofantagonists of a-amino-3-hydroxy-5-methylisoxazole-4-pro-pionate (AMPA)/kainate receptors and voltage-dependentcalcium channels. GABA was however absent from HCs inretinas prepared for electrophysiology, even after incubationat 37�C in Ames medium. As this is a prerequisite todetermine the physiological role of HC GABA release,retinas were incubated in various conditions with the aim tofavor this restoration. GABA was not taken up by HCs, butaddition of the GAD cofactor pyridoxal 5¢-phosphate (PLP)increased significantly GABA restoration in HC, in thepresence of either glutamate or glutamine, at physiologicaltemperature. This indicates that, though they don’t possessmembrane GABA transporters, mouse HCs can synthesizeGABA and contain GABA in vivo, and therefore appear tobe true GABAergic neurons. Assessments of lateral inhibi-tion in the outer retina – or of the global retinal processing –on slices or whole-mount mammalian retinas should thus beperformed in the presence of glutamine and PLP, to get closerto in vivo conditions. More generally, the neurotransmitterstatus of a given tissue prepared for electrophysiologyrecordings may differ significantly from the one reported byimmunohistology performed after intracardiac perfusion.

Materials and methods

AnimalsWe used adult mice (> 8 weeks) from several strains (Balb/cJ,

C57Bl/6J, 129SV/Pas, CD1, GAD65-eGFP transgenic mice, n = 5),

either purchased from Charles River (L’Arbresle, France) or bred

locally in the Institut Clinique de la Souris animal facility. Animals

were housed under a 12 h light/dark cycle and had access to food and

water ad libitum. Procedures involving animals and their care were

conducted in agreement with the French Ministry of Agriculture and

the European Community Council Directive no. 86/609/EEC, OJL

358, 18 December 1986. Generation of the transgenic mouse line

GAD65_3e/gfp5.5 30, expressing GFP under the control of the

GAD65 promoter was previously described (Lopez-Bendito et al.2004). Briefly, a 6.5 kb segment of the GAD65 gene that includes

5.5 kb of the 5¢-upstream region, the first two exons and a portion of

the third exon and the introns in between drives the expression of

GFP. Expression was confirmed by visualizing GFP fluorescence invivo, using GFP goggles (BLS Ltd, Budapest, Hungary), through the

fluorescence from neonate brains.

Tissue preparation for immunohistochemistryDepending on experiments, animals were either fixed by intracar-

diac perfusion (see below) or killed by cervical dislocation. In the

� 2010 The AuthorsJournal of Neurochemistry � 2010 International Society for Neurochemistry, J. Neurochem. (2011) 116, 350–362

GABA volatility in horizontal cells | 351

latter case, eyes were enucleated, globes were cut along the oraserrata and the anterior part of the eyeball was removed, and

remaining vitreous humor separated from the retina. Retina was then

detached from the sclera in 0.1 M phosphate buffer saline (PBS), pH

7.4, eventually incubated (see GABA restoration below) and fixed

by immersion for 2 h in 4% paraformaldehyde (PFA) in PBS.

Retinas were then processed either to 14 lm cryosections, after

cryoprotection in 30% sucrose PBS and embedding in Shandon

Cryomatrix (Anatomical Pathology International, Runcorn, UK),

using CM3000 cryostat (Leica Microsystems GmbH, Wetzlar,

Germany), or embedded in 3% agarose and sliced (200 lm) with

a VT1000S vibratome (Leica Microsystems GmbH).

Where indicated, tissue fixation was performed by intracardiac

perfusion. Animals were anesthetized with an intraperitoneal

injection of ketamine (70 mg/kg) and xylazine (5 mg/kg) and then

perfused with cold PBS supplemented with 50 lM 6-cyano-7-

nitroquinoxaline-2,3-dione (CNQX) and 100 lM cadmium ions

(Cd2+, from CdCl2), then with 4% PFA in cold PBS also with

CNQX and Cd2+. The eyes were removed and retinas isolated before

post-fixation (2 h in 4% PFA) then included and vibratome sliced.

GABA restorationFor GABA restoration experiments, the retina was dissected out

conventionally and then placed for at least 1 h 30 min at 37�C in an

incubating medium, constantly bubbled with a 95% O2/5% CO2

mixture, both for oxygenation and pH buffering. Depending on the

experiment, 200 lm thick vibratome-slices of retina were cut prior

to the incubation, and in other cases whole-mount retinas were

incubated before inclusion and slicing. Incubating media are

composed of artificial CSF (aCSF) with (in mM) NaCl 126, KCl

2.5, MgCl2 1.2, NaH2PO4 1.2, NaHCO3 18, CaCl2 2.4, Glucose 11,

supplemented with PLP (150 lM) and glutamine (1 mM) or

glutamate (1 mM). We also used as a comparison Ames’s medium

(Sigma-Aldrich, St-Louis, MO, USA) supplemented with glutamate

(1 mM) and CNQX (50–250 lM) or GABA (1 mM) and bicucul-

line (100 lM). After incubation, the tissue was transferred to a

glutamine/glutamate-free medium (for 30 min to 1 h 30 min) and

then fixed by immersion in 4% PFA. Room temperature was 22�C.

ImmunohistochemistryAll antibodies used in this study have been previously characterized,

or have staining patterns that match those previously reported in the

mammalian retina. All details regarding their characterization,

validation and use can be found under the experimental methods

section in Appendix S1 and in Table S1.

We used mainly the indirect fluorescence method to perform

immunohistochemical labeling of the retina. Retinal slices were

blocked for 2 h in a solution containing 10% normal goat serum

(NGS) and 0.5% Triton-X-100 in PBS. Primary antibodies were

diluted in 5% NGS and 0.5% Triton-X-100 in PBS and applied

overnight at 4�C. After washing in PBS, secondary antibodies [goat

anti-mouse or goat anti-rabbit Fab’2 coupled to Cy3 (Jackson

Laboratories, West Grove, PA, USA) or Alexa 488 (Molecular

Probes, Eugene, OR, USA), diluted 1/500 in 0.5% Triton-X-100 and

4¢,6-diamidino-2-phenylindole (DAPI) 1 lg/mL in PBS] were

applied for 2 h at 22�C. For both primary and secondary antibody

incubations, we made double-labeling with incubation solutions

containing two antibodies. Fluorescence pictures were acquired with

a Leica TCS SP2 confocal microscope with a 63· oil immersion

objective. Z-projections of stacks were made using the ImageJ

software (NIH, http://rsbweb.nih.gov/ij/). In all immunohistochem-

istry figures except Fig. 1(b), the 4¢,6-diamidino-2-phenylindole

(DAPI) staining (blue) was added to the merge images, to facilitate

the identification of the retinal layers.

For GAD65 and GAD67 immunostaining, we used in addition a

peroxidase/3,3¢-diaminobenzidine (DAB) amplification step. Retinal

slices were immersed into 3% H2O2 and 50% ethanol for 15 min at

22�C to quench endogenous peroxidases. After washing in PBS,

slices were blocked for 1 h in PBS containing 5% NGS and 0.5%

Triton-X-100. Primary antibodies were diluted in 0.5% Triton-X-

100 in PBS and applied overnight at 22�C. A biotinylated secondary

antibody (goat anti-mouse, BA-9200, Vector Labs, Burlingame, CA,

USA), diluted 1/500 in 0.5% Triton-X-100, was applied for 2 h at

22�C. Slices were incubated for 2 h in VECTASTAIN� Elite� ABC

Reagent (PK 6100, Vector Labs), washed in PBS, then incubated in

a DAB solution (D4168-50SET, Sigma-Aldrich).

In situ hybridizationDigoxigenin-labeled riboprobes, generated by in vitro transcription

(Dig-UTP from Roche, Mannheim, Germany) from DNA templates

using T7 (antisense) and SP6 (sense)-bacteriophage RNA Polyme-

rases (Promega, Madison, WI, USA), were used to detect GAD65

and GAD67 mRNAs. For GAD67, DNA template was a 634 bp

cDNA fragment of mouse GAD1 (GenBank NM_0080077), gift of

F. Guillemot, subcloned into a pGEM-T Easy vector. The templates

for transcribing RNA were made by linearizing recombinant

plasmids. For GAD65, DNA templates were kindly provided by

the Genepaint organization. They were synthesized by RT-PCR and

the initial PCR products were gel-purified and sequence–verified

before the transcription (see detailed protocol and information about

templates at http://www.genepaint.org).

Hybridization and detectionCryosections (12 lM) of non-fixed retina were collected on

RNAse treated Super-Frost Plus Slides (Fisher Scientific, Illkirch,

France). Fixation, carried out in a Leica Autosteiner XL, was in

4% paraformaldehyde for 20 min at 22�C. Slides were then

washed for 5 min in PBS, acetylated and dried by passing them in

successive ethanol solutions (70%, 90% and 100%). The hybrid-

ization steps were performed using a Tecan Genesis Freedom evoliquid handling platform (Tecan Deutschland Gmbh, Crailsheim,

Germany). Briefly, slides carrying retina sections were integrated

into flow-through chambers positioned into the Tecan Platform.

After a first step of proteinase K digestion (0.01 lg/mL in 50 mM

Tris, 5 mM EDTA, 0.05% Tween-20, pH 8.0) in PBS, sections

were pre-hybridized for 30 min in a Hyb-mix solution (Ambion,

Austin, TX, USA: B8807G). Sections were hybridized for 5 h

30 min in a humid chamber at 64�C in a solution consisting of the

pre-hybridization solution with the addition of the digoxigenin-

labeled RNA probe at a concentration of 300–600 ng/mL. Sections

were processed for the immunodetection of the digoxigenin label

using an anti-digoxigenin antibody coupled to peroxidase (Roche,

Penzberg, Germany) and revealed with a tyramide-biotin amplifi-

cation reaction. After the color reaction, sections were washed with

blocking solutions and cover-slipped with hydro-matrix (Micro-

Tech Lab, Graz, Austria).

Journal of Neurochemistry � 2010 International Society for Neurochemistry, J. Neurochem. (2011) 116, 350–362� 2010 The Authors

352 | S. Deniz et al.

Results

GAD65 promoter drives GFP expression in mouse horizontalcellsTransgenic mice expressing fluorescent proteins under thecontrol of cell-type specific promoters are useful tools toidentify cell populations in living tissues, facilitating theirstudies using electrophysiological or imaging techniques.Though HCs have been reported to be GABA immunoneg-ative in the adult mouse retina (Haverkamp and Wassle 2000;Schubert et al. 2010), we investigated the eventual GFPpresence in mouse HCs in a transgenic GAD65-eGFP mouseline (Lopez-Bendito et al. 2004). On retinal slices, a denseGFP signal was present in the inner half of the inner nuclearlayer (INL) (Fig. 1a), corresponding to amacrine cells andprobably some OFF bipolar cells (Kao et al. 2004), as therewas no colocalisation with Goa, a specific marker of ONbipolar cells (Figure S1). This pattern was similar to the onereported in a GAD67-eGFP knock-in mouse line (May et al.2008). In addition, some GFP expressing cells were alsopresent in the outer half of the INL, next to the outerplexiform layer (OPL), with a dense arborization in this

synaptic layer, with invaginating processes typical of HCs.Identification was confirmed by co-labeling with the HCmarker calbindin (Fig. 1a and b). Colocalization of GFP andcalbindin was not systematic, indicating that GFP wasexpressed in only a subset of HCs. We did not quantifyprecisely this population, but it was close to 10% of all HCs,with a tendency to have more GFP positive cells in theperiphery than toward the center of the retina.

Horizontal cells express GAD65 and GAD67As transgene expression may be ectopic, we checkedwhether HCs were expressing GABA synthesizing enzymes,using in situ hybridization with tyramide amplification tovisualize low levels of mRNA. For short durations(£ 45 min), GAD65 and GAD67 mRNA were detected onlyin cells in the inner half of the INL, as well as in a few cellsin the ganglion cell layer (data not shown). This correspondsto amacrine and displaced amacrine cells, well-describedpopulations of GABAergic neurons in the mouse retina(Haverkamp and Wassle 2000). For longer durations (1–2 h),GAD65 and GAD67 mRNAs were detected additionally in afew cells in the external part of the INL, in the two different

(a)

(b)

Fig. 1 GFP expression is driven by the GAD65 promoter in a subset

of mouse horizontal cells. Confocal images from cryosections of

retinas from transgenic GAD65-eGFP mice on a C57Bl/6J back-

ground, immunostained for calbindin (center panels and red in

merged images, right). The GFP signal is shown in the left panels

and in green in the merged images. (a) General view of the retinal

layers. (b) Detailed view of the horizontal cell processes. ONL, outer

nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer;

IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars:

40 lm (a) and 10 lm (b).



mice strains tested, Balb/cJ (Fig. 2a and b) and C57Bl/6J(data not shown), with a pattern similar to the distribution ofHCs.

To verify if GAD65 and/or GAD67 could also be detectedat the protein level in HCs, we performed immunohisto-chemistry with an antibody recognizing the two isoforms, onboth the C57Bl/6J and Balb/cJ mice, as well as on twoadditional strains, the 129SV/Pas and CD1, all these strainsbeing commonly used in physiological experiments and/or inthe construction of genetically modified mice. In all fourstrains, GAD65/67 immunostaining was stronger in the innerplexiform layer (IPL) as well as around the soma of cells inthe proximal part of the INL and, to a lesser extent, in theganglion cell layer (Fig. 2c for Balb/cJ, and Figure S2 for theother strains). HCs, identified with an anti-calbindin anti-body, were also labeled with the GAD65/67 antibody, thestaining being mainly detected in their perinuclear region(Fig. 2c for Balb/cJ, and Figure S2 for the other strains),

probably corresponding to the Golgi apparatus, as previouslyreported in neurons (Oertel et al. 1981), and visible also inamacrine cells. Based on the perinuclear labeling, at least70% of calbindin-positive cells were also immunoreactive forGAD65/67, depending on the strain considered: C57Bl/6J:70.0 ± 1.2% (one mouse, 27 slices, 1330 out of 1900calbindin positive cells); Balb/cJ: 88.9 ± 0.9% (two mice, 36slices, 1762 out of 1982 calbindin positive cells); CD1:70.5 ± 2.1% (three mice, 15 slices, 642 out of 910 calbindinpositive cells); 129 SV/Pas: 72.1 ± 1.5% (one mouse, 18slices, 841 out of 1167 calbindin positive cells) (Fig. 2d). Inaddition, we did not see any obvious difference in thepercentage of HCs immunoreactivity for GAD65/67 betweenthe central and peripheral region of the retina.

Horizontal cells contain GABAThe presence of GABA synthesizing enzymes in adultmouse HCs raised the question of whether GABA could be

(a)

(c)

(b)

(d)

Fig. 2 GAD expression in adult mouse horizontal cells. GAD65 (a)

and GAD67 (b) mRNAs expression on retinal slices from Balb/cJ

mice detected by in situ hybridization. While most of the signal was

located in the proximal part of the INL, corresponding to amacrine

cells, and to the GCL, a discrete labeling (black arrowheads) was

present in the distal part of the INL, with a distribution corresponding

to the repartition of horizontal cells. (c) A pan-antibody recognizing

both GAD65 and GAD67 (green) labels horizontal cells, identified by

calbindin immunoreactivity (red), mostly in their perinuclear region

(white arrowhead). A similar pattern is observed in amacrine cells

(white arrows), which processes are in addition heavily labeled in the

IPL. (d) Percentage of GAD immunoreactive cells reported to cal-

bindin positive cells in the OPL in several mouse strains. Almost 70%

of HCs were labeled with GAD antibody in all four strains (88.9% for

Balb/cJ mice, see text for details). Error bars represent SEM. Scale

bars = 20 lm.



present in these cells, despite previous reports of its absence(Haverkamp and Wassle 2000). A possible origin of thisapparent discrepancy is the fact that most of retinalimmunohistochemistry is classically performed on tissuefixed postmortem. It was reported that in the zebrafish, HCGABA content was lost rapidly upon depolarization(Kalloniatis et al. 1996); this could happen similarly inthe mouse, during the ischemic period of enucleation andanterior segment dissection, prior to fixation. To avoid thisphenomenon and thus to be closer to in vivo conditions, weperformed immunohistochemical analysis on tissue col-lected after intracardiac perfusion of the animal with 4%PFA. Immunolabeling for calbindin and GABA of retinasfixed according to this protocol was performed on 16animals (Balb/cJ: n = 7; C57Bl/6J: n = 3; 129SV/Pas:n = 3; CD1: n = 3). Results are shown in Fig. 3 forC57Bl/6J and Balb/cJ mice, and in Figure S3 for CD1 and129SV/Pas. As expected, amacrine cells and the sublaminaein the INL were strongly immunopositive for GABA. WhileHCs were not as strongly labeled, GABA could readily beseen in both HC somas and processes, especially in theBalb/cJ mice (Fig. 3b), in which 78.2 ± 2.8% HCs wereGABA immunopositive (558 out of 713 calbindin positive

cells, on 12 slices from three mice). For C57Bl/6J, only34.4 ± 2.7% HCs contained GABA (77 out of 223calbindin positive cells, on three slices from a singlemouse). GABA immunostainings in 129SV/Pas and CD1,though not quantified, were similar to what was observed inC57Bl/6J.

To favor further GABA detection in HCs in Balb/cJ retina,we tried to limit HC depolarization and GABA vesicularrelease during tissue preparation, by supplementing thefixative with 100 lM Cd2+, an inhibitor of voltage-depen-dent Ca2+ channels, and 50 lM CNQX, an inhibitor ofAMPA/kainate glutamate receptors to limit HC depolariza-tion. This resulted in a greater number of GABA-immuno-positive HCs, with staining both in somas and processes(Fig. 4a). GABA was present homogeneously all along theOPL, to a level closer to the one in amacrine cells (n = 4). Inaddition, some Muller glial cells were also labeled, thoughnot homogeneously along the retina. The high ratio ofGABA-containing HCs can be appreciated on a flatmountretina. On the 750 lm · 750 lm zone of intermediate retina(at mid distance between the optic nerve head and the retinalperiphery) shown in Fig. 4(b), 602 HCs were GABAimmunopositive out of 678.

(a)

(b)

Fig. 3 GABA is detected in horizontal cells after fixation by intracar-

diac perfusion in both C57Bl/6J (a) and Balb/cJ (b) mouse retinas.

While most of the GABA immunoreactivity (left panels, and green in

merged right images) was in the proximal part of the INL, in the IPL

and in the GCL, horizontal cells (arrowheads), identified by calbindin

immunoreactivity (center panels, and red in merged right images),

were also stained, to a higher degree in Balb/cJ than in C57Bl/6J

retinas. Scale bars: 50 lm.



GABA content of horizontal cells is volatile, and lost inelectrophysiological conditionsThese results supported the idea that GABA is rapidly lostfrom HCs in the minutes after animals were killed. As theretina isolation for electrophysiology is longer than forimmunohistochemistry, this suggested that GABA may beabsent from HCs in recording conditions (whole mountretinas or retinal slices). We thus checked if GABA waspresent in flatmount retina, either right after preparation orafter 1 h recovery at 37�C in classical electrophysiologysolutions, aCSF or Ames medium (Ames and Nesbett 1981),supplemented with 50 lM CNQX to favor horizontal cellpolarization, and thus limit their GABA release. Figure 5represents an immunolabeling performed on a Balb/cJ mouseretina after dissection and 1-h incubation in Ames medium at37�C. In these conditions, no GABA was detected in HCs,while amacrine cells and IPL were still strongly immunola-beled with GABA, as well as radial processes likely to beMuller glial cells (n = 3). Similar results were observed withincubation in aCSF at 22�C (n = 3) or at 37�C (n = 4) (datanot shown).

HC do not have a functional GABA uptake systemWhile the Ames medium contains many amino-acids,GABA is absent from its composition, which could hindera restoration of GABA in HC after retinal isolation ifuptake is a major source. To assess this hypothesis, wesupplemented the incubation medium, either aCSF at 22�Cor Ames at 37�C with 1 mM GABA. To limit HCdepolarization, and thus limit vesicular release and increasea potential electrogenic GABA uptake, the mediumalso contained 50 lM CNQX to block AMPA/kainatereceptors, and 100 lM bicuculline methiodide to blockGABAA receptors, as GABA, though classically aninhibitory transmitter, has been reported to depolarizemammalian HCs (Blanco et al. 1996; Varela et al. 2005b).Following 1-h incubation at 22�C in supplemented aCSF(Figure S4a, n = 3) or at 37�C in supplemented Amesmedium (n = 2), HCs were not immunopositive for GABA(Fig. 6a, Balb/cJ retina), while amacrine and Muller cellswere strongly labeled, as expected as they express theGABA transporters GAT-1 and/or GAT-3 (Johnson et al.1996).

(a) (b)

Fig. 4 Inhibiting vesicular release increase GABA immunoreactivity in

horizontal cells. Confocal images from a vibratome slice (a) or flat-

mount retina (b) from a Balb/cJ mouse, immunolabeled for GABA (top,

and green in merged bottom images) and calbindin (center, and red in

merged bottom images), after intracardiac perfusion of 4% parafor-

maldehyde in PBS, supplemented with 50 lM CNQX, to hyperpolarize

HCs, and 100 lM CdCl2 to block voltage-dependent calcium channels

and thus vesicular release. Scale bars: 100 lm in (a), 200 lm in (b).



HC can synthesize GABA from glutamate or glutamineAnother way to refill HCs is GABA neosynthesis fromglutamate, as GAD65/67 was detected in these cells.Glutamate can be taken up directly by HCs, which expressmembrane glutamate transporters (Rauen et al. 1996), orcould be synthesized from glutamine if these cells possess aglutamine import system. Both amino-acids are present in theAmes medium, glutamate at 7 lM and glutamine at 500 lM.As this medium was not sufficient to allow for GABArestoration in HC even at 37�C, we added 1 mM glutamate inaCSF, as well as 50 lM CNQX to block the depolarizingeffect of the excitatory amino acid. This allowed for a faintGABA immunoreactivity in HCs at 37�C (Fig. 6b, n = 2),but not at 22�C (Figure S4b).

Both GAD65 and GAD67 need PLP as a cofactor,GAD65 being predominantly found in the PLP-unboundinactive apo form (Kaufman et al. 1991), suggesting that thePLP concentration is not saturating in vivo. To favor GABAsynthesis, we thus supplemented further the incubationmedium with PLP. After 1 h at 37�C, with 1 mM glutamate,50 lM CNQX and 150 lM PLP, the GABA content of HCswas increased compared to previous conditions, both withaCSF (Fig. 6c, n = 5) or with Ames medium (Fig. 6d,n = 3). As our goal was to develop a protocol to replenishHCs to perform electrophysiology afterwards, we then triedto replace glutamate and CNQX by glutamine (1 mM), asthis could be added to the medium continuously, withoutblocking synaptic transmission. This allowed for a GABArefilling of HCs when added to aCSF at 37�C (Fig. 7a,n = 5). This refilling was stable in time, as GABAimmunoreactivity was still detected in HCs even when theincubated retina was placed 1 h 30 min in plain aCSF at22�C prior to fixation (Fig. 7b, n = 7). As Ames contains500 lM of glutamine, we supplemented this medium withonly PLP. This condition also favored the presence ofGABA in HC (Fig. 7c, n = 2). Supplementation of aCSF

with PLP alone at 37�C did also increase HC GABAcontent, though at a lesser degree (n = 3, Figure S5). Theseresults indicates that HCs can synthesize GABA in in vitroconditions provided PLP is added to the incubation medium,while an additional supplementation with glutamate or itsprecursor glutamine favors further the GABA refilling ofHCs.

Discussion

Horizontal cells contain GABA in vivoWe report here that GABA was not detected in mouse adultHCs following a classical tissue preparation for electrophys-iology, in line with the immunohistochemical characteriza-tion of the mouse retina (Haverkamp and Wassle 2000;Schubert et al. 2010). However, GABA-immunoreactivity inHCs was increased after intracardiac perfusion fixation,which should maintain the neurotransmitter pool closer toin vivo conditions. Adding pharmacological agents to thesolution further improved GABA-immunostaining of HCs,likely by preventing vesicular release. These observationswere specific to HCs, as GABAergic amacrine cells wereGABA-immunopositive in all experimental conditions, thusruling out an interaction between the different tissuepreparations and the efficacy of the GABA antibody. Thissuggests that the in vivo GABA HC content is quickly lostafter killing of animals.

More generally, these results point to a possible discrep-ancy between the neurotransmitter pools suggested byimmunohistochemistry and those effectively present in therecording conditions. In addition to the ischemic stressduring tissue preparation, differences in available metabolites(Rheims et al. 2009) and oxygenation (Hajos and Mody2009) may bias the physiological results obtained from slicesor isolated tissue.

Fig. 5 Horizontal cells do not contain GABA in recording conditions.

Confocal images of thick (200 lm) vertical slices from a Balb/cJ

mouse retina, immunolabeled for GABA (left, and green in merged

right images) and calbindin (center, and red in merged right images).

After dissection and isolation, the retina was incubated for 1 h at 37�Cin Ames medium, then fixed by immersion in PBS + 4% PFA and

sliced. Arrowheads in the left panel indicate the positions of the cal-

bindin positive somas from the center panel. Scale bar = 20 lm.



(a)

(b)

(c)

(d)

Fig. 6 Horizontal cells can synthetize GABA from glutamate. Confocal

images from vertical vibratome sections (50 lm) from Balb/cJ mouse

retinas immunolabeled for GABA (left, and green in merged right

images) and calbindin (center, and red in merged right images). (a)

When the isolated retina was incubated after dissection in Ames

medium supplemented with 1 mM GABA, 100 lM bicuculline me-

thiodide and 50 lM CNQX for 1 h at 37�C, the proximal retina and

Muller cell soma (arrowheads) and processes (arrows) were immu-

noreactive for GABA, but HC somas and processes were not labeled,

suggesting that there is no re-uptake of GABA in HCs. The outer

limiting membrane (OLM), formed by Muller cell processes, was also

strongly GABA immunoreactive. (b) When the incubating medium was

aCSF + 1 mM glutamate + 50 lM CNQX, a faint GABA immunore-

activity was seen in HCs. This immunoreactivity was increased with

the addition of 150 lM pyridoxal 5¢-phosphate, both when the medium

was aCSF (c) or Ames (d). Scale bars = 40 lm.



Horizontal cell GABA comes essentially from neosynthesis,not from uptakeMouse HCs are not able to take up GABA significantly, evenat 37�C (Fig. 6a), confirming that they don’t expressmembrane GABA transporters, as suggested in previousreports (Honda et al. 1995; Johnson et al. 1996; Casini et al.2006; Guo et al. 2009). This rules out a Ca2+-independent,transporter-mediated GABA release from HCs.

On the other hand, most adult mouse HCs expressGAD65 and/or GAD67, though at a much lower level thanamacrine cells (Figs 1 and S1). They are able to synthesizeGABA when placed in media supplemented with glutamateor glutamine. This synthesis is increased when PLP, acofactor of both GADs, is added to the incubation medium(Figs 6c,d and 7). GAD67 exists mostly in the PLP-boundactive form, to the contrary of GAD65, which is mostlyfound in the PLP-unbound, inactive form (Kaufman et al.

1991). Another indirect argument in favor of GAD65 overGAD67 is that the GAD65/67 immunolabeling in HCs wasconcentrated in the cell somas, in a ring-like structurereminiscent of the Golgi apparatus, as well as some smallpunctas in the OPL (Fig. 2c). Such a distribution is morelikely to correspond to GAD65, which is membrane-associated, while GAD67 is usually more widely distributedthroughout the neuron (Kaufman et al. 1991; Soghomonianand Martin 1998), as can be seen in amacrine cells, whichprocesses are strongly labeled in the IPL. However,antibodies specific for GAD65 and GAD67 did not allowconfirming a higher expression of GAD65 over GAD67.Both antibodies strongly labeled the amacrine cells, butthe signal in HCs could not be distinguished from thebackground, either with immunofluorescence (Figure S6aand b) or following peroxidase/DAB amplification (Fig-ure S6c and d). An alternate source of GABA could be its

(a)

(b)

(c)

Fig. 7 Horizontal cells can synthetize GABA from glutamine in the

presence of pyridoxal 5¢-phosphate. Confocal images from vertical

vibratome sections (50 lm) from Balb/cJ mouse retinas immunola-

beled for GABA (left, and green in merged right images) and calbindin

(center, and red in merged right images). (a) When the isolated retina

was incubated after dissection in aCSF supplemented with 1 mM

glutamine and 150 lm PLP for 1 h at 37�C, HC somas and processes

were immunoreactive for GABA. (b) The presence of GABA was not

transient, as HCs were still GABA immunopositive when incubated

vibratome slices (200 lm) were placed for 1 h in glutamine and PLP-

free medium prior to fixation. (c) In Ames medium, which contains

500 lm glutamine, adding 150 lm PLP for a 1 h incubation at 37�Cwas sufficient to restore GABA in HCs. Scale bars: 20 lm in (a),

15 lm in (b), 40 lm in (c).



synthesis from polyamines, as putrescine, spermine orornithine, some enzymes in the polyamines to GABApathways being dependent on PLP. However, polyamines donot seem to be present in HCs in the adult rabbit retina(Withrow et al. 2002), and the expression of ornithinedecarboxylase, the limiting step in putrescine synthesis, ispresent only at very low levels in the adult rat retina(Yamasaki et al. 1999). As the presence of glutamate orglutamine in the incubation medium favored the restorationof GABA in HCs in the presence of PLP, neosynthesis fromlow amounts of GAD65 and/or GAD67 is the most probablesource of GABA for HCs.

Differences in GABA HC content in mammalsAs retinas are frequently fixed postmortem for immunohis-tochemistry, the reported absence of GABA from adult HCsin some mammals, notably nocturnal rodents (Agardh et al.1986; Mosinger et al. 1986; Versaux-Botteri et al. 1989;Vardi and Sterling 1994; Fletcher and Kalloniatis 1997;Johnson and Vardi 1998; Haverkamp and Wassle 2000;Loeliger and Rees 2005; Schubert et al. 2010), may find itsorigin in the volatility of the neurotransmitter within thisneural population. Such volatility was also reported ingoldfish HCs, in which GABA stores can be depleted inminutes following depolarization (Kalloniatis et al. 1996).An opposite variation was described in the rabbit, in whichGABAwas absent from HCs when postmortem fixation wasperformed rapidly (� 1 min) but present when fixation wasperformed 10–40 min after death (Pow and Crook 1994). Itwas tentatively explained by an ongoing GABA synthesis, asGAD can function in ischemic conditions, while GABAcatabolism by transamination is dependent upon a-ketoglu-tarate availability, and thus aerobic conditions. Such adifference between mouse and rabbit could be explained bythe fact that the former has a retinal circulation, while therabbit retina is mostly avascular, and thus expected to beaffected less rapidly by ischemia. GAD could remain activelonger in the rabbit than in the mouse postmortem. Anotherunassessed source of discrepancy could be a differentialGABA transaminase activity.

Physiological consequence of glutamate/glutaminedependence of GABA synthesisWhether HCs synthetize their GABA physiologically fromglutamate or glutamine, the common source should beglutamate release by photoreceptors, either directly capturedby HCs via their glutamate transporters (Rauen et al. 1996),or subsequently as glutamine after transformation in Mullercells. As mouse HCs have a low capacity to synthesizeGABA, because of a low expression of GADs, this suggeststhat GABA release could be influenced by the recent localactivity. When photoreceptors are depolarized, as whenadapted to a given level of illumination, their glutamaterelease could lead to a higher GABA content in HCs, while

bright illumination or flickering stimuli for which photore-ceptors are more polarized could lower it. This wouldprovide a local, ‘recent history’ dependent, component to theinformation processing carried by HCs. Such a local rolemay depend on the local expressions of GAD65 and GAD67,and thus differ between species, being more prominent inGAD65 expressing zones, as in the macaque (Vardi et al.1994), the guinea pig (Guo et al. 2010) or in the rabbit visualstreak (Johnson and Vardi 1998).

Re-evaluating the role of GABA in the outer retinaThe lateral inhibition mediated by HCs was initially thoughtto be carried by GABA, from work in lower Vertebrates, butinconsistent pharmacology has casted doubts on such a role(reviewed in Kamermans and Spekreijse 1999; Piccolino1995), including in the macaque retina. This has led to thepropositions that HCs transmit their information throughchanges in the extracellular pH in the monkey (Davenportet al. 2008), as was previously proposed in amphibians(Hirasawa and Kaneko 2003), or through an ephapticcommunication involving hemigap junctions in the mouse(Xia and Nawy 2003), as previously proposed in lowerVertebrates (Kamermans et al. 2001; Kamermans and Fahr-enfort 2004). This does not preclude, however, that GABAmay play a role in lateral inhibition, in addition to thosemechanisms. Indeed, with the presence of GABA in HCs, allthe required elements for a GABA transmission areexpressed in the triadic synapse formed by HCs, bipolarcells and photoreceptors. HCs express the vesicular GABAtransporter VIAAT/VGAT in their terminals (Haverkampet al. 2000; Cueva et al. 2002; Jellali et al. 2002), as well asthe proteins required for vesicular release (Hirano et al.2005, 2007). While ionotropic GABA receptors do not seemto be systematically expressed on mammalian cone termi-nals, GABAA receptor subunits were found at the dendritictips of all bipolar cells in various mammalian retinas,including those of humans and macaques, with the strongeststaining being in apposition to horizontal cell processes(Haverkamp et al. 2000; Vardi and Sterling 1994; reviewedin Wassle et al. 1998), supporting the hypothesis of aGABAergic feedforward from HC to bipolar cells. Acriticism often raised against feed-forward was that such asignaling would modulate similarly the ON and OFFpathways, while a contrast-reinforcing process should exertan opposite action on the two channels. However, thedendrites of ON and OFF bipolar cells were demonstrated toexpress different chloride transporters (Vardi et al. 2000),allowing for a depolarizing action of GABA on ON dendrites(Varela et al. 2005a; Duebel et al. 2006). GABA could alsoact as an autocrine regulator as shown in the tiger salamander(Kamermans and Werblin 1992), as HC express GABAA

(rabbit, mouse and human, Blanco et al. 1996; Feigenspanand Weiler 2004; Picaud et al. 1998a) and/or GABAB (rat,Koulen et al. 1998) receptors.



The presence of all these elements on the pre- and post-synaptic sites is of course not a proof of the implication ofhorizontal cell GABA release in lateral inhibition. Electro-physiological recordings of HCs, their GABA sensitivesynaptic partners, and/or ganglion cell surround after incu-bation in glutamine/PLP containing medium should allow tore-evaluate the possible roles played by GABA in outerretinal processing.

Acknowledgements

This work was supported through HFSP grant RGY0004/2003 to

MJR, ANR GABARET to SP, Association francaise contre les

Myopathies (AFM) fellowship to EW, Universite Paris 6 fellowship

to SD, CNRS and Inserm. We thank Dr. Heinz Wassle and Nicholas

Brecha for their comments on the manuscript, and the Eurexpress

platform, especially Muriel Philipps and Violaine Alunni, for their

assistance with in situ hybridization experiments. Authors have no

conflict of interest.

Supporting information

Additional Supporting information may be found in the online

version of this article:

Appendix S1. Supplementary Materials and Methods.

Figure S1. GFP is not expressed in ON bipolar cells, but

probably in a subpopulation of OFF bipolar cells.

Figure S2. GAD65/67 expression in adult mouse horizontal cells

in three mouse strains.

Figure S3. GABA is detected in horizontal cells after fixation by

intracardiac perfusion in 129SV/Pas and CD1mouse retinas.

Figure S4. Horizontal cells cannot synthetize GABA from

glutamate at room temperature.

Figure S5. Incubation at 37�C in aCSF and PLP allows for a

partial GABA restoration in horizontal cells.

Figure S6. GAD65 and GAD67 expression in adult mouse retina.

Table S1. Antibodies used in this study.

As a service to our authors and readers, this journal provides

supporting information supplied by the authors. Such materials are

peer-reviewed and may be re-organized for online delivery, but are

not copy-edited or typeset. Technical support issues arising from

supporting information (other than missing files) should be

addressed to the authors.

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Mammalian retinal horizontal cells are unconventional GABAergic neurons

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