Page 1of50 AB_2261205; RRID: AB_10013783; RRID: AB_2201528 AB_2201528 RRID: AB_10013783; RRID: AB_2261205; AB_2314280; RRID: AB_10013483; RRID: AB_94936; RRID AB_94936; RRID: AB_10013483; RRID: AB_2314280; Acknowledgements: This work was supported by NIH R by NIH wassupported work This Acknowledgements: Associate Editor:Associate Dr.Meinertzhagen Ian A. Corresponding author: Tomomi Ichinose, M.D.,Ph.D Ichinose, Tomomi author: Corresponding RRID: AB_2314947; RRID: AB_2158332; RRID: AB_397957 RRID: AB_2158332; AB_2314947; RRID: RRID: AB_2313634; RRID: AB_2079751; RRID:AB_2086 RRID:AB_2079751; AB_2313634; RRID: retina D1Dopamine expression receptor cell typbipolaris Pershang Farshi Pershang School MIMedicine, 48201.Detroit, Molecular Behaviorand

Key words: BAC transgenic mice, immunohistochemistr mice, transgenic BAC words: Key DepartmentofAnatomy Celland Biology authorsThese *: contributed equally. Blindness (RPB) grants. grants. (RPB) Blindness (Visi P30EY004068 NIH (TI), Fund Startup (PDW), WSU

between this versionandtheVersionrecord. Pleasecitethis articleasdoi:10.1002/cne.23932. through thecopyediting, typesetting, pagination andproofreading process,whichmay lead todifferences This istheauthor manuscriptaccepted forpublicationandhas undergonefullpeerreview buthasnotbeen 3 , Ann Arbor,Ann MI 48109. ,

1* , Bozena FykKolodziej Bozena ,

Abbreviated Title: Dopamine receptor1 in retinal b in retinal receptor1 Dopamine Title: Abbreviated Accepted3135770836Phone: [email protected] Canfield, E. Detroit,540 MI48201 State Wayne University Medicine Schoolof ofAnatomy Dept. Celland Biology,Ophthalmology Article

This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology 1 , DepartmentofOphthalmology , 1*

, David M. Krolewski M. David , al Neuroscienceal Institute,University Michigan of M

. y, 01 EY020533 (TI), MidWest Eye Bank fund fund Eye Bank MidWest (TI), EY020533 01 on Core) and the Research to Prevent Prevent to Research the Core) on and in situ in 774; RRID: AB_2094841; RRID: RRID: AB_2094841; 774; ; RRID: AB_628142; RRID: RRID: RRID: AB_628142; ; : AB_2115181; RRID: AB_2248534; AB_2248534; RRID: AB_2115181; : 3 hybridization: RRID: AB_10000347; AB_10000347; RRID: hybridization: , Paul D. D. Paul Walker , e-specific in the mousein e-specific 2 , Wayne State UniversityState , SchoolWayne of ipolar cells cells ipolar 1 , Tomomi Ichinose , Tomomi edical 1,2 vision. daytime facilitate bipolar sculpts dopamine that indicate results Our pathways, such as color or motioncoded pathways color ar motioncoded as or such pathways, AI of (25%) subset a cells and horizontal including interne Other Drd1atdTomato. express not did cells cells. ON bipolar and 7 6, XBC, 52, type and cells nuclearlaye inner in the wasdetected fluorescence a cellsty in bipolar retinal in expressed is (D1R) mic transgenic BAC Drd1atdTomato we analyzed Here, however, it is not well understood how dopamine mod how dopamine well understood not is it however, dopa transmission, volume of Bymeans transmission. signaling transmits and cells amacrine dopaminergic dayti for keymolecule a is retina, dopamine the In Abstract

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons pedependent manner. Strong tdTomato tdTomato Strong manner. pedependent me vision. Dopamine is released by retinal byretinal released is Dopamine vision. me I amacrine cells. Diverse visual processing processing visual Diversecells. amacrine I cell performance in a typedependent manner to to manner typedependent a performance in cell In contrast, type 2, 3a, 51, 9, and rod bipolar bipolar rod and 9, 51, 3a, type 2, contrast, In 2 r and localized to type 1, 3b, and 4 OFF bipolar OFF bipolar and 4 3b, 1, type localizedto and r urons were also found to express tdTomato tdTomato express to werealso found urons either by conventional synaptic or by volume byvolume or synaptic byconventional either ulates visual signaling pathways in bipolar cells. cells. bipolar in visual pathways signaling ulates e thought to be initiated in retinal cells. retinal bipolar in initiated be to thought e mine modulates all layers of retinal neurons; neurons; retinal of layers all mine modulates e and found that dopamine D1 receptor receptor D1 dopamine that e and found Page 2of50

Page 3of50 Approximately a dozen bipolar cell types haverecen cell types bipolar dozen a Approximately Among the five types of dopamine receptors (D1likereceptors dopamine of types the five Among fullyinvestigated. been not of location the knowledge, of thisaccrual Despite and Werbli 1994; Wellis and (Maguire cells Werblin, activity of ganglion cells (Vaquero et al., 2001; O 2001; al.,et (Vaquero ganglioncells of activity al.,et 2009). Kothmann 2006; al., Urschel et 2002; t cells amacrine AII between connexin36 and 1999), 199 McReynolds, and Dong Dowling, 1985; and (Mangel 1996; Deng al., et (Caille somas in immunolabeling d possibly reexamined, been not has expression D1R methods and reported that the D1R was expressed in wasexpressed D1R the reported that and methods localization (1996) analyzed D1R and Veruki Wässle al.,et 1999; MoraFerrer 1996; and Veruki Wässle, and dopami photoreceptors in detected are receptors in ma expressed are D1(D1Rs) receptors receptors), 2008; Jin et al., 2015), coupling of horizontal cel horizontal of 2015), coupling Jin al., et 2008; t photoreceptors coupling between regulate to shown primari conveyed signaling dopamine to attributable in reported been dopamine ofhas effect modulatory in released is that isneurotransmitter a Dopamine Introduction

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons ls to alter the efficacy of retinal inhibitorymodu retinal efficacyof the alter to ls gata et al., 2012; Van Hook et al., 2012) and bipol and 2012) al., et Hook Van 2012; al., et gata dopamine receptors to specific retinal neurons has has retinal neurons specific receptors to dopamine 3 In the inner retina, dopamine modulates the the modulates retina, dopamine inner the In the retina during daylight conditions. The The conditions. during daylight retina the Stella and Thoreson, 2000; Witkovsky, 2004). 2004). 2000; and Thoreson, Stella Witkovsky, ly by volume transmission. Dopamine has been been has Dopamine transmission. volume ly by et al., 2006). 2006). al., et n, 1995; Ichinose and Lukasiewicz, 2007). Lukasiewicz, 2007). and Ichinose 1995; n, tly been elucidated in many species; however, in species; many elucidated tlybeen most types of retinal neurons, which is which neurons, retinal of types most o reduce rodmediated signaling (Deans et al., al., et (Deans signaling rodmediated oreduce : D1 and D5 receptors; D2like: D2, D3, and D4 D4 D3,and D2like:D2, receptors; D5 and D1 : o facilitate cone functions (Ribelayga et al., et (Ribelayga cone functions o facilitate nergic amacrine cells (Cohen et al., 1992; 1992; al.,et (Cohen cells nergicamacrine in the rat retina using immunocytochemical immunocytochemical ratretina using the in D2like while network, retinal of the nyneurons bipolar cell types 5, 6, and 8, but not in type 2. type not in but 8, and 6,5, types cell bipolar ue to difficulties associated withD1R associated difficultiesto ue 1; Hampson et al., 1994; Xin and Bloomfield, and XinBloomfield, 1994; al., et Hampson 1; lation lation ar ar

striatum (Ade et al., 2011) to investigate D1Rexpr investigate to 2011) al., et (Ade striatum (li mouse transgenic BAC Drd1atdTomato usedthe We bipolar on and effects dopaminergic characterized, expression receptor dopamine transmission, dopamine throughout cells including dendrites and axon termi and axon including cells dendrites throughout b in each expression characterize D1R to techniques 2005; al.,et Wäss (Haverkamp markers typespecific inner plexiform layer (IPL) where bipolar cell cell axon bipolar layerwhere (IPL) plexiform inner cell (DA) amacrine dopaminergicof types three that al., 2010; Volgyi et al., 2014). DA cell processes DA 2014). al., Volgyi 2010; et al., manner, indicating that dopamine regulates specific regulates thatdopamine indicating manner, thatD1Rs evidence found markers. typespecific We Contini et al., 2010). While these studies suggest studies these While 2010). al.,et Contini si the return that connections reciprocal make also 2004; Pignatelli and Strettoi, 2004; Helmstaedter e Helmstaedter 2004; Strettoi, and Pignatelli 2004; form be to thought neural pathways are These 2004). o pathwaysdepending neural separate into signaling r in the neurons secondorder the are cells Bipolar

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons receive excitatory inputs from ON bipolar an bipolar cells ON from inputs excitatory receive terminals are located (Zhang et al., 2007; Contini 2007; al.,et (Zhang located are terminals that bipolar cells are in position to be exposed t exposed be to position in cells are bipolar that etina and are responsible for encoding image image encoding responsible for are etina and essing cells in the retina. We employed bipolar ce bipolar employed retina. the We in cells essing 4 gnal to ON bipolar cells (Dumitrescu et 2009; al.,et (Dumitrescu bipolar cells ON to gnal t al., 2013; Euler et al., 2014). suggest Evidence 2014). al., et Euler t2013; al., nals, allowing us to investigate colocalization wit colocalization investigate to allowing us nals, cell functions remain to be elucidated. elucidated. be to remain cell functions s extend their processes into multiple layers of th layers into of multiple processes their extend s neural streams at the bipolar cell level. level. cell bipolar the streams at neural n features such as color or motion (Wässle, (Wässle, motion color or such as n features ipolar cell type. tdTomato was expressed wasexpressed tdTomato type. cell ipolar le et al., 2009) and singlecell dyeinjection singlecell dyeinjection 2009) and al., le et are expressed in a bipolar cell typespecific cell typespecific a bipolar in expressed are ed by distinct bipolar cell types (Ghosh et al., (Ghoshal., et types cell bipolar bydistinct ed in bipolar cells has not been well been cellsnot has bipolar in ne 6) developed for D1R research in the the researchin D1R developed for 6) ne

Page 4of50 o d et h h s s e ll Page 5of50 Animal protocols were approved by the Institutional bythe wereapproved protocols Animal Mice Mice Methods and Materials 5’3’ primers: Drd1atdTomato 5’3’ forward Drd1atdTomato primers: (PCR) meth reaction chain polymerase standard using 6, line as including thisstrategy, from generated et cDNA (Shuen withtdTomato sequence coding Drd1a sequence modi upstream kb 5’ of 185 plus gene Drd1a B Drd1a harbor mice These 16204). number (JAXstock (Bar Harbor Laboratories Jackson from wereobtained in used for (C57BL/6J) mice University. Wildtype

removed and placed in cooled, oxygenated dissecting oxygenated in cooled, placed and removed c using wereeuthanized daysmale) old; (28–60 Mice preparation Retinal colocalizatio D1R investigate to weregenerated YFP withDrd1atdTomato mice Double transgenic house. Colo generations. five than more breeding for house t GusGFP and Drd1atdTomato Heterozygous 013161). J from werepurchased (Clm1YFP) Clomeleon)1Gjau/J) al. Huang 1999; et al., et (Huang Margolskee byDr. Gus8.4green fluorescent expression. Drd1atdTomato

TTTCTGATTGAGAGCATTCG. A single 750 bp product ident product bp single 750 A TTTCTGATTGAGAGCATTCG.

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CTTCTGAGGCGGAAAGAACC and Drd1atdTomato reverse reverse Drd1atdTomato and CTTCTGAGGCGGAAAGAACC John Wiley& Sons reported by Ade et al. (2011). Mice were genotyped weregenotyped Mice (2011). al. et byAde reported 5 house heterozygous transgenic mice breeding, breeding, mice transgenic heterozygous house Animal Care and Use Committee of Wayne State State of Committee andUse Wayne Care Animal , 2003). Clomeleon 1 mice (B6. CgTg(Thy1 (B6. 1 Clomeleon mice , 2003). nies of Clm1YFP mice were also maintained in maintained werealso mice Clm1YFP of nies n with types7 and 9 bipolar cells, respectively. respectively. cells, 9 bipolar and withtypes7 n arbon dioxide and pneumothorax. Eyes were Eyes pneumothorax. dioxide and arbon HEPESbuffered solution containing (in mM): containing mM): (in solution HEPESbuffered , ME), as were Drd1atdTomato mice (line 6) 6) (line mice wereDrd1atdTomato ME), as , and GusGFP or Drd1atdTomato and Clm1 and Drd1atdTomato or GusGFP and ods on tail sample DNA using the the DNA following using tailon sample ods protein (GusGFP) mice were kindly donated werekindly (GusGFP)donated mice protein fied to replace the ATG codon plus 180 bp bp codon 180 plus ATG replacetothe fied AC (RP2347M2), which contains the entire entire the contains which (RP2347M2), AC ackson Laboratory (JAX stock number number (JAXstock Laboratory ackson al., 2008). Several transgenic lineswere transgenic Several 2008). al., ransgenic mice were maintained byin weremaintained ransgenic mice ified mice that were positive for werepositivefor that ified mice 137 NaCl, 2.5 KCl, 2.5 2.5 CaClKCl, NaCl, 2.5 137

Elite Kit, Vector Laboratories) in PBST for 1 hour hour 1 PBST for in Laboratories) Vector Kit, Elite werepro then sections Retinal 2 hours. for (PBST) in CA) 0.01Mphosp Burlingame, Laboratories, Vector bybiotinyla hours 18 followed for temperature room Clo (1:500; antidsRed polyclonal rabbit containing binding.S nonspecific reduce to temperature room serum alb bovine (1% in buffer blocking placed then fo (PB) buffer phosphate Min 0.1 peroxide hydrogen doublelabe for combination in or experiments (ISH) Retinal sections were processed for singlelabel im singlelabel were for processed sections Retinal hybridization In-situ immunohistochemistry. p PB, with 0.1M washed min, for 30 paraformaldehyde B Millipore, (EMD paper filter on was flattened and t from wasisolated retina the preparation, retinal histo on werecollected Cryosections MA). Waltham, witha Microm m 14–16 vertically at wassectioned tissue was 4em °C. The at sucrose overnight 30% in r sclera, the from wereseparated retinas fixation, p 0.1 PB, Min paraformaldehyde 4% using werefixed to wasmarked retina the experiments, some For cup. and cornea the stereomicroscope, dissecting a Using

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2 , 1.0 MgCl 1.0 , This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology 2 , 10 HEPES, 28 glucose, adjusted to pH 7.4 withNaO 7.4 pH to adjusted glucose, 28 HEPES, 10 , John Wiley& Sons he eyecup in oxygenated HEPESbuffered solution, solution, HEPESbuffered oxygenated in eyecup he insed several times in 0.1 M PB, and cryoprotected cryoprotected and PB, M 0.1 in several times insed and developed using 0.04% 3, 3’diaminobenzidine 3’diaminobenzidine 3, using 0.04% developed and 6 illerica, MA). Then, the tissue was fixed using 4% using 4% wasfixed tissue the MA). Then, illerica, ntech Laboratories, Inc., Mountain View, CA) at at CA) View, Mountain Inc., Laboratories, ntech munohistochemistry (IHC) and and (IHC) munohistochemistry cessed with avidin/biotin reagents (VectaStain (VectaStain reagents withcessedavidin/biotin ted goat antirabbit secondary antibody (1:1000; (1:1000; antibody secondary antirabbit goat ted ling. For IHC, sections were immersed in 0.3% 0.3% in wereimmersed sections IHC, For ling. umin, 0.1% Triton X100, 0.1M PB) for 1 hour at hour 1 PB) for 0.1M X100, 0.1% umin, Triton ections were incubated in fresh blocking buffer buffer inblocking wereincubated fresh ections HM 525 cryostat (Thermo Fisher Scientific, FisherScientific, cryostat(Thermo 525 HM r 30 min to quench endogenous peroxidases and and peroxidases endogenous quench to min 30 r bond slides (Fisher Scientific). For wholemount wholemount For (FisherScientific). slides bond and (EMS) FreezingMedium in Tissue bedded lens were quickly removed to obtain the eye the obtain to werequickly removed lens identify dorsal and ventral sides. The eyecups eyecups The sides. ventral and dorsal identify hatebuffered saline (PBS), 0.1% Triton X100 X100 0.1% Triton (PBS), saline hatebuffered H 7.4, for 30 min at room temperature. After After temperature. room at min 30 7.4, Hfor H 7.4 and processed for freefloating and processed 7.4 H in situ in hybridization hybridization H. H. Page 6of50 Page 7of50 Axiophot microscope fitted with a MicroFire camera camera MicroFire witha fitted microscope Axiophot A in 10 mM TrisHCl, 0.5 M NaCl, pH 8 at 37 °C for 37 at for pH °C 8 NaCl, M 0.5 TrisHCl, 10 in mM A sections. Signal denoting D1R mRNA hybridization wa hybridization mRNA D1R denoting Signal sections. s the confirm to controls processed also shown). We t in signal of and lower levels tubercle, olfactory within heavy showed results labeling The striatum. p D1R cRNA the positive As a control, development. NY) and stored Rochester, Health, (Carestream film in wereloaded sections visualize hybridization, To and airdried. alcohol, of concentrations were washed °C. Sections 70 at 1 hour for SSC 0.1× SSC (2×, of in decreasingconcentrations incubation i wererinsed sections hybridization, After hours. a humidifie into loaded werecoverslipped, Sections 2.5×10 p and mM sodium 50 dithiothreitol, mM tRNA,10 yeast su dextran 10% (50% buffer formamide, hybridization and for airdried alcohol of concentrations graded Sect 15 min. pH 8) for (TEA, triethanolamine 0.1M acetylated and 7.0) citrate, pH sodium M 0.015 and c salinesodium in 2× wererinsed sections ISH, For lig All FisherScientific). (Thermo medium mounting i cleared alcohol, of concentrations graded through hydrog with0.006% (DAB) hydrate tetrahydrochloride

6 dpm of the the of dpm

Accepted 35Article Slabeled D1R cRNA probe (accession# NM010076; nucl NM010076; (accession# probe D1R cRNA Slabeled

This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons he cerebral cortex and septal nucleus (data not (datanot septal nucleus and cortex cerebral he n 2× SSC and incubated in 200 g/ml ribonuclease ribonuclease 200 g/ml in incubated and SSC 2× n 30 min. Each slide was covered with 70 l slidewith70 Each was covered min. 30 to a film cassette with Biomax MR autoradiographic autoradiographic MR cassette withBiomax ato film 7 n xylenes, and coverslipped with Permount® withPermount® coverslipped and xylenes, n the caudate/putamen, nucleus accumbens, and and accumbens, nucleus caudate/putamen, the ht microscopy images were captured by a Zeiss Zeiss bya werecaptured images microscopy ht d chamber, and placed in an oven at 55°C for 18 55°C for oven at an in and placed dchamber, in dehydrated water, distilled in werewashed ions itrate (SSC; 1×SSC is 0.15 M sodium chloride chloride sodium M 0.15 is 1×SSC (SSC; itrate 1 hour. Stringent washes were performed by wereperformed washes Stringent hour. 1 at room temperature in 0.25% acetic anhydride in in anhydride inacetic 0.25% roomtemperature at (Carl Zeiss, Oberkochen, Germany). Oberkochen, Zeiss, (Carl 1×, and 0.5×) at room temperature followed by followed temperature room 0.5×)at and 1×, pecificity of D1R mRNA expression in retinal in retinal expression D1R mRNA pecificity of for 4–10 days in a darkroom before film film before darkroom in a days 4–10 for robe was hybridized to thin sections of mouse mouse of sections thin to was hybridized robe lfate, 3× SSC,1×Denhardt's solution, 0.1 mg/ml mg/ml 0.1 solution, SSC,1×Denhardt's 3× lfate, in distilled water, dehydrated in graded dehydrated water, distilled in en peroxide. Sections were then weredehydrated then Sections peroxide. en hosphate buffer, pH 7.4) containing containing 7.4) pH buffer, hosphate s detected in sections incubated with incubated in sections detected s eotides 14842196). 14842196). eotides

35 S captured at a resolution of 1024×1024 102 or pixels 1024×1024 of resolution a at captured water 20× or 63× 63× oil, a using Germany) Wetzlar, c witha was viewed preparation The Technologies). u coverslipped and washed Sections were 1:200–1000. hours.The 2 for room temperature CA) at Carlsbad, conjugated with Al secondaryantibodies, of mixture re incubation, antibody °C.Following primary 4 at with preparations and wholemount temperature room wereinc Sections in PBS. 100 0.5% TritonX and NDS genera Primary temperature. antibodies room at hour s donkey normal 3% containing solution a in blocked preparations wholemount and sections thin Retinal characterization Antibody and Immunofluorescence d PA) for 16–28 (Polysciences, Warrington, emulsion autoradiograph on verified hybridization successful the and rehydrated Sections residue. werethen pen o concentrations in graded weredehydrated sections for paraformaldehyde 4% using development postDAB proce were first sections andISH, IHC combined For D1R mRNA. endogenous of detection probe cRNA D1R (Figure 1B2B3). probe antisense cRNA D1R labeled wit pretreated sections in or probe sense cRNA D1R (Figure but 1B1), probe antisense cRNA D1R labeled

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons tinal tissues were washed and incubated in a in incubated and werewashed tissues tinal 8 ic film. Sections were dipped inK5D Ilford weredipped Sections film. ic exa 488, 568, 594, or 647 (Life Technologies, Technologies, (Life 647 or 594, 568, 488, exa 4×256 pixels, with line and frame average setup setup average line and frame with pixels, 4×256 were washed several times in 0.1 M PBS and and PBS Min 0.1 several times werewashed dilution for secondary antibodies ranged from secondaryantibodies dilution for 1 for in PBS 100 0.5% TritonX and (NDS) erum with asdescribed procedurewas performed ISH water immersion objective lens. Imageswere lens. objective immersion water onfocal microscope (TCS SP2 or SP8, Leica, Leica, SP8, or (TCS SP2 microscope onfocal ssed for IHC as described and then fixed again again then and fixed as described IHC ssed for ays prior to development and coverslipping. and development to aysprior pap remove in xylenesto placed and alcohol f h RNAse followed by hybridization to byhybridization followed RNAse h ted in different host species were diluted in 1% in 1% specieswerediluted host in different ted not in retinal sections hybridized to to hybridized retinal sections in not filter paper attached were incubated for 3 days 3 for wereincubated attached paper filter These results provide evidence for specific specific for evidence provide results These ubated with primary antibodies overnight at overnight at with antibodies primary ubated sing ProLong Gold antifade reagent (Life reagent (Life antifade Gold sing ProLong 15 minutes. Following extensive rinsing, rinsing, Followingminutes. 15 extensive 35 35 Slabeled Slabeled S Page 8of50 Page 9of50 After electrophoretic separation, proteins weretra proteins separation, electrophoretic After diluted 1:1000 in 1× TBST with 4% milk for 1 hour a 1 hour milk for with4% TBST in 1× 1:1000 diluted no (cat. (HRP) peroxidase horseradish to conjugated incub was the membrane washes, several After milk. mo monoclonal antibody, in primary 4°C at overnight (TBST) fo 20 TBS/Tween 1× in dry8% milk in blocked provided are used antibodies primary the of Details other. each signals from separate to and scanning was sequential the tissue, triplelabeled th m wasand 0.3 images stack zstep 3.for The to

(BioRad laboratories, Hercules, CA) for 90 minutes 90 CA) for Hercules, laboratories, (BioRad protei µg 80 and 23227) no. cat. Scientific, Fisher weredetermine concentrations Protein homogenized. bu (RIPA) assay radioimmunoprecipitation ice–cooled (n=2 C57BL/J6 (n=2) and Drd1atdTomato from Retinas blot and isolationWestern Protein previ compared to and werecharacterized antibodies JCN in (RRIDlisted and are characterized been have v from werepurchased antibodies All respectively. stronge obtain to wereused and antidsRed antiGFP

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons n samples were run on 10% acrylamide gels. acrylamide 10% wereonrun samples n nsferred onto polyvinylidene (PVDF) membranes membranes (PVDF) polyvinylidene onto nsferred performed to eliminate crosstalk between channels channels between crosstalk eliminate to performed 9 endors or gifted from scientists. All antibodies antibodies All scientists. gifted or from endors in Table 1 and Table 2. In transgenic mouse lines, mouse transgenic In2. 1and Table in Table t room temperature. Protein bands were detected weredetected bands Protein room temperature. t at a constant voltage of 150 V. Membrane wasMembrane V. 150 of voltage constant a at e zoom factor was 2.5 or 3.0. For double or Foror double 3.0. or was2.5 efactor zoom use antiD1R, diluted 1:500 in 1× TBST with 4% with4% in 1× TBST 1:500 diluted antiD1R, use . 554002, BD Biosciences, San Jose, Jose, CA) San BD Biosciences, . 554002, ous reports (Figures 1D, 5B). 5B). (Figures reports 1D, ous ). Dopamine D1 receptor, CtBP, and ribeye CtBP, receptor, D1 Dopamine ). ated in secondary goat antimouse antibody secondaryantimouse in goat ated r GFP/YFP signals and tdTomato fluorescence, tdTomato fluorescence, and signals GFP/YFP r ffer (Thermo Scientific, cat.no. 89900) and and cat.no. 89900) Scientific, (Thermo ffer r 1 hour at room temperature then incubated incubated then room temperature at 1 rhour d using the BCA Protein Assay Kit (Thermo Kit Assay Protein BCA the dusing ) wildtype mice were dissected and placed in placed and weredissected mice wildtype )

CaCl NaHCO and stage a chamber microscope on a to transferred For neurobiotin temperature. room box at oxygenated chopper handmade a thickness)using µm (250 slices fil of a piece on placed eyecup, the from isolated using oxygenat weremade preparations slice Retinal injection Neurobiotin CA). Jose, San Simple, (Protein imager Mi (WBKLS0100, substrate HRP chemiluminescent using Laboratories), and goat antiChAT antibody (1:200, (1:200, antibody antiChAT goat and Laboratories), Life (1:200, Tech 488 Alexa streptavidinconjugated usingwas 4% fixed preparation slice the injection, photogra to camera CCD usingthe wascaptured image afImmediately M. 7–11 of resistances had and CA) M with a P1000 FL) Sarasota, Instruments, Precision wer Electrodes cell. entire the filled neurobiotin w recordings QImaging). Wholecell (Retiga2000R, wi equipped UK) Sussex, East Scientifica, 2000, Pro slices byv in retinal cell stained somata tdTomato solutio pipette the in wereincluded Lab) Vector %, sulforhodam dye, A fluorescent mOsm). (269 withKOH CO 2 (pH 7.4 at 30°C). The intracellular solution cont solution intracellular The 30°C). at 7.4 (pH 2 , 10 HEPES, 1.1 EGTA, 10 NaCl, 1.0 MgCl NaCl, 1.0 10 1.1 HEPES, EGTA, 10 , 3

Accepted MO), waswhichconti Louis, St. (Sigma, mOsm) (294 Article

This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons e pulled from borosilicate glass (1B150F4; World (1B150F4; glass borosilicate World from pulled e 2 ter membrane (HABG01300, Millipore) and cut into into cut and Millipore) (HABG01300, ter membrane , 5 ATPMg, and 1.0 GTPNa, adjusted to pH 7.2 7.2 pH to adjusted GTPNa, 1.0 and ATPMg, 5 , iewing them with an upright microscope (Slicescope (Slicescope microscope uprightwith an them iewing 10 paraformaldehyde for 30 min, incubated withincubated min, 30 for paraformaldehyde n. Wholecell patch recordings were made from from weremade recordings patch n. Wholecell nologies), rabbit antiDsRed (1:500, Clontech Clontech (1:500, rabbit antiDsRed nologies), EMD Millipore) overnight at room temperature. temperature. room at Millipore) EMD overnight th a charge coupled device (CCD) camera (CCD) coupledcamera device charge a th ained the following (in mM): 111 Kgluconate, 1.0 1.0 Kgluconate, 111 mM): (in the ained following icropipette Puller (Sutter Instruments, Novado, Novado, Instruments, (Sutter Puller icropipette ed HEPES buffered solution. The retina was retina solution. The buffered HEPES ed ere continued for 10–15 min to ensure that the the that ensure to min 10–15 for continued ere perfused with Ames' medium buffered withbuffered withmedium Ames' perfused injection, each slice preparation was preparation eachslice injection, ter wholecell recording, a sulforhodamine B sulforhodamine a wholecell recording, ter . The retinal preparations were stored in an in an werestored retinal preparations . The ine B (0.005%, Sigma), and neurobiotin (0.5 (0.5 neurobiotin and Sigma), (0.005%, B ine ph the live retinal preparation. After dye livepreparation. retinal the ph llipore) and imaged on a Fluorochem E E Fluorochem a on imaged and llipore) nuously bubbled with 95% with95% Onuouslybubbled 2 and 5% 5% and Page 10of50 Page 11of50 Alexa 647 antigoat (Life Technologies) for 2 hours for (Life Technologies) antigoat 647 Alexa

intensities of both markers and tdTomato across som across tdTomato and markers both of intensities OFF For analysis. used mathematical also images. We Data analysis Data coverslipped. and Technologies) waswhichs slide, histobond a on placed carefully images and by analyzing series of single optical se single optical of series and by analyzing images colocalizati fluorescence and tdTomato markers Cell deconvo werethen images confocal 3D The coverslip. spe (RI), refractiveindex immersion lens objective wasa spherical aberration deconvolution, image For deconvolu the used MD). Rockville, We Cybernetics, and X3 AutoQuant using wasperformed analysis Image tdTomato mi three obtained images from retinal nine tdTomatopos grains per of numbers The ISH. and IHC th from layer(INL) inner the nuclear across somata centertdTomatopo of the over circle positioned µm wasanal ISHexperiments the in expression mRNA D1R sec with donkey was incubated preparation the Then,

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons 11 cimen mounting medium RI and distance from the the distance from and RI medium mounting cimen ealed with antifade reagent (ProLong Gold, Life Life Gold, (ProLong reagent withantifade ealed at room temperature. The slice preparation was slice preparation The temperature. room at ctions (0.3 µm thick) from collected collected thick)from µm (0.3 ctions e vertical retinal sections processed for combined combined for processed sections retinal vertical e sitive (n=171) or tdTomatonegative (n=103) (n=103) or sitive(n=171) tdTomatonegative on was initially examined by rotating the 3D 3D the byrotating examined wasinitially on <0.05). (alpha ttest bypaired compared and ce utodetected for each channel based on channel on based each for utodetected as using AutoQuant X3. Brightness of images images Brightness of X3. AutoQuant as using tion, 3D, and colocalization analysis functions. analysis functions. colocalization and 3D, tion, ondary antibodies; Alexa 568 antirabbit and and 568 antirabbit Alexa antibodies; ondary luted. luted. bipolar cells, we measured fluorescent fluorescent cells, we measured bipolar itive or negative cell were averaged across across wereaveraged negative itivecell or ImagePro Premier 3D software (Media (Media software 3D Premier ImagePro yzed by counting silver grains within a a 6.6 within silver grains yzedbycounting z stack stack

rp sn alb Matlab. using graph 90°. T wasrotated channel when one images of that is The 1. coefficient correlation of maximum The (, = ) (, equation: the using coe correlation 2D levels. 0 to werereduced levels separat are (0.3 twoof colors µm) sections digital of Images CtBP 2011). al., et Zinchuk 2011; al., et Mat custom a using coefficient analysis correlation colocalizatio puncta CtBP such as objects small For 100%). andplott cell markers, intensity of the maximum to fluoresc Arbitrary levels. subsaturated at wasset √ ( (,) , ) (,)

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons ed into two grayscale images. Background noise noise Background images. twograyscale into ed ence of both markers and tdTomato was normalized tdTomato markersand both of ence 12 correlation coefficient of images was compared to wascompared images of coefficient correlation puncta and HCN1 were excised (3 × 3 µm). Single ×3 µm). (3 wereexcised HCN1 and puncta ed across the cell (edges designated 0% and and 0% designated cell (edges the across ed lab (MathWorks, Natick, MA) code (TI wrote) (Soto (Soto (TI wrote) code MA) Natick, (MathWorks, lab fficient program compares two images dot by dot bydot dot twoimages compares program fficient n analysis, we performed twodimensional cross cross twodimensional we performed analysis, n he correlation coefficient was plotted in 3D mesh mesh 3D in wasplotted coefficient correlation he Page 12of50 Page 13of50

transgenic reporter mouse to investigate celltype investigate to reporter mouse transgenic daytime func retinal for iskey molecule a Dopamine mouse Drd1a-tdTomato Results using using waslocalized tdTomato whether determine To (ONL). No tdTomato wasfoun 1A). (Figure IPL the and (OPL) dendr well layeras as cell (GCL), ganglion and INL wasobser tdTomato of expression retina, the Within 2013). the express to known striatum the of neurons spiny D1 driven bythe tdTomato fluorescence express mice re the of cells tdTomato in primarily expressed are o werehighly grains concentrated the Nevertheless, axons/dendri through transport mRNA to attributable observ Grains n=3 mice). ttest, paired (p<0.0001, grain ± 3.49 to 0.2 compared soma tdTomatopositive IN the of neurons In 1C1–C2). (Figure soma positive werec expression mRNA D1R endogenous of indicative (Figursections retinal the on wastested probe The 35

Accepteddoubl retinal sections on probes D1R cRNA Slabeled Article

This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons 13 specificity of D1R expression in the retina. the These in D1R expression of specificity tina. tina. ed in the area of tdTomatonegative cells might be might cells tdTomatonegative of area the in ed e 1B1B3). Silver grains within the emulsion layer emulsion the withinSilver grains 1B1B3). e ites and axons in both the outer plexiform layer outer plexiform the in both and axons ites tion. We used the Drd1atdTomato line 6 BAC BAC line 6 Drd1atdTomato used the tion. We endogenous D1R (Ade et al., 2011; Thibault et al., et Thibault 2011; al., et (Ade D1R endogenous ver tdTomatopositive cells, indicating that D1Rs that indicating cells, vertdTomatopositive ved in cell bodies of many neurons located in the the in located neurons manyof cell bodies in ved tes (Kindler et al., 2005), or background noise. noise. background or 2005), al., et (Kindler tes L, 10.18 ± 0.3 grains were associated with wereassociated grains ± 0.3 10.18 L, s found over tdTomatonegative cells cells tdTomatonegative over s found R gene locus, as demonstrated in medium in medium demonstrated as locus, gene R d in photoreceptors in the outer nuclearlayer outer in the in photoreceptors d to authentic D1R neurons, we performed ISH ISH we performed D1R neurons, authentic to oncentrated over the majority of tdTomato majorityof the over oncentrated e labeled for tdTomato immunoreactivity. immunoreactivity. tdTomato labeled for e

2014). OFF bipolar cell types can be identifiedwit be can cell types bipolar OFF 2014). withtdT colocalization determine to order in somas fluorescence. withtdTomato colocalization terminal and dendr ramify processes cells their These cells. Thirteen types of bipolar cells have been identifie been have cells bipolar of types Thirteen cells bipolar cone OFF D1Rexpre investigating offers for advantages mouse endog express cells tdTomatopositive that indicate retina. vs.peripheral central across fluorescence 1FI)(Figure and f preparation retinal wholemount also We withtdTomatoimmunofluorescence. combined th identify to makingimpossible it 1E2E4) (Figure diffi was However, signal less frequent. weremuch types of ON bipolar cells, and a single rod bipolar rod single a cells,and ON bipolar of types bipolar cell markers labeled particular bipolar cel bipolar particular labeled cell markers bipolar ce OFF bipolar examine to techniques immunolabeling OPL and IPL, with stronger immunoreactivity seen in seen immunoreactivity withstronger and IPL, OPL withproc colocalized wasmainly immunostaining D1R o part and the OPL in observed was immunoreactivity a (Jarvieet reports withprevious consistent be to tissu retinal mouse in this antibody of specificity cells tdTomatoexpressing analyzedalsowhether We

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons e was assessed by immunoblot (Figure 1D) and found found 1D)(Figure and byimmunoblot wasassessed e ls as well as subsets of amacrine cells andganglio cells amacrine of subsets well as as ls d, including five types of OFF bipolar cells,seven bipolar OFF of types five including d, l., 1989; Luedtke et al., 1999). D1R D1R 1999). al., et Luedtke l., 1989; Taken together, both the ISH and IHC results results IHC and the ISH both together, Taken cell type (Helmstaedter et al., 2013; Ichinose et Ichinose 2013; al., et (Helmstaedter type cell h specific antibodies (Wässle et al., 2009). We use 2009). al., et We (Wässle antibodies specific h 14 e morphologies of cells expressing D1Rs unless unless D1Rs expressing cells of emorphologies ound similar distributions of cellular tdTomato tdTomato cellular of distributions similar ound ites in the IPL, which obscured bipolar cell axon axon bipolar cell obscured which the IPL, in ites omato. Thus, we analyzed bipolar cell dendrites and and cell dendrites bipolar we analyzed Thus, enous D1R, and that the tdTomato transgenic transgenic tdTomato the andthat D1R, enous INL regionthe of soma in the cultobserve to the the ssing cells of the retina. the of cells ssing f the IPL (n=3 mice, Figure 1E1E4). Punctate Figure Punctate 1E1E4). (n=3 mice, IPL the f colocalized with D1R immunoreactivity. The The immunoreactivity. D1R with colocalized esses of tdTomatoexpressing cells both in the the in both cells tdTomatoexpressing of esses ll types that colocalize with tdTomato. All OFF OFF All colocalizewith tdTomato. ll types that examined tdTomatoexpressing cells using tdTomatoexpressing examined s3 stratum; examples of nonlocalization nonlocalization of examples stratum; Page 14of50 al.,

n n d d Page 15of50 and 4 were D1Rexpressing bipolar cells, whereas ty whereas cells, bipolar were D1Rexpressing 4 and bipolar OFF in expressed was D1R suggest data these cells (p=0.2 for type 2, p=0.09 for type 3a, paired type 3a, 2, type for p=0.09 for (p=0.2 cells cells weresi and 3a 2 type of intensities tdTomato

Type 4 cells were labeled with calsenilin (Csen), w (Csen), withcalsenilin cells werelabeled 4 Type tdTomat wereall which RIIβ, (PKA) kinase A protein cells,n=3mic (22 negative tdTomato wereall which Ty colocalization. tdTomato determine to wereable were select that wereused antibodies other Second, Figure 2F). n=4 mice; cells, negative(78 tdTomato labeled both cells, OFF bipolar 2 Type 2E). Figure waswithconfirmed fl that observation an tdTomato, cells bipolar OFF 1 All type 2A2D). (Figure cells colabeling (Syt2b) 2b cells. Synaptotagmin bipolar antibod (NK3R) receptor 3 antineurokinin an First, (p<0.01 for all types, types, paired all for (p<0.01 intensit significantly higher exhibited cells 4 and 60%to loc cell (40 the centerof the from obtained loc 0%to (20 cells of outside levels fluorescence cell ma bipolar OFF of colocalization verifythe To 3E3F). Figure

Accepted Article t test) indicating that these cells colocalizetdTom cells these that test)indicating This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons y in the inside as compared to outside of the cell cell the of outside to compared as inside ythe in

(*, NK3Ronly) were found to colocalize with colocalizewith to were found NK3Ronly) (*, t ation in Figure 2E, 2F, 3B, 3D, and 3F) to 3F) levels 3D, and 3B, 2E, Figure 2F, in ation rkers with tdTomato, we compared baseline baseline we compared withtdTomato, rkers 15 ation in Figure 2E, 2F, 3B, 3D, and 3F). Type 1, 3b 1, Type 3F). 3D, and 3B, 2F, 2E, Figure in ation milar between baseline (outside) and the inside of and the inside (outside) baseline between milar test) indicative of no colocalization. Collectivel colocalization. no of indicative test) hich were all tdTomato positive (81 cells, n=3 mice cells,n=3 positive(81 tdTomato wereall hich y was used to identify both type 1 and 2 OFF OFF 12 and type both identify to ywasused with NK3R and Syt2b antibodies, were all wereall antibodies, Syt2b and withNK3R was employed to distinguish type 2 OFF bipolar OFF bipolar type 2 distinguish to wasemployed

uorescence intensity analysis (18 cells, n=2 mice; mice; n=2 cells, intensity(18 analysis uorescence pe 3a bipolar cells were labeled with HCN4, withHCN4, cells werelabeled bipolar 3a pe e; Figure 3A3B). Type 3b cells were labeled with werelabeled cells 3b Type Figure 3A3B). e; o positive (56 cells, n=3 mice; Figure 3C3D). Figure mice; cells,n=3 (56 o positive ive for single types of OFF bipolar cells, and we cells,and bipolar OFF of types single ive for pes 2 and 3a were D1Rnegative. and were D1Rnegative. 3a 2 pes cells in a type dependent manner: types 1, 3b, 3b, 1, types manner: dependent type a in cells ato protein. In contrast, contrast, In protein. ato y, , , ; ;

We labeled five type 5 cells; three cells colocaliz cells three 5 cells; type labeled five We (Ghosh et al., 2004). Different sets of type 5 cell type sets 5 of Different 2004). al., et (Ghosh choline a ON the and the depth) IPL (~40% of border cells whose ON bipolar as cells defined are 5 Type cells. ON bipolar in expression neu mice, reporter transgenic of combination witha 6 cells type for onlyare available immunomarkers withcolocalization O tdTomato of investigation The cells bipolar ON with wider axon terminals. Finally, type 5XBC cell 5XBC type Finally, terminals. wideraxon with terminals. axon had ramified narrowly and kinetics 2014). Ty al., et (Ichinose analyses physiological 5t cells type in ofsets 3 distinguished 2014). We 200 Pourcho, and FykKolodziej 2004; al., et (Ghosh To investigate whether type 5 cells colocalized wit colocalized cells 5 type whether investigate To colocalize with tdTomato (Figure 4B). The former re former The 4B). (Figure withtdTomato colocalize cells express Na express cells 52 type to transientlylight similar to responded reported by originally as fashion, a monolayer in

+

Accepted in submission). (paper channels HCN and channels Article

This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons s have been recognized in many species of the retin the of species many recognizedin been have s cells. In addition, we found that type 52 and XBC and 52 thattype we found addition, In cells. ed with tdTomato (Figure 4A). Two other cells did n did other cells Two (Figure 4A). tdTomato with ed he mouse retina using morphological and and using morphological retina mouse he pe 51 cells responded to light with sustained withlightsustained to responded cells 51 pe Helmstaedter et al. (2013). These cells also These al.(2013). et Helmstaedter 16 h tdTomato, we initially tried neurobiotin injectio neurobiotin we initiallytried tdTomato, h s exhibited the widest pattern of axon ramification axon of widest the pattern exhibited s Type 52 cells were transiently responding cells cells responding weretransiently cells 52 Type N bipolar cell types was challenging because because waschallenging types cell bipolar N (Wässle et al., 2009). We overcame this limitation this overcame limitation 2009). al., et We (Wässle robiotin injections, and IHC to characterize D1R D1R characterize to IHC and injections, robiotin axon terminals ramify between the ON–OFF the ramify between terminals axon sembled type 5XBC and the latter looked like the the like looked latter the type 5XBCsembled and 7; Helmstaedter et al., 2013; Ichinose et al., et Ichinose 2013; al., Helmstaedter et 7; cetyltransferase (ChAT) band (stratum 3 or S3) S3) 3or (stratum band (ChAT) cetyltransferase Page 16of50 n. ot ot

a a Page 17of50

same layer near ON ChAT band. Similarly, a portion Similarly, a band. ChAT ON layernear same Ct counted also withHCN1. We colocalized cells XBC immunoreactivity (15.3 ± 1.2%, n=12 fields) is likeis n=12 fields) 1.2%, ± (15.3 immunoreactivity minor 5H). The and 5D (Figure withHCN1 colocalize sign withHCN1 layercolocalized the in puncta CtBP immunoreactivi strong HCN1 where IPL the layerof a colocal cells also 51 type whether investigated We (Fig D1Rs express XBCs type and 52 that indicating comple signaling strong HCN1 in submission). (paper and52 type that found cells and 5 type individual cell mar bipolar 5ON istype HCN1 a that suggested coef correlation by2D was whichconfirmed 5D), and the area in puncta CtBP majorityof that the found antibodyw CtBP2 synapse marker. ribbon a antibody, w colocalized immunoreactivity HCN1 whether examine Immunostaining with the Hyperpolarizationactivated withthe Immunostaining cell this identifyto approach additional an sought immunoreactivity was observed in the entire wit IPL the entire in observed was immunoreactivity if cells th and 5 type ofsubset a for marker wasa e Cangiano 2003; al., et (Muller 5A) (Figure ramify the near localization HCN1 revealed antibody (HCN1) inefficienc the of However, because cell. 51 type

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons type. type. ese cells colocalized with tdTomato. HCN1 HCN1 withtdTomato. colocalized cells ese XBCs colocalized with bright HCN1 signaling signaling HCN1 withbright colocalized XBCs ly from type 51 bipolar cells because type 52 type and 52 because cells bipolar 51 type lyfrom y of neurobiotin injection to label type 5 cells, w type 5 label to injection neurobiotin yof 17 colocalized with strong HCN1 signaling (Figure 5C, 5C, (Figure signaling HCN1 withstrong colocalized t al., 2007). We tested whether HCN1 signaling signaling HCN1 whether tested 2007). al., t We ized with tdTomato. We counted CtBP2 puncta in in CtBP2 countedpuncta withizedtdTomato. We h strong signals in the the in signals strong h aling and a portion of the puncta did not not did puncta the of portion a and aling Cyclic Nucleotidegated potassium channel 1 1 channel potassium Cyclic Nucleotidegated ker. We conducted neurobiotin injection to to injection neurobiotin conducted ker. We of CtBP (18.7 ± 1.4%, n=10 fields) did not not did n=10 (18.7fields) CtBP 1.4%, ± of ure 5E, 5G). 5G). 5E, ure ficient analysis (Figure 5F). This result This analysis5F). (Figure ficient ty was observed. We found that the majority of of the majority that found wasobserved. We ty portion of CtBP puncta without HCN1 HCN1 without puncta CtBP of portion tely colocalized with tdTomato fluorescence, fluorescence, withtdTomato telycolocalized ON ChAT band where axon terminals of type type 5 of terminals axon where band ChAT ON as verified (Methods, Figure 5B1B2). We 5B1B2). Figure We (Methods, verified as BP puncta with tdTomato fluorescence at the at withtdTomato fluorescence puncta BP ith bipolar cell terminals, we used CtBP2 we used CtBP2 cell terminals, bipolar ith s3 s3 stratum (Figure 5A). To 5A). (Figure To stratum e e

and 6 of bipolar cells (Figure 6A1). Entire structu Entire (Figure cells 6A1). bipolar of 6 and withtdTomat cells bipolar type of 6 Colocalization 51 type that suggest results these together, Taken axon terminals of type 6 cells were observed in the wereobserved cells type of 6 terminals axon Helmstaedter et al., 2013), and our tdTomatotarget and our 2013), al., et Helmstaedter to compared number cellsin fewerare 9 8 and Type n=3mice (117cells, tdTomato for werenegative RBCs 7ON type bipo that indicating 3 mice), 96%, cells, ty majorityof The 6C1C2). (Figure colocalization exami and strains GusGFP line6 and Drd1atdTomato (Figure weakly labeled are (RBCs) cells bipolar rod t which in 2003) al., etHuang 1999; al., et (Huang co tdTomato and cells 7 bipolar type investigate To D1Rs. cells express bipolar ON 6 type 6 type of staining withneurobiotin wasconsistent crosscorrelation withconfirmed2D is This A1A2). coloca terminals axon 6 type Syt2bpositive 2009). 9 bipolar cells receive synaptic inputs exclusively inputs receive synaptic cells bipolar 9

and the puncta vs. tdTomato (P=0.08), suggesting th suggesting (P=0.08), tdTomato vs. puncta the and No difference 5I). (Figure withtdTomato colocalize

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons res of type 2 cells were intensely labeled, whileo labeled, cells type wereintensely 2 res of from genuine Sopsinexpressing cone cone Sopsinexpressing genuine from o was examined using Syt2b, a marker for types 2 2 types for a marker Syt2b, using wasexamined o cells (n=4 cells, data not shown), indicating that that shown),indicating cells, not data (n=4 cells pe 7 GFP cells were tdTomato positive (91 / 95 / 95 (91 positive tdTomato cells were GFP 7 pe ype 7 bipolar cells are strongly GFP positive and and stronglypositive are GFP cells 7bipolar ype 18 lar cells express D1Rs. In contrast, all GFP positi all contrast, GFP In D1Rs. cells lar express s were observed between CtBP puncta puncta CtBP vs. HCN1 wereobserved s between inner portion of the IPL (Figure 6A) (Wässle et al et (Wässle 6A) (Figure IPL the of portion inner coefficient analysis (Figure 6B). This observatio This 6B). (Figure coefficient analysis 6C). We generated double transgenic mice with transgenic mice double generated 6C). We line mouse GusGFP we the used localization, cells did not express D1Rs. D1Rs. express not did cells lized with tdTomato (n=3 mice, 20 fields; Figure 6 6 Figure fields; 20 (n=3 mice, lizedwithtdTomato ed neurobiotin injections did not label them. label not Type did injections neurobiotin ed at type 51 cells did not colocalize with tdTomato. with colocalize not did cells 51 type at other types (Haverkamp et al., 2005; 2005; al., et (Haverkamp types other ). ). ned evidence for GFP and tdTomato tdTomato and GFP for evidence ned Page 18of50 nly ve n n ., .,

Page 19of50

expressing cells attaching to genuine S conetermin S genuine to attaching cells expressing n do negativeand tdTomato are cells bipolar 9 type cells,4 (n=15 expression withtdTomato colocalize dendri extended cells 9 bipolar thattype confirmed wasobserved YFPwhich in fluorescence line, mouse the and tdTomatoDrd1a mice from transgenic double process tdTomatolabeled the whether investigate To inset). 7A, (Figure tdTomatosta detected side. ventral the on We cones genuine indicating 7B), (Figure retina ventral the 7A) (Figure sections retinal in dorsal werelabeled c tdTomatoexpressing of processes investigated and antib Sopsin used an 2005). al., et We (Haverkamp ce Mopsinexpressing genuine cones are of majority v the located on are cones mixed Sopsinexpressing (Szel et aredistributed unevenly cones expressing Scon genuine the to processes their extended cells 2005). al., et (Scones) (Haverkamp photoreceptors 8A), which is consistent with the results in Figure in results the with consistent is which 8A), tdTomato colocalizewith not did RBCs (RBCs). cells (PKCα) alpha an C kinase protein a weused Finally, 7D (Figure cells 7or horizontal and 4,3, 2, types

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons ,E) (Breuninger et al., 2011). 2011). al., et (Breuninger ,E) 6C and a previous report (Veruki and Wässle, 1996) and (Veruki Wässle, report previous a and 6C Scones on the dorsal side and mixedS and M and mixedS and side dorsal the on Scones , whereas many cones were labeled with Sopsin in withSopsin werelabeled cones manywhereas , 19 tes toward the genuine Scones, and did not and not did Scones, genuine the toward tes mice, Figure 7C1C3). These results indicate that that indicate results These 7C1C3). Figure mice, al., 1992; Haverkamp et al., 2005). Both M and BothMand 2005). al., et Haverkamp 1992; al., ot express D1Rs. Processes of tdTomato of Processes D1Rs. express ot als were from other types of bipolar cells, such as cells,such bipolar of othertypes were als from e terminals. In the mouse retina, M and Sopsin retina,Mand mouse the In terminals. e We therefore investigated if tdTomatoexpressing tdTomatoexpressing if investigated therefore We tibody to examine D1R expression in rod bipolar bipolar rod in D1R expression examine tibodyto expressing cells (104 cells, n=3 mouse; Figure Figure cells, n=3 mouse; (104 cells expressing ody to label genuine Scones in the dorsal retina dorsalin the retina Scones genuine label to ody lls, while the minority are genuine Scones Scones genuine minorityare whilethe lls, ined processes attached to the genuine Scones Scones genuine the to attached processes ined entral side of the retina. In the dorsal retina, t the retina, dorsal In retina. the ofside entral es were from type 9 bipolar cells, we generated we generated cells, bipolar type 9 were es from ells. A small number of the Scone terminals terminals Scone the of number small A ells. in type 9 cells (Haverkamp et al., 2005). We 2005). al., et We 9cells type (Haverkamp in Clomeleon 1 (Clm 1YFP/CFP) transgenic transgenic 1YFP/CFP) 1Clomeleon (Clm he

. formations and the majority of rings were not fille ringswerenot the majorityof and formations wholemou In 2014). al., Volgyi 1987; et and Wässle, ringlikestruct and form (TH) hydroxylase tyrosine surrounded bydop are cells amacrine AII that known to perform decided 8C1C3). Figure in We arrowheads cells werepositive dab1labeled of ~25% However, that suggesting 8C1C3), Figure (arrows in tdTomato dab1immun of the majority that showed results Our dab1immuno whether investigated 2000) and Curran, We used disabled1 (dab1) antibody to specifically antibodyto (dab1) useddisabled1 We B3).

or rod bipolar cells. Three groups of type 5cells type of groups Three cells. bipolar rod or cell bipolar in D1Rs together, wereexpressed Taken gap junctions in horizontal cells are controlled by controlled cells are in horizontal junctions gap a AII and cells horizontal whether examined also We cells amacrine AII and cells Horizontal colo not did cells type 51 whereas D1Rs, expressed immunoreactive horizontal cells colocalized with td with colocalized cells horizontal immunoreactive tdTomato to compared cells as horizontal in weaker ce horizontal label antibodyto calbindin useda We by ON bipolar cell activity (Hampson et al., 1992; 1992; al., et activity cell (Hampson bipolar byON

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons expressed D1Rs differently: XBCs and type 52 cell type 52 and XBCs differently: D1Rs expressed d with tdTomato cells (asterisks in Figure 8D1, D2) 8D1, Figure in cells (asterisks withtdTomato d dopamine and AIIAII couplings might be controlled be might couplings AIIAII and dopamine Bloomfield and Volgyi, 2009; Kothmann et al., 2012) al., et Kothmann and Volgyi, 2009; Bloomfield 20 ures around AII somas at the INL/IPL border (Voigt (Voigt border INL/IPL the at somas AII around ures Tomato (n=16 cells, 100%, cells,100%, (n=16 Tomato lls. Although tdTomato expression was relatively expression tdTomato lls. Although label AII amacrine cells in mouse retina retina and (Rice in mouse cells amacrine AII label nt retinal preparations, we observed TH ringlike ringlike TH we observed preparations, retinal nt expression in bipolar cells, calbindin cells, in bipolar expression types 1, 3b, 4, 6, and 7 but not in types 2, 3a,9 3a,9 2, in types but not and 7 6, 4, 3b, 1, types macrine cells colocalized with tdTomato because because withtdTomato colocalized cells macrine for tdTomato (10 out of 41 cells, n=2 mice; n=2 cells, mice; 41 of out (10 tdTomato for oreactive cells were not colocalized with with colocalized werenot cells oreactive calize with D1RtdTomato fluorescence. fluorescence. calizeD1RtdTomato with aminergic fibers that are immunoreactive for immunoreactivefor are that aminergic fibers AII amacrine cells do not express D1Rs. express cellsnot do amacrine AII another experiment to confirm this result. It is It result. this confirm to experiment another labeled AII cells colocalized with tdTomato. withtdTomato. cells AII colocalized labeled n =2 mice; Figure 8B1 Figure =2mice; Page 20of50 . . s s

. . Page 21of50

while about 25% of AII cells were immunoreactive fo AII wereimmunoreactive cells of 25% whileabout the together, D3). Taken 8D1, Figure in (triangles (n=7 fields) ringformations the of % ~25 However,

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons 21 majority of AII amacrine cells were D1R negative, negative, wereD1R cells amacrine AII majorityof were occupied by tdTomatopositive somas somas bytdTomatopositive wereoccupied r tdTomato, indicating that they express D1Rs. D1Rs. they express that indicating tdTomato, r

may contain “extra” contain may via recombination genome the into mouse introduced technology is(BAC) chromosome artificial Bacterial mouse transgenic BAC line-6 Drd1a-tdTomato Discussion mainly processes in the IPL (Figure 1E2). These These fi 1E2). the(Figure IPL in processes mainly (Figures 2 neurons in most axon terminals and somas tdTomato retina. the of cells tdTomatoexpressing (F tdTomato fluorescence with colocalized also that as cells, tdTomatopositive in colocalization mRNA colocalizedD1R signals verifythat to antibody D1R linehasbeen not mouse Drd1atdTomato the Because retina. the thisof mous advantage sowe took and 2011) al., et D1Rexpressing to limited is fluorescence tdTomato in the co expression tdTomato the of fidelity alter Further experiments Sfxn1. gene, extra an contains receptor immunoreactivity in the rat retina retina and fou rat the immunoreactivity in receptor wellbeen s not has bipolar cells in expression D1R cells bipolar retinal in expression receptor D1 reti the in cells analyzing D1Rexpressing for tool

′ Accepted Article Th gene. the to original target in addition genes

This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons rrect cell population (Ting and Feng, 2014). 2014). Feng, and (Ting population cell rrect na. na. nd that type 5, 6, and 8 cone bipolar cells, and cells,and cone bipolar 6,8 type and 5, that nd 22 ndings demonstrate that this mouse line is a a lineisuseful thatthis mouse demonstrate ndings fluorescence was observed in the dendrites, dendrites, in the wasobserved fluorescence tudied. Veruki and Wässle (1996) analyzed D1 D1 (1996)analyzed and Veruki Wässle tudied. with tdTomato (Figure 1). We found evidence for evidence for found (Figure 1). tdTomato We with well as processes labeled with the D1R antibody D1R antibody withthe labeled processes well as a method by which large regions of DNA can be DNAbe can of regions bylarge which method a igure 1). These data confirm D1R expression in in expression D1R confirm data These 1). igure e to examine celltype specific D1R expression in in expression D1R celltype specific examine to e revealed that this unwanted extra gene does not not does gene extra thisunwanted that revealed cells as previously confirmed in the striatum (Ade (Ade striatum in the previously as confirmed cells , 3, 4, 6 and 8) while the D1R antibody stained antibodystained D1R whilethe and 8) 6 4, 3, , mediated genetic engineering. However, BACs BACs However, engineering. genetic mediated used for retinal research, we used ISH and a a and we used ISH research, retinal for used e Drd1atdTomato line line 6 Drd1atdTomato e Page 22of50

Page 23of50 AII amacrine cells were not immunoreactive. Assum immunoreactive. werenot cells amacrine AII

horizontal cells were D1R immunoreactive, whereas t whereas immunoreactive, wereD1R cells horizontal and mouse retinas, our results are consistent with consistent are retinas,results our mouse and because of low efficiency of filling neurobiotin in neurobiotin filling lowof of efficiency because expression, subset tdTomato for werenegative cells thawe Although found 4). (Figure cells bipolar XBC verified this with observation of bright HCN1 signa HCN1 bright of withobservation this verified t indicated analysis colocalization marker) synapse aofsubset marker for a serve as can antibody HCN1 Therefore, we initially used an intracellular intracellular dye ( an used we initially Therefore, because immun challenging was cell analysis bipolar u werecharacterized cell types bipolar OFF cells. transgenicmic and Drd1atdTomato markers type cell of markers established (2009) colleagues and Wässle and Wäss Veruki cells since bipolar in localization our To protein. this cells express bipolar some and D1Rto tran linked signaling regulator downstream a (2007 al. et clearlydetermined. Witkovsky werenot in immunoreactivity D1R and observed retinas mouse al. et NguyenLegros (1999)and al. et MoraFerrer ne cells are RBCs, TH type 2, whereas positive, are colocalization (paper in submission). Using HCN1Using i in submission). (paper colocalization

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons type 5 cells. We, therefore, examined whether thewhether examined therefore, cells. 5 type We, neurobiotin)injection technique to analyze type 5 5 type analyze to technique neurobiotin)injection le (1996). (1996). le 23 ling, neurobiotininjected XBCs, and type 52 cells 52 type and XBCs, neurobiotininjected ling, sing immunoreactive fluorescence markers. ON ON markers. singfluorescence immunoreactive this previous report: type 6 and a subset of type 5 type subset a of 6 and type report: previous this knowledge, no studies have characterized D1R D1R characterized have studies no knowledge, hat HCN1 is a type 5 cell marker (Figure 5). We 5). (Figure marker 5 cell a istype We HCN1 hat ) investigated the immunoreactivity of DARPP32, DARPP32, of immunoreactivity the investigated ) mmunoreactivity as a marker, we found that XBCs we that found a marker, as mmunoreactivity t three type 5 cells were positive and two type 5 5 twotype cells and 5 type were positive t three (1997) used a D1R antibody in goldfish, rat, and and rat, goldfish, D1R in a (1997)antibody used gative. smission, and found that horizontal, AII amacrine, amacrine, AII that horizontal, and found smission, characterization was not conclusive onlyconclusive wasnot characterization oreactive markers were only available for type 6. 6. type for available wereonly markers oreactive ing that bipolar cell types are equivalent in rat in equivalent are types bipolar cell that ing ype 2 and rod bipolar cells, and ChAT, TH and TH ChAT, cells, and bipolar rod 2 and ype type 5 ON bipolar cells. CtBP2 (ribbon (ribbon CtBP2 cells. ON bipolar 5 type e, we investigated D1R expression in bipolar bipolar expression in D1R we investigated e, bipolar cell types. In combination with bipolar withbipolar combination cellIn types. bipolar INL cells and the IPL; however, the cell types the IPL; however, the and cells INL and and

D1Rs were expressed in cone bipolar cells but not i but not cells bipolar cone in wereexpressed D1Rs discussed are network bipolarin cell the dopamine summariz are properties typedependent cell bipolar dopa Howmatrix. does cell bipolar the in pathways th indicating manner, typedependent in a expressed ex cell bipolar modulates dopamine that demonstrate bydopamine werereduced cells,which bipolar from gammaaminobutyr (1995)recorded and Wellis Werblin applicatio bydopamine wereincreased cells bipolar thatglutam (1994)demonstrated and Maguire Werblin dopamine and functions cell Bipolar Haverkamp). Silke withDr. communication cellov staining weaker bipolar the to attributable T (4 mice). tdTomato werenegativefor cells 9 type in 3 cells mice). (n=91 withtdTomato colocalized G 9). 1 (type Clomeleon (typeor 7) Gus8.4GFP and we genera cells, 9bipolar and 7 analyzing type For cells type 5 of twosets those, Among 2014). al., mo the in wereshown 5 cells type of sets different a but tdTomato, with cells colocalized 52 type and with the fact that dopamine is released in mesopic in released mesopic is dopamine that fact withthe

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons er the ClmYFP mice generations (personal generations mice ClmYFP the er expressed D1Rs while another did not. did whileanother D1Rs expressed 24 In Clomeleon 1 double transgenic mice, all fifteen all fifteen mice, transgenic 1 double Clomeleon In use retina (Helmstaedter et al., 2013; Ichinose et et Ichinose 2013; al., et (Helmstaedter retina use nother type 51 ON bipolar cells did not. Three Three not. cells did ON bipolar type 51 nother to photopic conditions. However, a subset of cone cone of However, subset a conditions. photopic to he small number of type 9 cells is likelyis 9cells type of small number he ted double transgenic mice with Drd1atdTomato withDrd1atdTomato mice transgenic ted double below. n rod bipolar cells (Figure 9), which is consistent is which cells 9), (Figure bipolar rod n mine modulate bipolar cell visual signaling? The The signaling? visual cell bipolar mine modulate n, and this effect was mediated via D1Rs. Also, Also, via D1Rs. effect wasthis mediated and n, ed in Figure 9, and the potential functions of potential functions the and 9, Figure in ed signaling through D1Rs. Both results Bothresults D1Rs. through signaling at dopamine modulates visual processing processing visual modulates dopamine at citatory signaling. We found that D1Rs are D1Rs are that found signaling. We citatory usGFPpositive type 7 bipolar cells clearly 7 bipolar type usGFPpositive ate puffevoked responses in salamander OFF OFF salamander in responses puffevoked ate ic acid (GABA) puffevoked inhibitory currents inhibitory currents puffevoked (GABA) acid ic Page 24of50

Page 25of50 AIIAII coupling is regulated both by dopamine and and bydopamine both regulated is coupling AIIAII

Similarly, dopamine might not play a role in gap ju in gap role a play not might dopamine Similarly, colorvision. rolein a major have do ma types not cell bipolar D1Rexpressing 2014). and Scones from both receive inputs cells bipolar bipolar cells whereas type 1 cells are Mopsin sele Mopsin are cells typewhereas 1 cells bipolar c colorcoded designated cell types are bipolar Two inputs. rol a play not signaling D1R might thus, and types, inputsty conemixed and Rod 9). (Figure 2006) al., an 1 whiletypes 2008) al., Haverkamp et 2007; al., receive 7 and 4, 3a, 3b, Types signaling. dominant a play might dopamine cells, input bipolar rod/cone cones from both inputs receives mixed cells bipolar 1995). Massey, (Mills and dopamine than rather cel amacrine with AII connections junction gap make dynamicallyregulated are that synapses electrical 8D). This might indicate that there are multiple re there multiple are that indicate might This 8D). expressD1Rs cells amacrine AII % 25 of that showed al et (Kothmann (NMDA)receptors methylDaspartate

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons gulation systems of AIIAII couplings. couplings. of AIIAII systems gulation 25 nction regulation in bipolar cells. Gap junctions junctions Gap cells. in bipolar regulation nction ctive green OFF bipolar cells (Figure 9). All othe All 9).(Figure cells OFF bipolar green ctive rodand cone of changing dominancy in the e by illumination. Although ON cone bipolar cells cells conebipolar ON Although byillumination. Mcones (Breuninger et al., 2011; Ichinose et al.,et Ichinose 2011; al., (Breuningeret Mcones pes do not match D1Rexpressing bipolar cell cell bipolar not D1Rexpressing do match pes d 2 receive inputs only from cones (Haverkamp et cones(Haverkamp from inputsonly receive d 2 cone to roddominant switchingfrom in role mixed inputs (Mataruga et al., 2007; Tsukamoto et et Tsukamoto 2007; al., et (Mataruga inputs mixed in mixed D1Rs expressed If rods. are and ON bipolar cell activity through postsynaptic N postsynaptic cell activity ON through bipolar ells. Type 9 cells are Sconeselective blue ON ON blue Sconeselective cells are 9 Type ells. tch chromatic pathways; thus dopamine may not may not dopamine pathways;thus chromatic tch ls, nitric oxide appears to be the key regulator key the regulator be to appears oxide nitric ls, , while others do not express D1Rs (Figure 8C (Figure D1Rs not express do whileothers , ., 2009; Kothmann et al., 2012). Our results Ourresults 2012). al., et Kothmann .,2009; are are r 2013; Borghuis et al., 2013; Ichinose et al., 2014) al., et Ichinose 2013; al., et Borghuis 2013; modulating bipolar cell visual signaling. Because Because cell signaling. visualbipolar modulating channels voltagegated GPCRregulated possess cells Althoug 2013). al., Puthusseryet Pan, 2000; 1998; among expressed heterogeneously are channels gated Detailed maps of temporal tuning in bipolar cells h bipolar in temporaltuning of maps Detailed processing. temporal processing, visual properties, channel own byits tightlyis regulated significance of understanding D1R distribution acro distribution D1R understanding of significance andHel Ichinose 2014; et al., (Ichinose laboratory th of the findings integrates 9 Figure 2005). al., Slaug and respectively tuning, (Awatramani temporal t responses sustained and transient twotypes: into (Surmeier et al., 1992; Cantrell et al., 1997; Catt 1997; al., et 1992; al., Cantrell et (Surmeier wh phosphorylation (GPCR)mediated receptor coupled Volta 2014). al.,et Smith 2008; al., et (Mojumder re mammalian the in withobservations consistent is bipolar cells, D1Rs are expressed in transient but but in transient D1Rs expressed are cells, bipolar tempo between acorrelation have found we may data, stim steplight to response vs.sustained transient resulting in reduced lightevoked responses in a su in a responses lightevoked reduced in resulting

Ichinose and Lukasiewicz (2007) demonstrated that d that demonstrated Lukasiewicz and (2007) Ichinose

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons e present study with previous results from our from results previous study with e present erall, 2000; Cantrell and Catterall, 2001). Voltag Catterall, 2001). Cantrell and erall, 2000; channel activity might modulate timedomain of timedomain modulate activity channel might . Bipolar cell light responses are roughly are divided cell responses light . Bipolar gegated channel activity is regulated byGprotein activity regulated channel is gegated ulation. With the inclusion of the D1R expression D1R expression the inclusion of the ulation. With lmer, 2015). This summary highlights the the highlights summary This 2015). lmer, the time course of voltagegated channel activation channel course voltagegated of time the 26 not sustained cells. Conversely, D1Rs are are D1Rs Conversely, cells. sustained not ave been described by several groups (Baden et al., et (Baden groups byseveral described been ave o steplight stimuli reflection of high and low and high of reflection stimuli steplight o bset of bipolar cells in the salamander retina. Th retina. salamander the cells in bipolar of bset ss the different types of bipolar cells as related asrelated cells bipolar typesthe of ss different h we do not currently know which types of bipolar bipolar of types which currently know wenot do h tina using electroretinogram (ERG) analysis (ERG) electroretinogram using tina hter, 2000; Euler and Masland, 2000; Ichinose et 2000; Ichinose Masland, and Euler 2000; hter, opamine reduces the voltagegated Na voltagegated the reduces opamine ral properties and D1R expression. In ON ON In expression. D1R and properties ral , this may be the target of dopamine for for dopamine of target the be , this may ich can be a target of dopamine modulation modulation dopamine of a target be can ich bipolar cells (Connaughton and Maguire, and cells (Connaughton bipolar + current, current, Page 26of50 e

to to is

Page 27of50

signaling in bipolar cells plays a role in in temporal role cellsa plays bipolar in signaling OFF bipola transient not but sustained in expressed Parkinson’s disease exhibit a deficit in se a flicker deficit exhibit disease Parkinson’s and/o and motion conemediated multiple strengthens pathw signaling visual of sculpting in the dopamine previouslyrealize retina than the mouse of network more for a evidence provide results our summary, In 1997; al., et (Djamgoz cells responding transiently

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons nsitivity, which might be attributable to dysfuncti to attributable be might which nsitivity, processing. Consistent with this notion, patients withthis notion, Consistent processing. 27 Jackson and Owsley, 2003). Owsley, 2003). and Jackson ays. This suggests that dopamine supports and and supports dopamine suggeststhat This ays. d, suggesting more important modulatory roles for for modulatory roles important more suggesting d, r cells. These results strongly suggest that D1R D1R that stronglysuggest results These cells. r complex D1R localization within the bipolar cell cell bipolar the within localization complex D1R r flickersensitive pathways. pathways. r flickersensitive onal onal with All authors had full access to all the data in the data the all to access had full authors All

Role of authors authors of Role interest competing financial no declare authors The Statement Interest of Conflict manuscri the on comments insightful for Lukasiewicz als are Authors support. injection neurobiotin his CoreFaci in Vision K. and Vistisen Barret R. thank for the gift of dab1 antibody, and AA Hirano for h for Hirano AA and antibody, dab1 of gift the for Margolskee R.F. antibody, the TH and mice tdTomato Univ State (Wayne Kapatos G. thankwouldto like We Acknowledgements Other BF equally contributed. PF and and TI. DMK, BF, Obtai and TI. PDW analysis: Statistical and TI. PDW manuscript the Critical of revision TI. manuscript: interpretat Analysis and DMK, and TI. BF, PF, data: Study analysis.data the accuracyof the and data

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons study and take responsibility for the integrity of integrityof the responsibility take for studyand for important intellectual content: PF, BF, DMK, PF, content: intellectual important for o grateful to Drs. ZH Pan, DQ Zhang, and PD PD Zhang, DQ and ZH Drs. Pan, to grateful o er gift of the NK3R antibody. We also wouldt like also antibody. NK3R We the ofgift er 28 lity for their experimental support and N. Challa N. f Challa and support experimental their lityfor concept and design: PDW and TI. Acquisition of Acquisition and TI. PDW design: and concept s. ion of data: PF, BF, PDW, and TI. Drafting of the the Drafting of and TI. PDW, BF, PF, data: ofion ned funding: PDW and TI. Technical support: PF, PF, support: and TI. PDW Technical ned funding: pt. pt. ersity) for generously providing the Drd1a the generouslyproviding ersity)for for the gift of GUS8.4GFP mice, DQ Zhang Zhang DQ mice, ofgift the GUS8.4GFP for the the Page 28of50 o o or or Page 29of50 BloomfieldVolgyiSA,B. diverse 2009. The function Berens T, Baden P,Bethge Euler Spikes T. M, 2013. Borghuis BG, BG, Borghuis MarvinJS,Looger JB. Demb LL, 2013.T Caille I, BlochCaille B, Dumartin 1996.UltrastructuraB. Breuninger T, Breuninger T, HaverkampPuller EulerC, S, T. 2011. Cangiano L,Cangiano Gargini C, DellaSantinaGCL, Demontis Connaughton VP, Connaughton Maguire 1998.DifferentialG. expre Cohen AI, Cohen Todd RD,Harmon O'Malley S, KL. 1992.Pho StructureCatterallWA. 2000. andregulation vol of Cantrell AR, Cantrell CatterallWA. 2001.Neuromodulation of Deans GoodenoughDeans MR,Volgyi B, Bloomfield DA, SA, P Lin Contini Kobayashi B, M, Okano K, Masland H, RH, Cantrell AR, Cantrell Smith RD,GoldinScheuer AL, T, Catter

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology l localizationl dopamineD1 of receptor immunoreacti aryalinlocalizationrats anddopamineD1 of re D2 tribution of hyperpolarization-activated tribution of andcyclic John Wiley& Sons all 1997.DopaminergicWA. sodium modulation of cur tage-gatedCa2+ channels.Annu BiolCell Rev Dev 16 bipolar Chromatic cell pathwaysinthe retin mouse alroles andregulation ofneuronal gap junctions i in cellsmammalianbipolar support temporal layerin , Cervetto , L. 2007.High-pass filtering ofinput si wo-photonimaging ofnonlinear glutamate dyrelease tinalcells: bipolar buildingelementary ofblocks Raviola SynapticE. input 2010. of cellON-bipolar channels: Na+ unexpectedan cellular form plastof f cellsbipolar in a mammalianJNeurophysi retina. Improved TransgenicBAC Fluorescent Reporter Line eenlight,dopaminerelease couand horizontal cell ssionvoltage-gated and of K+ currentsCa2+ inbipo ent andsustained ent responses inganglion cells th of toreceptors toreceptors retinas of mouse possess receptorsD4 c e H.2004. Typesbipolar e of inthe cells reti mouse modulation of gap modulation junction of permeability between ama Neurobiology of retinal Neurobiology dopamineof inrelation deg to aul DL. aulDL. 2002.Connexin36 is essentialfor transmiss Ectopic retinal Ectopic bipolarON synapsesin cell OFF the Comp Comp Neurol 501(6):891-903. 1362. polar cell. ONE PLoS 2(12):e1327. 12093-12097. Spiny Frontiers Neurons. insystems neuroscience 5 ci ci 33(27):10972-10985. beling.Neuroanat JChem 32(2-4):101-116. ergicinnervation.Brain Res 730(1-2):17-31. 29 d ganglionmelanopsin cells. NeurolJ Comp 517(2):2 uron36(4):703-712. urol urol 518(11):2035-2050. ionofspecific sitesinthe sodiumchannel alpha gnals by theIh nucleotide-gated ceptors ceptors striatal on nthe retina. Nat vision. Rev Nat s s onto the vity in vity rat a. JNeuroscia. :521-555. ol 83(4):1817- ol na.J Comp g inner of the e retina. e J pling inthe icity.RevNat lar cellsinthe inner plexiform rentin ionrod- of namicsat enerative forSensitive oupled to oupledto crine cells crine :32. 26-244. 26-244. Ichinose T, Lukasiewicz PD. 2007. Ambient light reg Ambientlight 2007. PD.LukasiewiczT, Ichinose HelmstaedterM,Briggman JainSC,Turaga KL,V, Seu S, Haverkamp WässleH, DuebelKunerJ,Augustine T, Ichinose Fyk-Kolodziej Ichinose T, B, J.2014. RolesCohn of Ichinose Hellmer Ichinose T, CB. 2015.Differential signalin Hampson VaneyEC, R, Weiler 1994.pH-gated dopa DI. Haverkamp S, Haverkamp MichalakisS, E, SeeligerClaes Hu MW, Haverkamp S, Haverkamp Specht D, ZaidiMajumdarNF,S, Brandst Ichinose ShieldsCR, Ichinose T, Lukasiewicz 2005.Sodiu PD. Huang L, ShankerHuang L, YG, DubauskaiteJ, ZhengYanJZ, W, Huang L, MaxHuang L, M, MargolskeeRF, SuMaslandH, Eu RH, Jackson GR, Owsley Jackson C. 2003.Visualdysfunction, neu Jarvie KR,Booth Jarvie G, BrownNiznik HB. EM, 1989.Glyc Luedtke RR, Luedtke GriffinConroySA, SS, Jin X, A, Pinto Kothmann WW, Kothmann SC, J.Massey O'Brien 2009. Dopamine-s H,WangS, Kindler Richter Tiedge H. D, 2005. RNAt JinChuangNG, MassonRibelaygaAZ, CP.PJ, 2015. Ro Kothmann WW, Kothmann EB, Trexler CM,Whitaker W, MasseyLi Maguire G, F.Maguire Werblin 1994.Dopamine enhances glu a Mataruga A, Mataruga Kremmer E, MullerF.2007.Type and 3a Mangel SC, Mangel JE.Dowling 1985.Responsiveness andrec Mills SL, MasseyMills SC.1995. propertiesDifferential Mojumder Mojumder SherryDK, DM, LJ. Frishman 2008. Contribu of the of retina. mouse JNeurosci 25(22):5438-5445. 507(1):1087-1101. cellsbipolar ofretinathe expressmouse calsenili Nat Neurosci2(12):1055-1062.Nat The Journalneuroscience The of : journal the official mammalian retina. Biol Sci Proc 255(1342):67-72. Neurosci Neurosci 26(19):5248-5255. CNGA3(-/-)bipolar cone mice: cells react the on mi the retina.the mouse Journal of Physiology J Neurosci JNeurosci 27(17):4756-4764. alpha o, G and alphabeta o, in3, beta G 4 retinal bipol ON Ggamma13 colocalizes Ggamma13 withgustducin receptintaste plexiform layerin plexiform the mouseNature retina. 500(746 in ganglion cells. JNeurosciin ganglion cells. 25(7):1856-1865. 6759. activity-dependentplasticity ofgap junctional cou amacrine cell uncoupling. cell amacrine J Neurosci 29(47):14903-1 21:223-245. dopamineinmouse retina. JPhysiol 593(7):1597-163 parathyroid gland. parathyroid PharmacolMol 36(4):566-574. murine murine antibodiesmonoclonal specificrat forthe D 101(2):170-187. pathway in pathway retina.the mouse NeurolJ Comp 502(6):1 darkness anddopamine. darkness Science 229(4718):1107-1109. JNeuroscisalamander retina. 14(10):6094-6101. 377(6551):734-737. mammalianelectroretinogram.flash JPhysiol 586(10

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology In Press Sesack1999.SR.Immunoblot and immunohistochemical g andgreceptorglutamate compositions in biOFF the ON cone bipolarON cell cone subtypes in temporal i coding of two gapof two pathways junctional AII madeby amacrine John Wiley& Sons m channels m intransient retinal cellsbipolar enhan ulates sodium channel activity to dynamically contr dynamically activityto sodiumchannel ulates ransportandlocal control Annu of translation. Rev rodegenerativediseases, andaging.Clin 21( Neurol oprotein nature oprotein dopamine of receptorsD1 in bra the ng W. HS,Denk reconstruction 2013.Connectomic of mphries P, Biel mphries Feigenspan P, Synaptic M, plaA. 2006. Euler FengG, GJ, T. 2005.The primordial,blue-co eptive field eptive size of horizontalcarp cells are redu type bipolarconeOFF 3b cells forprovidethe alte d electrical couplingdelectrical controlled is by circadian a Rosenzweig S, SpielmanS, Rosenzweig AI, MaxM,Margolskee 19RF. tamate-gated ionic tamate-gated currentinbipolarOFF cells of minergic minergic horizontal modulation of cell gap junction atter JH,atterWasco W, WässleS.Tom H, Dieck 2008.Typ ler T. 2003.G T. ler proteinsubunit gamma coexpr G is 13 tion of voltage-gated sodiumtion of channels b-wave the to . SC, O'BrienSC, J.2012. Nonsynaptic NMDAreceptors med timulated dephosphorylationtimulated connexin of mediates36 of the Society forof Neuroscience 34(26):8761-8771. nand cones contact as as rods.well NeurolJ Comp ar ar cells.NeurolJ Comp 455(1):1-10. plingAIIamacrine inthe cell network. JNeurosci ssinginput andform cone synapses ectopic rod with 30 1aandD1b dopaminereceptor subtypes. JNeuroimmun 1):168-174. 4911. or cells and or IP3 mediates responsesbitterto denat 1. ):2551-2580. 123-1137.

nthe retina. mouse ce ce visualresponses ced by prolonged clock and clock polarcell intypes Cell BiolCell Dev ol retinalsignaling. ol ne ne colorsystem cells. Nature 3):709-728. rnativerod the the tiger sticityin comparison of in and s in s theinner essed G essed with 32(20):6747- of of the e 4 4 cone OFF e 99. 99. iate iate AII s. Js. onium. Page 30of50 ol ol Page 31of50 Mora-Ferrer YazullaS, C, Studholme Haak-FrendsKM, Muller F, ScholtenF, Muller IvanovaA, E, Haverkamp S, Kremm Pignatelli V, Pignatelli Strettoi E. 2004.Bipolar cells ofth Pan ZH.2000. Pan expressionDifferential ofhigh- andt Ogata G, Stradleigh G, Ogata TW, PartidaIshidaGJ, AT. 2012 SimonJ,Nguyen-Legros A, Bloch I, B. Caille 1997. ShuenM,JA,ChenGlossB, N.Calakos2008. Drd1a-t Curran Rice DS, 2000. T. Disabled-1 is in expressed Puthussery T,Puthussery Venkataramani Gayet-PrimoSmithJ,S, Ribelayga C, Ribelayga CaoY, Mangel circadian SC.2008. The Smith BJ, CoteBJ, Smith Tremblay PD, F.2014. DopamineD1 re Thibault D, ThibaultFortinD, F,Loustalot GM,Bourque MJ, Tru Ting JT, FengG. TingJT, 2014.Recombineering strategies fo Szel A, RohlichA,Szel P,Caffe AR, AguirreG Juliusson B, Tsukamoto Tsukamoto Y, K, Morigiwa Ishii IwatsukiM, Takao M, AltrockS, tom Dieck WD, Kessels Qualmann MM, B, Re Surmeier DJ, SurmeierDJ, EberwineWilsonJ,CaoCJ, Stefani Y, SL, Jr.,Stella Thoreson WB. 2000.Differential mod Vaquero CF, Vaquero Pignatelli A, PartidaIshidaGJ, AT. 20 Soto F, BleckertF, A, Soto R, Lewis Y, Kang Kerschensteine Veruki ML, Veruki WässleH.1996. Immunohistochemical loca Voigt T, WässleH. Voigt 1987. Dopaminergicinnervation o Urschel S, HoherS, Urschel Schubert AlevT, T, Sohl C, G, Wor Van Hook VanHook Wong KY,BersonMJ, 2012.DM. Dopaminergic goldfish retina. JComp retina. goldfish Neurol 411(4):705-714. bipolar cells of the ratcellsbipolar ofretina.the JNeurophysiol 83( receptors inadult retinalreceptors rat J ganglion cells. Co retina retina Neurosciof Vismammals. 14(3):545-551. retinal bipolar retinal cells andsynapticconcentrated at 424(2):327-338. 476(3):254-266. bipolar cellsbipolar augment input magnocellularto visual 16059. medium spiny neuronsspiny medium in thedirect patand indirect segregation insegregationthe developing usingstriatum tr BAC classes classes of inthecones retina. Neurolmouse J Comp BrandstatterJH. 2005.Molecular dissection ofthe striatonigral neurons. striatonigral Proc USciAcad Natl AS 89( andbipolar conerevealedON bycells ectopic metab andRIBEYE isBassoon essential assembly for the of Frontiers andbeyond. inbehavioral neuroscience 8: D2 dopamine receptorsD2 and JNeurosciEur cAMP. 12(1 excitatory synapses excitatory retinal onto ganglion cells dur Neurosci Neurosci 35(4):507-518. network adaptation network inretinal JNeu ganglion cells. daily rhythms inphotopicrhythms daily vision.Chronobiology int 4128. 8(11):2286-2297. mouse retinas. Jmouse Neurosci 27(23):6261-6267. communication between communication AII cells.amacrine JBiol Ch A-mediated kinase phosphorylation connexin36 of in

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology e e mousegeneretina: a gun, Jmorphological study. , Van , T. Veen 1992.Uniquetopographic separationo 01. dopamine-Aand protein kinase A-dependent mech Immunocytochemicallocalization ofdopamine receD1 A, KitaiA,1992. ST. Dopaminereceptor subtypescoloc ulation ofrod andcalciumconecurrents intiger s type AIIamacrine in cells retina.the mouse JCom John Wiley& Sons rD, CraigAM, Wong RO. increase2011. Coordinated sdorferP,Asahara T, R, Dermietzel R, Weiler Wille wo types of low-voltage-activated wo of types calciumcurrents .Dopamine andfull-field illumination activate D1 clockinthe retina controlscoupling. rod-cone Neu r developingr nextgeneration transgenic f BAC tools deau LE. and2013. Evaluation of dopamineD1 D2 rec dTomatoBAC transgenic forsimultaneous mice visual K,NakanishiFukuda S, Y. novel2007.A connection fcellsIIamacrineA in mammalianretina. JNeuros Taylor RG, WR. 2013.NaV1.1 inchannelsaxon initi ceptorsmodulate ONcone Nav channelscell bipolar er E, E, Kaupper HCNUB. 2003. are channels diexpressed cho 1999. cho M. Dopamine D1-receptorimmunolocalizatio gusBraunerFejtovaH, D, O, Bracko GundelfingerA, lization ofdopamine lization receptorsD1 inrat retina. Eu mp Neurol mp 520(17):4032-4049. 1):513-527. modulation of ganglion-cell modulation of photoreceptors in rat. terminals. Eur JNeurosciterminals.Eur 17(10):2084-2096. 21):10178-10182. ingNeuraldevelopment. Dev 6:31. rosci21(21):8624-8635. photoreceptorsynapse:ribbon physical interaction ernational:1-11. pathways primate inthe retina. JNeurosci 33(41): 31 complex.ribbon Jthe Biol Cell 168(5):825-836. ansgenicmice. ONE PLoS 8(7):e67219. 325(3):327-342. 111. hways basal hways of the ganglia. JNeurosci 28(11):2681- em 281(44):33163-33171. otropic glutamate otropic receptor (mGluR7) in 7 mGluR6-def mouse retina resultsmouse indecreased gap junctional 0):3537-3548. CompNeurol alamanderby retina pNeurol and D2-D5-type cke 2006.Protein K. f two spectral ron ron 59(5):790-801. alize inalize rat inrod andcone or or optogenetics ci ci 7(12):4115- ininhibitory and between rods al segmentsal of ptorsinthe anismfor r Jr Neurosci eptor eptor ization of to controlto ED, ED, fferentiallyin JEur nin 16045- of 2685. icient Zhang DQ, Zhou ZhangTR, DQ, DG. 2007.FunctionalMcMahon heter D, BloomfieldSA. Xin 1999.Dark-and light-induced WitkovskyP,Svenningsson Yan H,L, P, Bateup Silve WitkovskyP. 2004.Dopaminefunction. and retinal D WerblinWellis DP, FS. 1995.Dopamine GAB modulates Zinchuk V, Wu Wu ZinchukGrossenbacher-ZinchukV, Y, O, Stefani E. B, DebertinVolgyi Balogh G, M, E, Popovich Kovacs- Wässle H, PullerWässle F,C, Muller Haverkamp S.2009.Co H. Wässle 2004.Parallelprocessing inthe mammalia

Comp Neurol Comp 405(1):75-87. retina. JNeurosciEur rodent 25(11):3233-3242. release transmitter from bipolar cell interminals JNeurosci retina. 29(1):106-117. multiple roles multiple invision.J Neurosci 27(3):692-699. enhanced backgroundenhanced reduction proteinproximit with 6(10):1554-1567. innervation to AIItoinnervationamacrine in rabbitretcells the

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons changes incoupling between in horizontal cells ma rR. localization2007.Cellular and function of DA Oller T. 2014. Compartment-specifictyrosine hydrox ne ne contacts, mosaics,andterritories of bipolar ce nNat retina. NeurosciRev 5(10):747-757. ocOphthalmol 108(1):17-40. 2011. 2011. spatialQuantifying correlations fluoresce of Ac receptors Ac inhibition mediating ofcalcium entry ina.Neuroscience 270:88-97. tiger tiger JNeuroscisalamander retina. 15(71):4748Pt

ogeneity of retinal ogeneity dopaminergicneuronsof underlying 32 y index andindex y correlationcoefficient estimations. Na RPP-32 in RPP-32 the lls inlls the mouse mmalianJ retina. ylase-positive ylase-positive nt using markers intoand -4761. their their t t Protoc Page 32of50 Page 33of50

overlaid with overlaid ( (B3). incubation probe antisense cRNA D1R labeled with processed Radioac D1R mRNA. detects that probe antisense cRNA Drd1atdTomat the Autoradiogram from (B1) staining. tdTomato wasde section. atransverse in expression Dr from retinal preparations Analysis of 1 Figure Legends Figure negative for tdTomato (#). (B) A magnified view of A (B) magnified (#). tdTomato for negative werel cells (*). 2 Type withtdTomato colocalized identified cells, 1 cells. Type OFF bipolar 2 and b OFF 1 inand 2 type expression tdTomato 2 Figure homog was cells tdTomatoexpressing of distribution wascap image preparation. The retinal wholemount box(E the in from Highpower image (E4) processes. was fre D1R immunoreactivity (E3) layer. INL the in entire IPL with a stronger immunoreactivity seen in seen immunoreactivity stronger witha IPL entire conebipola of process dendriticon detected mainly antib withdsRed wasenhanced fluorescence tdTomato represent dimers in various states of glycosylation states of various in dimers represent molec larger kDa. The 4566 between D1R, for weight antibod with D1R mouse immunolabeled (TOM) tdTomato bl (D) boxC1. the in Western from image Highpower in situ in 35

Slabeled D1R Slabeled

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This article isprotected by copyright. All rights reserved. cRNA sense probe (B2) and pretreated with RNAse pri withRNAse pretreated (B2) and sense probe cRNA Journal ofComparativeNeurology John Wiley& Sons 35 Slabeled D1R cRNA probe (low power image). (C2) (low image). power probe cRNA D1R Slabeled with NK3R but not Syt2b antibodies, were antibodies, not Syt2b but withNK3R d1atdTomato transgenic mice. (A) tdTomato tdTomato (A) mice. transgenic d1atdTomato abeled with both NK3R and Syt2b, but were but Syt2b, and NK3R withboth abeled 33 (Jarvie et al., 1989; Luedtke et al., 1999). (E1) (E1) 1999). al., et Luedtke 1989; (Jarvieal., et a type 1 cell soma. All magnified views in this this an views in All soma. magnified 1 cell type a

s3 quently colocalized with tdTomato positive cell withtdTomato quentlycolocalized r cells in the OPL and cell processes within the the within processes cell and OPL the cellsr in eneous across the retina (n=2 mice). (n=2 mice). retina the across eneous tected using antiDsRed antibody and DAB and DAB antibody antiDsRed using tected 3). (FI) tdTomato fluorescence in the INL in in in the INL fluorescence (FI)tdTomato 3). ipolar cells. (A) NK3R antibody labeled type type 1 labeled antibody (A) NK3R cells. ipolar ot of mouse retinas from wild (WT) and Drd1a wildand (WT) retinas from mouse of ot tured from different regions of the retina. The regions the of different from tured o retinal section processed with processed section retinal o C1) tdTomato expression (DAB staining) isstaining) (DAB tdTomato expression C1) ular weight bands (~90150 kDa) kDa) may(~90150 weightbands ular stratum. Staining was hardly detected in somas somas in Stainingdetected was hardly stratum. ody. (E2) Punctate D1R immunoreactivity was immunoreactivity D1R Punctate ody.(E2) tivity is not observed in control slices slices control in observed tivitynot is y. The arrow shows the reported molecular molecular reported y.the arrow The shows 35 or to or SlabeledD1R 35 S

d axon terminals did not colocalize with tdTomato. C withtdTomato. not did colocalize terminals axon withtdTo colocalized terminals wideaxon with cell typofsets different labeled Neurobiotin 4 Figure fluorescen tdTomato confirmed measurement intensity s cell OFF bipolar 4 type that views show Magnified bipolar cells OFF type 4 (Csen)labeled Calsenilin tdTomato fluorescenc revealed measurement intensity colocal thatare cells OFF 3b bipolar type of somas werepositiv cells OFF bipolar type 3b RIIβlabeled fluore tdTomato no confirmed measurements intensity term viewaxon shows (A2) A magnified cell. bipolar (*). tdTomato colocalizewith not cells did bipolar b OFF 3 inand 4 type expression tdTomato 3 Figure (n=10). withtdTomato colocalize measuremen intensity (F)Fluorescent withtdTomato. entir the across wasobserved fluorescence tdTomato bipo type 1 For Methods. in the described as (n=8) bipolar cel and tdTomato of intensities Fluorescent fluore withtdTomato colocalization no showed cells

of type 1 and type 2 cells. Only the type 1 cell type c 1 the Onlycells. type 2 1and type of digital single excised arefrom figures subsequent

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons olocalized with tdTomato. (D) Three somas of type type somas2 of (D) Three withtdTomato. olocalized (A1) A magnified view shows a soma of a 3a OFF OFF a3a of view soma a shows A magnified (A1) e 5 ON bipolar cells. (A) A type 5XBC ON bipolar bipolar type ON 5XBC A (A) cells. ON bipolar 5 e 34 section images (0.30.5 m thickness). (C) Somas (C) Somas thickness). m (0.30.5 images section mato. (B) A type 5 ON bipolar cell with compact withcompact cell bipolar ON (B) type 5 A mato. were colocalized with tdTomato (*). (E1E2) (E1E2) (*). withtdTomato werecolocalized l markers were measured across somas (inset) (inset) somas across weremeasured markers l e for tdTomato (*). (C1C2) Magnified views show Magnified (C1C2) (*). tdTomato for e ized with tdTomato fluorescence. (D) Fluorescent (D) Fluorescent fluorescence. withizedtdTomato hAT band staining is shown for the the IPL sublayers. for shown is staining band hAT lar cells, NK3R labeled the cellular edges while edges cellular the labeled NK3R cells, lar scence. Scale bars indicate 10 µm. (E) µm. 10 indicate bars Scale scence. omas colocalized with tdTomato. (F) Fluorescent (F)Fluorescent withtdTomato. colocalized omas inals of a 3a OFF bipolar cell. (B) Fluorescent(B) cell. OFFaof3a bipolar inals ipolar cells. (A) HCN4 labeled type 3a OFF OFF 3a type (A)labeled HCN4 cells. ipolar t showed that type 2 cell somas did not did somas cell 2 that type showed t e soma. Some type 1 cells were not labeled labeled werenot cells 1 Some type soma. e scence in HCN4labled cells (n=10). (C) PKA (C) PKA cells (n=10). HCN4labled in scence e in type 3b OFF bipolar cell somas (n=10). (E) cell(n=10). somas bipolar type OFF 3b in e ce in type 4 OFF bipolar cell(n=11). somas bipolar 4 inOFF type ce Page 34of50

Page 35of50 All panels are single digital section images (0.3 µ (0.3 images single section digital are panels All

(D) High power view showing HCN1 staining and CtBP CtBP and staining HCN1 view showing power (D)High sy ribbon a with overlaps signaling (C) HCN1 2005). withdiffer retina in mouse reported immunostaining similarpatte showed (B2;1) Table Systems Synaptic anti IPL. Both the throughout labeled are terminals th in labeled are in photoreceptors synapses ribbon sy ribbon labeled antibodies CtBP2/Ribeye antibody. ed outer IPL the the near in wasobserved signaling 5 type labels antibody thatHCN1 Evidence 5 Figure puncta were(955 withtdTomato. colocalized not or the as wereexpressed CtBP (I) puncta animals). n=3 HCN1 with colocalized not or withHCN1 colocalized were puncta (H) CtBP (lower panel). 90º wasrotated no whereas panel), upper images; (n=20 wasdetected coefficient analys (G) crosscorrelation 2D random. i (lower panel) coefficient correlation the reduced colocaliz withHCN1 puncta (n=25 staining withHCN1 signal HCN1 and puncta analysis CtBP of coefficient immediatelynext staining withtdTomato colocalized withbipolar colocalized that HCN1 indicating HCN1, stainin strong HCN1 the of side another toside one

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons ndicating that HCN1/CtBP2 colocalization was not wasnot colocalization HCN1/CtBP2 that ndicating m thick). From a through d, images were taken from weretaken images d, through From a thick). m 35 CtBP2 from Santa Cruz (B1) and antiRibeye from from antiRibeye and Cruz (B1) Santa from CtBP2 is of tdTomato and HCN1 signaling. Correlation Correlation signaling. HCN1 and tdTomato of is ent Ribeye antibody (Figure 1; (tom Dieck et al., al., et Dieck (tom 1; (Figure Ribeye antibody ent e OPL, while ribbon synapses in in bipolar synapses cell whileribbon OPL, e g every 1 µm. Many CtBP puncta colocalized withcolocalized puncta Many CtBP geveryµm. 1 ge of the ON ChAT band. (B) Verification of CtBP CtBP of Verification (B) band. ChAT ON the of ge analyzed from 9 images, n=3 animals). n=3animals). 9 images, from analyzed napse marker, CtBP, showing labeled puncta. puncta. showing labeled CtBP, marker, napse to the ON ChAT band. (F) 2D crosscorrelation crosscorrelation (F)2D band. ChAT ON the to cell terminals. (E) Strong HCN1 staining also also staining HCN1 Strong (E) cell terminals. ing. The majority of CtBP2 puncta colocalized CtBP2 puncta majorityof The ing. cells and colocalized with tdTomato. (A) HCN1 withtdTomato. colocalized and cells expressed as the proportion of puncta puncta of proportion as the expressed napses in mouse retinal sections. Puncta of Puncta of sections. retinal in mouse napses (1217 puncta were analyzed from 12 images, images, 12 analyzedfrom were puncta (1217 rn that are also consistent with the withthe thatconsistent alsoare rn proportion of puncta colocalized with tdTomato tdTomato with colocalized puncta of proportion ation; upper). A 90° rotation of the HCN1 image HCN1 image the of rotation 90° A upper). ation; correlation was observed when one channel onechannel when wasobserved correlation puncta from the dashed box illustrated in B. in B. illustrated box dashed the from puncta (B) ((B) fluore withtdTomato terminals axon type 6 viewsof c 6 cells type of terminals axon Only somasaxons). stained type 6 axon terminals (n=20 fields). ( (n=20 fields). terminals 6axon type stained

labeled axon terminals of type 6 cells and enti and the cells type 6 of terminals axon labeled coloca cells bipolar 7ON and type 6 Both 6 Figure Figure 8 Rod bipolar cells, horizontal cells, cells, and cells,horizontal bipolar Rod 8 Figure vesseblood BV, CP, pedicle. (Csen). cone dendrites par werealso terminal Scone genuine a to attached bipo type OFF 3b a is which PKARIIβ expressed cones in seen image magnified and (arrow colocalized not tdTomato positive 9 and Type tdTomato. express not with tdToma mice Doubletransgenic –C3) (C1 opsin. indicati observed, werefrequently cells expressing terminal the to wereattached processes fluorescent th indicating observed, wereoccasionally terminals tdTo and cells Sopsinexpressing Genuine 7 Figure vessel. blood withtdTom colocalized cells ON bipolar 7 type that withGu mice Doubletransgenic (C1C3) correlation. colocalized with tdTomato (arrows). (C1C3) AII ama (arrows).AII (C1C3) withtdTomato colocalized colocalizewithtdTomato. not did which withPKCα, left

Accepted ArticletdTo showed that coefficient correlation cross 2D )

This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons right AII amacrine cells. (A) Rod bipolar cells werelabe cells bipolar (A) Rod cells. amacrine AII re structure of type 2 OFF bipolar cells (dendrites cells bipolar OFF type 2 structure of re ) A 90° rotation of the green channel resulted inresulted n channel green the of rotation )A 90° 36 ng that they were mixed cones with Sopsin and M and withSopsin cones mixed they were that ng ato (C3). * indicates doublelabeled cells, BV, cells,BV, doublelabeled indicates * (C3). ato at they were genuine Scones. tdTomato Scones. weregenuine they at lized with tdTomato fluorescence. (A1) Syt2b fluorescence. lizedwithtdTomato scence in a single digital section (0.3 µm thick). µm (0.3 section digital single in a scence olocalized with tdTomato. (A2A3) Magnified Magnified (A2A3) withtdTomato. olocalized (B1–B3) Calbindinlabeled horizontal horizontal cells Calbindinlabeled (B1–B3) (see inset). (B) In the ventral retina, retina, Sopsin ventral the (B) inset). In (see bottom row). (D) tdTomato processes S near processes tdTomato row). (D) bottom sGFP(C1) and Drd1atdTomato (C2) revealed (C2) revealed Drd1atdTomato and sGFP(C1) tially colocalized with type 4 OFF bipolar cell OFF 4 withtype tiallycolocalized l; CP, cone cone pedicle. CP, l; crine cells immunolabeled with disabled1 (Dab withdisabled1 immunolabeled crinecells mato processes. (A) In the dorsal retina, Scone retina, dorsal the In (A) processes. mato processes associated with Scone pedicle were pedicle withScone associated processes to and Clm1 revealed that type 9 cells (*) did cells (*)did 9 that type revealed Clm1 and to lar cell marker. (E) tdTomato processes processes tdTomato (E) larmarker. cell mato fluorescence colocalized colocalized withsyt2b fluorescence mato Page 36of50 led o Page 37of50

(asterisk). (D3) ~25 % of ringlike formations were ringlikeformations % of ~25 (D3) (asterisk). rings Most (D2) border. IPL the INL/ at formations retinal a mount flat In (D1) (arrowheads). tdTomato (arrows). ~25 withtdTomato colocalized werenot 1) Masland, 2000; Ichinose et al., 2014; Ichinose and and Ichinose 2014; al., et Ichinose 2000; Masland, * 2014); al., et Ichinose 2011; al., et (Breuninger 200 Haverkamp al., et 2007; al.,et Tsukamoto 2007; recei types *, unknown u.k.: cell function. bipolar Gray: no cells, bipolar and 9,rod 3a, 51, 2, type 52, 4, 3b, XBC, 1, type positive, tdTomato Orange; e showing Drd1atdTomato scheme Summarized 9 Figure

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This article isprotected by copyright. All rights reserved. Journal ofComparativeNeurology John Wiley& Sons **, temporal properties of each type (Euler and and (Euler type each of properties temporal **, ving mixed rod and cone inputs (Mataruga et al., et inputs(Mataruga and cone rod vingmixed t studied, type 8. The table summarizes each type type each summarizes table The 8. studied, type t 37 filled with tdTomatopositive cells (triangle). (triangle). cells withtdTomatopositive filled were not filled with tdTomato labeled cells labeled withtdTomato filled werenot preparation, TH staining revealed ringlike revealed staining TH preparation, Hellmer, 2015). 2015). Hellmer, 6, and 7 bipolar cells. Black; negative, tdTomato Black; 7bipolar cells. and 6, % Dab1positive cells were labeled with with werelabeled cells Dab1positive % 8); **, chromatic characterization in each type type each in chromatic characterization 8); **, xpression in each type of bipolar cell. bipolar cell. type of each in xpression

Acceptedretinal of Figure preparations Analysis fromDrd1 1 Article

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a-tdTomato transgenic mice.transgenic a-tdTomato

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Acceptedlabelsantibody FigureHCN1 type Evidence55 that Article

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cells and colocalized and cells with tdTomato.

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Accepted7 type and 6 bipolarFigure ON Both 6 cells colocal Article

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ized with tdTomato fluorescence. withizedtdTomato

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FiguretdTom Genuine7 and S-opsin-expressing cells

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atoprocesses.

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FigureRod8 cells,bipolar cells, A horizontal and

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II amacrine II cells.

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Page 46of50 Page 47of50 TH Sy Ribeye α PKC RIIβ PKA OPN1SW NK3R N114/10 clone HCN4 cloneN70/28 HCN1 GFP dsRed Dab1 Receptor DopamineD1 CtBP2 ChAT clone 40A5 Calsenilin/DREAM CalbindinD Antibody antibodies1 Primary Table

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This article isprotected by copyright. All rights reserved. -

418 418 of - terminusof umanOPN1SW - mino acids mino acids mino 778 - - terminusof PC12 PC12 cells 672 rat 417 of P - mino acidsmino rotein 28 from from 28 Journal ofComparativeNeurology

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Journal ofComparativeNeurology MTKHPAKRLGCGPEGERDVREHAFFRRIDWEKLENREIQPPFKPKVCGKG Human FYLFKNISSVGPWDGPQYHIAPVWAFYLQAAFMGTVFLIGFPLNAMVLVA OPN1SW,Human TPHTAWYSEQASLEMREAAATEIRRAITGRIPESLRNCVNKEFEVTSAPWS CtBP, Human 369 Immunogen John Wiley& Sons

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receptors in a subtype-dependent manner. manner. subtype-dependent in a receptors Bot cells. bipolar in retinal localization receptor i mice, authors transgenic BAC Drd1a-tdTomato Using Text Abstract Graphical

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h ON and OFF bipolar cells express D1 D1 express cells bipolar and OFF ON h nvestigated dopamine D1 D1 dopamine nvestigated Page 50of