Diverse GABAergic neurons organize into subtype-specific … · 1 Diverse GABAergic neurons...

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DiverseGABAergicneuronsorganizeintosubtype-specificsublaminae

intheventrallateralgeniculatenucleus

UbadahSabbagh1,2,GubbiGovindaiah3,RachanaD.Somaiya1,2,RyanV.Ha4,JessicaC.Wei5,

WilliamGuido3,MichaelA.Fox1,4,6,7,#

1CenterforNeurobiologyResearch,FralinBiomedicalResearchInstituteatVirginiaTechCarilion,Roanoke,VA2GraduatePrograminTranslationalBiology,Medicine,andHealth,VirginiaTech,Blacksburg,VA3DepartmentofAnatomicalSciencesandNeurobiology,UniversityofLouisvilleSchoolofMedicine,Louisville,KY4SchoolofNeuroscience,VirginiaTech,Blacksburg,VA5NeuroSURF,FralinBiomedicalResearchInstituteatVirginiaTechCarilion,Roanoke,VA6DepartmentofBiologicalSciences,VirginiaTech,Blacksburg,VA7DepartmentofPediatrics,VirginiaTechCarilionSchoolofMedicine,Roanoke,VA

#Correspondence: MichaelA.Fox([email protected])

Runningtitle:Celltype-specificlaminaeintheventralLGN

Keywords:geniculate,GABAergic,thalamus,visual,retinal,circuit

InthisPDF: MainText,GraphicalAbstract,Figures1-7,SupplementalTable

Wordcount: 7181

Acknowledgments

ThisworkwassupportedinpartbythefollowinggrantsfromtheNationalInstitutesofHealth:EY021222(M.A.F),

EY030568 (M.A.F), NS105141 (M.A.F.), NS113459 (U.S.), and EY012716 (W.G.). The AAV1-Cre virus

(pENN.AAV.hSyn.Cre.WPRE.hGH) was a gift from J.M. Wilson. We thank Dr. A.S. LaMantia (Fralin Biomedical

ResearchInstitute,VirginiaTech)forthoughtfulcommentsonthemanuscript.

Conflictsofinterestdisclosure:Theauthorshavenoconflictsofinteresttodeclare.

Abbreviations:RGC,retinalganglioncells;LGN,lateralgeniculatenucleus;dLGN,dorsallateralgeniculate

nucleus;vLGN,ventrallateralgeniculatenucleus;IGL,intergeniculateleaflet;SC,superiorcolliculus;SCN,

suprachiasmaticnucleus;IHC,immunohistochemistry;ISH,insituhybridization;AAV,adeno-associatedvirus;

RRID,researchresourceidentifier.

.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (whichthis version posted May 4, 2020. . https://doi.org/10.1101/2020.05.03.073197doi: bioRxiv preprint

https://doi.org/10.1101/2020.05.03.073197

http://creativecommons.org/licenses/by-nc-nd/4.0/

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ABSTRACT

In the visual system, retinal axons convey visual information from the outsideworld to dozens of distinct

retinorecipientbrainregionsandorganizethatinformationatseverallevels,includingeitheratthelevelofretinal

afferents, cytoarchitecture of intrinsic retinorecipient neurons, or a combination of the two. Two major

retinorecipient nucleiwhich are densely innervated by retinal axons are the dorsal lateral geniculate nucleus

(dLGN),whichisimportantforclassicalimage-formingvision,andventralLGN(vLGN),whichisassociatedwith

non-image-formingvision.Theneurochemistry,cytoarchitecture,andretinothalamicconnectivityinvLGNremain

unresolved,raisingfundamentalquestionsofhowitreceivesandprocessesvisualinformation.Toshedlighton

theseimportantquestions,welabeledneuronsinvLGNwithcanonicalandnovelcelltype-specificmarkersand

studiedtheirspatialdistributionandmorphoelectricproperties.Notonlydidwefindahighpercentageofcellsin

vLGNtobeGABAergic,wediscovered transcriptomicallydistinctGABAergiccell typesreside in the twomajor

laminaeofvLGN,theretinorecipient,externalvLGN(vLGNe)andthenon-retinorecipient,internalvLGN(vLGNi).

WithinvLGNe,weidentifiedtranscriptionallydistinctsubtypesofGABAergiccellsthataredistributedintofour

adjacentsublaminae.Usingtrans-synapticviraltracingandinvitroelectrophysiology,wefoundcellsineachthese

vLGNesublaminaereceivemonosynapticinputsfromtheretina.Theseresultsnotonlyidentifynovelsubtypesof

GABAergiccellsinvLGN,theysuggestthesubtype-specificlaminardistributionofretinorecipientcellsinvLGNe

may be important for receiving, processing, and transmitting light-derived signals in parallel channels of the

subcorticalvisualsystem.


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INTRODUCTION

Informationaboutthevisualworldiscapturedbytheretinaandtransmittedbyretinalganglioncells(RGCs)

to a diverse array of retinorecipient nuclei, including those in thalamic, hypothalamic, and midbrain regions

(Flemingetal.2006;Gaillardetal.2013;Monavarfeshanietal.2017;Morin&Studholme2014;Marterstecketal.

2017).Thereisanorganizationallogictotheselong-rangeretinalprojectionswhereRGCs,ofwhichtherearemore

thanthreedozenmorphologicallyandfunctionallydistinctsubtypes,projecttodistinctandsometimesmutually

exclusiveretinorecipientregions(Hattaretal.2006;Berson2008;Dhandeetal.2011;Dhandeetal.2015;Kayet

al. 2011; Osterhout et al. 2011; Yonehara et al. 2009).Many of these retinorecipient nuclei are critical to the

executionofspecificvisualbehaviors.Forinstance,retinalinputstothedorsallateralgeniculatenucleus(dLGN)

areimportantforimage-formationanddirectionselectivity,thosetothesuperiorcolliculus(SC)areimportantfor

gazecontrol,thosetopretectalnucleiareimportantforpupillaryreflexesandimagestabilization,andthosetothe

suprachiasmaticnucleus(SCN)areimportantforcircadianphotoentrainment(Dhandeetal.2015;Piscopoetal.

2013;Seabrooketal.2017).NotonlydoRGCsprojecttodifferentretinorecipientnuclei,butprojectionsofdistinct

RGCsubtypesarealsosegregatedwithinasingleretinorecipientregion.Forexample,ithaslongbeenappreciated

thatprojectionsfromtranscriptomicallydistinctipsilateralandcontralateralRGCsterminateindistinctdomains

ofmostrodentretinorecipientnuclei(Godementetal.1984;Morin&Studholme2014;Muscatetal.2003;Jaubert-

Miazzaetal.2005;Wangetal.2016).

Along-standingobjectiveofvisualneuroscientistshasbeentocharacterizecell-typespecificcircuitsinthese

retinorecipientregions,intermsofbothinputsfromRGCsandoutputstodistinctdownstreambrainregions.For

example,thatdistinctsubtypesofRGCsterminateindifferentsublaminaeoftheSC(Dhande&Huberman2014;

Hubermanetal.2008;Hubermanetal.2009;Kimetal.2010;Marterstecketal.2017;Oliveira&Yonehara2018).

Post-synaptic to these retinal inputs are at least four morphologically and functionally distinct classes of

retinorecipientneuronswhicharestellate,horizontal,wide-field,andnarrow-fieldcells(Gale&Murphy2014;Gale

& Murphy 2016). Identifying subtype-specific retinocollicular circuitry facilitated the discovery that specific

collicularcell typesparticipateindifferentaspectsofvisuallyguidedbehavior(Hoyetal.2019;Reinhardetal.

2019;Shangetal.2018;Shangetal.2015).

ThedLGN,whichprocessesandrelaysclassical image-formingvisual information toprimaryvisualcortex,

shares an organizational feature with SC in the kinds of retinal afferents it receives, where subtype-specific

arborizationofRGCaxonsbeenclearlycharacterizedandformsso-called“hiddenlaminae”(Reese1988;Martin

1986; Hong & Chen 2011). These hidden layers have been revealed by methods which individually label

functionallyandmorphologicallydistinctclassesofRGCsusingtransgenicreportermouselines(Hubermanetal.

2008;Hubermanetal.2009;Kayetal.2011;Kimetal.2010;Kimetal.2008).ThedLGNispopulatedbyjustafew

typesofretinorecipientneurons,whichincludethreeclassesofthalamocorticalrelaycells(X-like,Y-like,andW-

like)and1-2classesofGABAergicinterneurons(Arcellietal.1997;Jaubert-Miazzaetal.2005;Kraheetal.2011;


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Leistetal.2016;Lingetal.2012).Whiletheirorganizationisnotasorderedastheirretinalafferents,classesof

dLGNrelaycellsexhibitsomeregionalpreferencesintheirdistribution,whereasinterneuronsareevenlydispersed

throughoutthenucleus(Kraheetal.2011).Celltype-specificcircuitryandfunctionhasalsobeendemonstratedin

dLGN,whereW-likerelayneuronsreceiveinputfromdirection-selectiveRGCsandinturnprojecttothesuperficial

layersofmouseprimaryvisualcortex(Cruz-Martínetal.2014).

Whileourunderstandingofsubtype-specificcircuitshasfacilitatedfunctionalstudiesofSCanddLGN,there

remainmanyretinorecipientregionsaboutwhichsuchfoundationalinformationisunknown.Onesuchregionis

theventralLGN(vLGN),aportionofventralthalamusthatneighborsdLGNandissimilarlyinnervatedbyretinal

axons.Althoughlessstudied,ithasbeenshownthatvLGNisremarkablydistinctfromitsdorsalcounterpartinits

transcriptome,proteome,cytoarchitecture,andcircuitry(Harrington1997;Suetal.2011;Monavarfeshanietal.

2018;Sabbaghetal.2018).Infact,distinctsubtypesofRGCsprojecttovLGNanddLGN,andthemajorityofdLGN-

projectingRGCclassesfailtosendcollateralaxonsintovLGN,despitehavingtopassbyit(orthroughit)onthe

way to dLGN (Huberman et al. 2008;Huberman et al. 2009;Kim et al. 2008). Retinal axons that target vLGN

terminateinalateralsubdivisionknownastheexternalvLGN(vLGNe),whichiscytoarchitectonicallydistinctfrom

theinternalvLGN(vLGNi)whichreceiveslittle,ifany,retinalinput(Gabbott&Bacon1994;Harrington1997;Niimi

etal.1963;Sabbaghetal.2018).TheidentityofretinorecipientcellsinvLGNeremainslargelyunknown,although

itislikelytoincludeGABAergiccells(Huangetal.2019),whichrepresenttheprevalenttypeofneuroninvLGN

(Gabbott&Bacon1994;Harrington1997;Inamuraetal.2011).

Here,toaddressthesegaps,wesoughttodeterminethecell-typespopulatingthevLGN,andtheirconnectivity

toretinalafferents.WeassessedvLGNneurochemistryandcytoarchitecturebylabelingcellswithcanonicaland

novel cell type markers. We found a richly diverse and tightly organized cellular landscape in vLGN, where

transcriptomicallydistinctcelltypesaredistributedinlaminarsubdomains,whichappeartoreceivemonosynaptic

inputsfromtheretina.OurfindingsnotonlyidentifyanovelorganizationofretinorecipientcellsinvLGN,they

suggestthisordermaybeimportantforreceiving,processing,andtransmittingdistinct light-derivedsignalsin

parallelchannelsofthesubcorticalvisualsystem.


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MATERIALSANDMETHODS

Animals

WildtypeC57BL/6micewereobtainedfromJacksonLaboratory.Weobtainedthefollowingmicefrom

Jackson Laboratory: Pvalb-Cre (JAX #: 008069, RRID:IMSR_JAX:008069), Gad2-Cre (JAX #: 010802,

RRID:IMSR_JAX:010802), Sst-Cre (JAX #: 028864, RRID:IMSR_JAX:028864), Sun1-stop-GFP (JAX #: 021039,

RRID:IMSR_JAX:021039), Thy1-STOP-YFP (JAX #: 005630, RRID: IMSR_JAX:005630), ROSA-stop-tdT (JAX #:

007909,RRID:IMSR_JAX:007909)andGAD67-GFP(JAX#:007673,RRID:IMSR_JAX:007673).Animalswerehoused

inatemperature–controlledenvironment,ina12hrdark/lightcycle,andwithaccesstofoodandwateradlibitum.

Bothmales and femaleswere used in these experiments. GenomicDNAwas isolated from tails genotyping as

previously described (Su et al. 2010) using the following primers: yfp: fwd-AAGTTCATCTGCACCACCG, rev-

TCCTTGAAGAAGATGGTGCG; cre: fwd-TGCATGATCTCCGGTATTGA, rev-CGTACTGACGGTGGGAGAAT; sun1: fwd-

CTTCCCTCGTGATCTGCAAC,mut_rev-GTTATGTAACGCGGAACTCCA,wt-rev: CAGGACAACGCCCACACA; tdt: fwd-

ACCTGGTGGAGTTCAAGACCATCT, rev-TTGATGACGGCCATGTTGTTGTCC. Animals were maintained and

experimentsconductedincompliancewithNationalInstitutesofHealth(NIH)guidelinesandapprovedprotocols

oftheVirginiaPolytechnicInstituteandStateUniversityInstitutionalAnimalCareandUseCommittee(IACUC).

Unless otherwise stated, n= number of animals and a minimum of three age-matched wildtype (and, where

transgenicreporterswereused,ofsamegenotype)animalswerecomparedinallexperimentsdescribed.

Immunohistochemistry(IHC)

Micewereanesthetizedusing12.5μg/mLtribromoethanolandtranscardiallyperfusedwithPBSand4%

paraformaldehyde(PFA;pH7.4).Extractedbrainswerekeptin4%PFAovernightat4°C,andthenincubatedfor

atleast48hin30%sucroseinPBS.FixedtissueswereembeddedinTissueFreezingMedium(ElectronMicroscopy

Sciences,Hatfield,PA,USA)andcryosectionedat30μmsectionsonaLeicaCM1850cryostat.Sectionswereair-

driedontoSuperfrostPlusslides(FisherScientific,Pittsburgh,PA,USA)andweredriedfor15minbeforebeing

incubatedinblockingbuffer(2.5%bovineserumalbumin,5%NormalGoatSerum,0.1%Triton-XinPBS)for1h

atroomtemperature(orat22°C).Primaryantibodiesweredilutedinblockingbufferatthefollowingdilutionsand

incubated on tissue sections at 4°C overnight: Calb1(Swant, CB-38a, 1:1000) and Pvalb (Millipore-Sigma,

MAB1572, 1:1000). Sections were then washed three times PBS and incubated in anti-mouse or anti-rabbit

fluorescently conjugated secondary antibodies (Invitrogen Life Technologies, RRID:SCR_008410) diluted in

blockingbuffer(1:1000)for1hat22°C.Tissuesectionswerethenwashedatleast3timeswithPBS,stainedwith

DAPI(1:5000inwater),andmountedusingVectashield(VectorLaboratories,Burlingame,CA,USA).

Riboprobeproduction

Riboprobesweregeneratedaspreviouslydescribed (Su etal. 2010;Monavarfeshani etal. 2018).Gad1

(Gad1-F:TGTGCCCAAACTGGTCCT;Gad1-R:TGGCCGATGATTCTGGTT;NM_001312900.1;nucleotides1099-2081),


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Lypd1 (Lypd1-F: AAGGGAGTCTTTTTGTTCCCTC; Lypd1-R: TACAACGTGTCCTCTCAGCAGT; NM_145100.4;

nucleotides 849-1522), Arx (Arx-F: CTGAGGCTCAAGGCTAAGGAG; Arx-R: GGTTTCCGAAGCCTCTACAGTT;

NM_007492.4; nucleotides 1830-2729), Ecel1 (Ecel1-F:CGCGCTCTTCTCGCTTAC; Ecel1-

R:GGAGGAGCCACGAGGATT; NM_001277925.1; nucleotides 942-1895), Nxph1 (Nxph1-

F:ATAGGACAGGGCTGTCACCTTA; Nxph1-R:TTACTGAGAACAAGCTCCTCCC; NM_008751.5; nucleotides 1095-

1695),Spp1(Spp1-F:AATCTCCTTGCGCCACAG;Spp1-R:TGGCCGTTTGCATTTCTT;NM_001204201.1;nucleotides

309-1263), Sst (NM_009215.1; nucleotides 7-550), and Penk (Penk-F: TTCCTGAGGCTTTGCACC; Penk-R:

TCACTGCTGGAAAAGGGC;NM_001002927.3;nucleotides312-1111)cDNAsweregeneratedusingSuperscriptII

ReverseTranscriptaseFirstStrandcDNASynthesiskit(#18064014, Invitrogen)accordingtothemanufacturer

manual,amplifiedbyPCRusingprimersdesignedfortheabovegenefragments,gelpurified,andthenclonedinto

apGEM-TEasyVectorusingpGEM-TEasyVectorkit,(#A1360,Promega)accordingtothekitmanual.Anti-sense

riboprobes against target genes were synthesized from 5 µg linearized plasmids using digoxigenin-(DIG) or

fluorescein-labeleduridylyltransferase(UTP)(#11685619910,#11277073910,Roche,Mannheim,Germany)and

theMAXIscriptinvitroTranscriptionKit(#AM1312,Ambion)accordingtothekitmanual.5µgofriboprobe(20

µl)werehydrolyzedinto~0.5kbfragmentsbyadding80µlofwater,4µlofNaHCO3(1M),6µlNa2CO3(1M)and

incubatingthemixturein60°C.RNAfragmentswerefinallyprecipitatedinethanolandresuspendedinRNAase-

freewater.

Insituhybridization(ISH)

ISHwasperformedon30μmthincryosectionsasdescribedpreviously(Sabbaghetal.2018).Sectionswere

firstallowedtoairdryfor1hratroomtemperatureandwashedwithPBSfor5mintoremovefreezingmedia.They

werethenfixedin4%PFAfor10min,washedwithPBSfor15min,incubatedinproteinaseKsolutionfor10min,

washedwithPBSfor5min,incubatedin4%PFAfor5min,washedwithPBSfor15min,incubatedinacetylation

solutionfor10min,washedwithPBSfor10min,incubatedinPBS-diluted0.1%tritonfor30min,washedwith

PBSfor40min,incubatedin0.3%H2O2for30min,washedwithPBSfor10min,pre-hybridizedwithhybridization

solutionfor1hr,beforebeinghybridizedwithheat-denaturedriboprobesat62.5°Covernight.Sectionswerethen

washedfor5timesin0.2XSSCbufferat65°C.SlideswerethenwashedwithTBS,blocked,andincubatedwith

horseradish peroxidase-conjugated anti-DIG (#11426346910, Roche) or anti-fluorescent antibodies

(#11207733910, Roche) overnight at 4°C. Lastly, bound riboprobes were detected by a tyramide signal

amplificationsystem(#NEL753001KT,PerkinElmer).

Anterogradeaxonandmono-synaptictracing

IntravitrealinjectionofcholeratoxinsubunitB(totraceretinalterminals)wasperformedaspreviously

described(Monavarfeshanietal.2018;Suetal.2011).Briefly,micewereanesthetizedwithisoflurane,and1μlof

1mg/mlfluorescentlyconjugatedAlexa-647-CTB(Invitrogen,C34778)wasinjectedwithafineglasspipetteusing

apicospritzer.After3days,animalsweresacrificedandtranscardiallyperfusedwithPBSfollowedbyPFA.


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AsimilarintravitrealinjectionofAAV2/1-hSyn-Cre-WPRE-hGH(2.5×1013GC/mL,herereferredtoasAAV-

Cre)wasusedtomonosynapticallylabelretinorecipientneuronsinthevLGN.1.2μlofAAV-Creviruswasinjected

either monocularly or binocularly at an approximate 45° angle relative to the optic axis.

pENN.AAV.hSyn.Cre.WPRE.hGH was a gift from James M. Wilson (Addgene viral prep #105553-AAV1;

RRID:Addgene_105553). Animalswere sacrificed and perfusedwith PFA as described above 6-10weeks after

injection.

Transcriptomicanalyses

RNAsequencingexperimentsondevelopingWTvLGNanddLGNwasdescribedandpublishedpreviously

(Monavarfeshanietal.2018).

Invitroslicepreparationandwholecellrecording

In vitro recordings were conducted on genetically labeled vLGN neurons using methods described

previously(Hammer etal.2014).Micewereanesthetizedwith isoflurane,decapitatedandbrainswererapidly

immersedinanice-cold,oxygenated(95%O2/5%CO2)solutioncontainingthefollowing(inmM):26NaHCO3,

234sucrose,10MgCl2,11glucose,2.5KCl,1.25NaH2PO4,2CaCl2.Coronalsections(270um)containingdLGNand

vLGNwerecutonavibratomeandplacedinachambercontainingartificialcerebralspinalfluid(ACSF;inmM:126

NaCl,2.5KCl,1.25NaH2PO4,2.0MgCl2,26NaHCO3,2CaCl2,2MgCl2and10glucose,saturatedwith95%O2/5%

CO2,pH7.3)at32°Cfor30minandthenatroomtemperature.Individualslicesweretransferredtoarecording

chambermaintainedat32°Candperfusedcontinuouslyatarateof2.5ml/minwithoxygenatedACSF.Borosilicate

pipetteswerepulledusingatwo-steppuller(Narishige)andfilledwithasolutioncontainingthefollowing(inmM):

117 K-gluconate, 13 KCl,1MgCl2, 0.07 CaCl2, 0.01 EGTA, 10 HEPES, 2 Na-ATP, and 0.4 Na-GTP (pH 7.3, 290

osmol/L).Forallrecordings,biocytin(0.5%,Sigma)wasincludedintheinternalsolutionforintracellularfilling

and3-Dneuronreconstructionusingconfocalmicroscopy(Charalambakisetal.2019;El-Danafetal.2015;Krahe

etal.2011).Thefinaltipresistanceoffilledelectrodeswas6to8MΩ.

Wholecellpatchrecordingsweremadeincurrentandvoltageclampusinganamplifier(Multiclamp700B,

MolecularDevices), filtered at 3-10kHz, digitized (Digidata 1440A) at 20kHz and stored on computer. Pipette

capacitance, series resistance, input resistance, and whole-cell capacitance were monitored throughout the

recordingsessiondevices.

To examine the intrinsic membrane properties of vLGN neurons, the voltages responses triggered by

currentstepinjection(-120to+200pA,20pApulses,600ms)wererecordedatrestingmembranelevels.Synaptic

responseswererecordedinvoltageclamp(holdingpotentialof-70mV)andevokedbyelectricalstimulationofthe

optictract(OT)usingbipolartungstenelectrodes(0.5MΩ;A-MSystems)positionedjustbelowtheventralborder

ofvLGN.OTstimulationconsistedofa10Hz(10pulses)traindeliveredatanintensity(25to200µA)thatevoked

amaximalresponse(Hammeretal.2014;Jaubert-Miazzaetal.2005).


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Quantificationandimaging

ToquantifytheGABAergicneuronsasapercentageoftotalcellsinvLGNanddLGN,welabeledandcounted

Gad1+ (by ISH), Gad2+ (by Gad2-Cre::Sun1-Stop-GFP transgenic reporter), and GAD67+-GFP (by GAD67-GFP

transgenicreporter)neuronsanddividedbyallcellscountedinthatsectionbyDAPIcounterstaining.Toquantify

thedensityofagivenGABAergicsubtypeinthetwomainsubdomainsofvLGN,welabeledwiththerespective

subtypemarker(afterintravitrealCTBinjection)andcountedcellsintheretinorecipient(CTB+)vLGNeandnon-

retinorecipient(CTB-)vLGNiandnormalizedtotheareaoftherespectivevLGNsubdomain.Areasweremeasured

bymanualoutliningoftheborderofvLGNeorvLGNiusingImageJsoftware(version1.52n,NIH).Boundariesof

vLGNordLGNweredeterminedbyDAPIcounterstaining.Allquantificationwasperformedonthreebiological

replicates,countingatleastthreevLGNsectionspermouse.

Toquantifythespatialdistributionofcell-typemarkerexpressionacrosstheentirevLGN,wedevelopeda

customlinescanscript(khatScan)thatrunsinImageJwhichoverlaysthevLGNwithequallyspacedlines.Weopted

forthisapproachovermanuallydrawinglinestoavoiduserbias.Abriefsummaryofhowthisscriptworks:to

determinethecurvatureofthevLGNinaparticularimage,khatScanpromptstheusertodrawalinealongtheoptic

tractadjacenttothevLGN,thenautomaticallydrawslinesofasetlengthandnumberguidedbythatcurveand

plotsthesignalintensityacrossthexcoordinatesofeachline.Theseintensitiescanthenbeaveragedtodetermine

wherethereisaspecificenrichmentforthatmarkerinthevLGN.Allimagingforquantificationwasperformedon

aconfocalZeissLSM700microscopeat20xmagnificationand0.5digitalzoom.


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RESULTS

IdentificationofdistinctsubtypesofGABAergiccellsinvLGN

We first determinedpreciselywhatproportionof vLGN cellswereGABAergic. In thebrain,GABAergic

neurons express Gad1 and/or Gad2, genes which encode glutamate decarboxylases (GAD67 and GAD65

respectively),thebiosyntheticenzymesnecessaryfortheproductionoftheneurotransmitterGABA.Wetherefore

tooktwocomplementaryapproachestolabelGABAergicinterneurons:weperformedinsituhybridization(ISH)

withriboprobesgeneratedagainstGad1mRNAandwecrossedGad2-Cremice(inwhichtheCrerecombinaseis

expressed under the control of Gad2 promoter) to Rosa-Stop-Sun1/sfGFP mice to transgenically label Gad2-

expressing cells with a GFP-tagged nuclear protein (Mo et al. 2015). Both approaches revealed a dramatic

enrichmentofGABAergiccellsinvLGNcomparedtodLGN,asexpectedfrompreviousstudies(Figure1a-i)(Langel

etal. 2018;Yuge etal. 2011).We found that>25%of cells invLGNwereGad1+ (Figure1c).A slightlyhigher

percentage of vLGN cells (~40%) were GFP+ in Gad2-Cre::Sun1-Stop-GFP mice (Figure 1f). This increased

percentagecouldbeattributable to the limitationsofmRNAdetectionby ISHandthereforerepresentsamore

accuratepictureoftheoverallpopulationofGABAergiccellsinvLGN.Thissameapproachlabeledlessthan10%

ofcellsindLGN,anumberinlinewithpreviousreports(Arcellietal.1997;Suetal.2020;Evangelioetal.2018).

Severalgroups,includingourown,havepreviouslyusedaGAD67-GFPtransgeniclinetolabelmost(ifnot

all) GABAergic cells in dLGN (Charalambakis et al. 2019; Su et al. 2020; Seabrook et al. 2013). However few

GABAergicneuronsarelabeledinvLGNofthesemice(Figure1g).Infact,wefoundlessthan5%ofcellsinvLGN

wereGFP+inGAD67-GFP(Figure1i).ThedramaticallyfewerGFP+cells,comparedtothenumberofGABAergic

cellsobservedbylabelingwitheitherGad1ISHorGad2-Cre::Sun1-Stop-GFP,isduetothefactthattheGAD67-GFP

labelsonlyasubsetofthalamicGABAergiccells–likelylocalinhibitoryinterneurons–invisualthalamus(Suetal.

2020).

Together,theseresultssuggestedthatmultiplesubtypesofGABAergiccellsexistinvLGN,unlikeindLGN

whereGABAergiccellsarerelativelyhomogenous(Leistetal.2016;Jageretal.2016;Kalishetal.2018).Forthis

reason,wenextaskedwhetherwecouldidentifynovelmolecularmarkerstocharacterizetheheterogeneityof

GABAergicneuronsinvLGN.WeassessedgeneexpressionprofilesinthedevelopingmousevLGNanddLGNin

previouslygeneratedRNAseqdatasets(Monavarfeshanietal.2018;Heetal.2019).Ourrationalewastoidentify

candidatecelltypemakersbyfocusingourattentionongeneswhichwere:a)enrichedinvLGNbutnotdLGN,b)

expressedbyGABAergiccellsinotherbrainregions,and/orc)expressedwithdifferentdevelopmentalpatterns

which could indicate expressionbydifferent subsets of neurons.To characterize if anyof these genes labeled

distinctpopulationsofneuronsinvLGN,wegenerated>40riboprobestoperformISH(TableS1).Wealsotook

advantageofavailablecell-specificreportermiceandantibodiesforthisscreen.


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Fromthisunbiasedriboprobescreen,weidentifiedtwogenes,Nxph1andArx,whoseexpressioninvLGN

wasrestrictedtocellsinlargelynonoverlappingdomains.Nxph1,whichencodesthea-NrxnligandNeurexophilin-

1,wasexpressedinthemostlateralportionofvLGN(Figure1j-k).Arx,whichencodesthehomeoboxtranscription

factorAristalessRelatedHomeoboxprotein,wasexpressed inthemostmedialportionofvLGN(Figure1l-m).

Double-ISH,withGad1riboprobes,revealedthatbothNxph1andArxmRNAsweregeneratedbyGABAergiccells

invLGN(Figure1k,m,n-o).To testwhetherNxph1 andArxmarkedGABAergiccell types invLGNeandvLGNi

respectively,welabeledretinalganglioncellarborsinvLGNbyintraocularinjectionoffluorescentlyconjugated

Cholera Toxin Subunit B (CTB) (Figure1p). Indeed,we found thatNxph1+ neurons reside in vLGNe andArx+

neurons reside invLGNi (Figure1q).These results further suggested that transcriptionallydistinct subsetsof

GABAergicneuronsexistinvLGNanddemonstratedcellulardiversityinbothlaminaeofvLGN–vLGNeandvLGNi

(Figure1r).

WenextsoughttodeterminewhetherGABAergicneuronsresidingwithinvLGNeorvLGNicouldbefurther

subdividedintodistinctneuronalsubtypes.SeveralofthegenesidentifiedasdifferentiallyenrichedinthevLGN

comparedtodLGNwerecanonicalmarkersofinhibitoryinterneuronsinotherpartsofthebrain(i.e.Somatostatin,

Calbindin,Parvalbumin)(Figure2a-c)(Tremblayetal.2016;Limetal.2018).TolabelSomatostatin-expressing

cells,weusedtwoapproaches:aSst-Cre::Rosa-Stop-tdTreportermouseinwhichallSst+cellsweretransgenically

labeledwithtdTandriboprobesagainstSstmRNA.BothapproachesrevealedsparseSst+cellsdistributedbroadly

acrossboththevLGNandtheintergeniculateleaflet(IGL),asliceofretinorecipienttissueseparatingvLGNfrom

dLGNinmice(Figure2danddatanotshown).BycouplingGad1ISHinSst-Cre::Rosa-Stop-tdTtissue,wefoundthat

>85%ofSst+cellsgeneratedGad1mRNAandwerethereforeGABAergicneurons.Bylabelingretinalaxonswith

CTBandquantifyingthedensityoftdT+cellsinvLGNeandvLGNi,wedeterminedthatSst+neuronswereevenly

distributedinvLGNeorvLGNi(Figure2e-h).Next,weimmunolabeledCalbindin-expressingneuronsandfound

thatCalb+cellswereinbothIGLandvLGN,andmostwerealsoGABAergic(Figure2i-k).WhenwelabeledvLGNe

andvLGNiwith intravitreally injectedCTB,we found thatCalb+ cellswerepreferentiallydistributed in vLGNi,

althoughtheyrepresentonlyafractionofthecellsinthisregion(Figure2l-m).Finally,welabeledParvalbumin-

expressingneuronsbygeneratingaPvalb-Cre::Thy1-Stop-YFPmouseinwhichallPvalb+cellsweretransgenically

labeled with YFP (Figure 2n). We observed a modest number of Pvalb+ cells in vLGN, but unlike the broad

distributionofSst+andCalb+neurons,Pvalb+cellswereconcentratedinwhatappearedtobealayerofvLGNand

wereabsentfromIGL(Figure2n).BycouplingGad1ISHinPvalb-Cre::Thy1-Stop-YFP,wefoundthat>90%ofPvalb+

cellsgeneratedGad1mRNAandwerethereforeGABAergic(Figure2o-p).Whenwelabeledretinalprojectionsin

vLGNe with CTB and Pvalb+ GABAergic neurons by immunolabeling, we determined that Pvalb+ cells were

exclusivelypresentinvLGNe(Figure2q-r).ItwasnoteworthythatSst+,Calb1+,andPvalb+cellswerelargelyabsent

fromtheneighboringdLGN,suggestingthattheseGABAergiccelltypeswereuniquefrompreviouslystudieddLGN

interneurons.


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Sinceneither thePvalb+ neurons (whichpreferred thevLGNe)norCalb+ neurons (whichpreferred the

vLGNi)labeledallofthecellsintheirrespectivesubdomains,wehypothesizedthatanevenricherheterogeneity

ofGABAergiccellsexisted.ThisledustoaskwhethertherewereothertypesofGABAergicneuronswhichexhibited

similarvLGNsubdomainpreferences.Usingourriboprobescreen,weidentifiedfouradditionalgenesthatwere

generatedbyregionallyrestrictedsubsetsofcellsinvLGN:Spp1,Penk,Lypd1,andEcel1.Thetranscriptsforallof

thesegeneswereenrichedinvLGNcomparedtodLGN(Figure3a-d)andweregeneratedbyGad1+GABAergic

neurons(Figure3e-x).

RiboprobesgeneratedagainstSpp1,whichencodestheextracellularglycoproteinOsteopontin,revealed

Spp1+cellsweresparselypresentinthevLGN(andabsentfrombothIGLanddLGN).Interestingly,Spp1+cellswere

distributedinastratifiedfashionwithinvLGNe,justasweobservedforPvalb+cells(Figure3e,h-i).ISHforPenk,

whichencodesProenkephalin,revealedthatPenkmRNAwaspresentinasubsetofvLGNcellsandinmanyIGL

neurons (Figure 3j-k). Like what we observed for Spp1+ and Pvalb+ neurons, Penk+ neurons also appeared

distributedinastratifiedfashion.LabelingretinalafferentswithCTBrevealedthatbothSpp1+andPenk+neurons

werelocatedintheretinorecipientvLGNe,althoughitwasuncleariftheywerepresentinthesameregion(Figure

3m-n). Finally, ISH for Lypd1, which encodes LY6/PLAUR Domain Containing 1, and Ecel1, which encodes

EndothelinConvertingEnzymeLike1,revealedthatthesegenesexhibitedsimilarcellularexpressionpatternsin

vLGNandIGL(Figure3o,t).Lypd1+andEcel1+cellsweresparselydistributedintheIGLanddenselypopulated

twodistinctandseparateregionsof thevLGN,occupyingboththe lateral-mostregionofvLGNeandtheentire

vLGNi(Figure3r-s,w-x).BasedonsimilaritiesofexpressionpatternsinbothvLGNandIGL,itseemedlikelythat

Ecel1andLypd1mRNAsweregeneratedinthesamesubsetsofGABAergicneurons.

Taken together, these experiments reveal novel markers of transcriptomically and regionally distinct

subsetsofGABAergicneuronsinvLGN.Importantly,notonlydidthesecellshaveregionalpreferences,butPvalb+,

Penk+,Spp1+,Ecel1+,andLypd1+neuronseachappearedtobeorganizedintosegregatedstratathatspanthedorso-

ventralaxisofthevLGN.

TranscriptomicallydistinctGABAergicneuronsorganizeintoadjacentsublaminaeofvLGNe

We next asked whether these were in fact mutually exclusive groups of cells and whether they

corresponded to distinct vLGNe sublaminae. We started by assessing whether Spp1 and Pvalb mRNAs were

generatedbythesameneuronsoroccupiedthesamedorso-ventralzone.PerformingISHonPvalb-Cre::Thy1-Stop-

YFPtissuerevealedthatSpp1mRNAwasgeneratedbysome,butnotall,Pvalb+cells(~50%Spp1+neuronswere

Pvalb-)andviceversa(~25%Pvalb+neuronswereSpp1)(Figure4a-d).Spp1+Pvalb+,Spp+Pvalb-,andPvalb-Spp1+

cellsallappearedtoresidewithinthesamesublaminaofvLGNe.Toquantitativelyassesscellulardistributionin

vLGN,wedevelopedanautomatedscriptinImageJtomeasurefluorescentintensityalongthemedial-lateralaxis

of vLGN (Figure 4c’’). Fluorescent signals at each medial-lateral coordinate were averaged along the entire


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dorsoventralextentofvLGN(andbetweenbiologicalreplicates)andthequantifieddataidentifiedthesamespatial

regionofvLGNaspopulatedbothSpp1+andPvalb+cells(Figure4e).

Next,weaskedwhetherPenkwasgeneratedbyeitherSpp1+orPvalb+cells.Forthis,weuseddoubleISHor

geneticreporterlines.Inbothcases,wewereunabletoidentifyasingleoccurrenceinwhichPenk+neuronsco-

expressed either Pvalb or Spp1 (Figure 4f-h and data not shown). Moreover, these experiments clearly

demonstratedthatPenk+cellswerenotonlytranscriptomicallydistinctfromSpp1+andPvalb+cells,butalsowere

presentinanadjacentsublamina.LinescananalysisoffluorescencefromPenkandSpp1doubleISHexperiments

confirmedthatthesepopulationsofGABAergicneuronswerepresentindistinctsublaminae(Figure4i).Next,we

tookatriplelabelingapproach(doubleISHforEcel1+andPenk+neuronsinthetransgenicPvalb-Cre::Thy1-Stop-

YFP)totestwhetherthesublaminaepopulatedbyPvalb+andPenk+cellsweredistinctfromthosepopulatedby

Ecel1+ cells (Figure4j-m).Again,weobservednoEcel1+Pvalb+ orEcel1+Penk+ neuronsusing thismethod, and

quantitativeanalysisofEcel1/Penk/Pvalbexpressionpatternsalongthemedio-lateralextentofvLGNrevealedat

leastthreedistinctsublaminaeinvLGNe(Figure4i).

LabelingofEcel1+,Pvalb+andPenk+cellsatoncedidnotlabelallGABAergiccellsinvLGN.Infact,itappeared

asifthespacebetweenthelateral-mostlayerofEcel1+cellsandtheSpp1+layermayrepresentanotherlayerof

GABAergiccellswhichwefailedtoidentifywithourriboprobescreen(arrowinFigure4m).Totestthisidea,we

performedtwotriplelabelingexperiments:oneinwhichwelabeledEcel1andSpp1mRNAinGad2-Cre::Sun1-Stop-

GFPtissue,andoneinwhichwelabeledEcel1andGad1mRNAsinPvalb-Cre::Thy1-Stop-YFPtissue(Figure4n-w).

Inbothcases,weidentifiedGABAergiccellsbetweentheEcel1+layerandtheSpp1+Pvalb+layer.Linescananalysis

confirmeda small populationofGad2+Ecel1-Spp1- neuronsbetween the sublaminae containingEcel1+ orSpp1+

neurons(Figure4q-r,v-w),suggestingtheexistenceofatleastafourthsublaminainvLGNewithyet-to-be-defined

GABAergicneurons.

WerecognizedthatourspatialregistrationofGABAergicsubtypesintodistinctsublaminaeinvLGNemight

havebeenanartifactofthecoronalplaneofsection.Todeterminewhetherthesesublaminaewereinfacttrue

structuralcomponentsofvLGN,weperformedaxial(horizontal)sectionsofPvalb-Cre::Thy1-Stop-YFP(Figure5a-

b).ByperformingdoubleISHonthistissue,wefoundthat1)Spp1+andPvalb+cellsresideinthesamelayer(anda

populationofcellsexpressbothtranscripts),and2)Ecel1+,Pvalb+,andPenk+cellspopulatedistinctsublaminaeof

vLGNe (Figure 5c-k). Taken together, these data demonstrate, for the first time, that the vLGNe contains

heterogeneouspopulationsoftranscriptomicallydistinctGABAergiccelltypesthatmapontoatleastfouradjacent

sublaminae(thatarenotappreciablewithconventionalhistochemicalstainingapproaches).

GABAergicneuronsinthefoursublaminaeofvLGNeareretinorecipient

The laminarsegregationof transcriptomicallydistinctcell types invLGNraises thepossibility that this

organizationfunctionstoparsedifferentstreamsofvisualinformation.ThisledustohypothesizethatGABAergic


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cellsindistinctsublaminaeinvLGNeweredirectlyinnervatedbyretinalaxons.Infact,whileretinorecipientcells

in dLGN are well defined, the retinorecipient neurons in vLGN are largely unknown. To anterogradely label

retinorecipient cells invLGN,we intravitreally injecteda trans-synaptic adeno-associatedvirusexpressingCre

recombinase(AAV2/1-hSyn-Cre-WPRE-hGH,referredtohereasAAV1-Cre)intoRosa-Stop-tdTmice(Zinggetal.

2017)(Figure6a-b).Thistrans-synapticviraltransfectionstrategyhaspreviouslybeenshowntoaccuratelymap

monosynaptic connections and, in our hands, resulted in sparse tdT+ labeling of cells in retinorecipient brain

regions (Figure 6B)(Zingg et al. 2017). Using this approach, we trans-synaptically labeled 53 retinorecipient

neurons in vLGN (n=12 animals).Oncewe identified tdT+ cells in vLGN,we assessed their spatial localization

relative to the sublaminaedescribed above andperformed ISH to determinewhether the distinct subtypes of

GABAergic neurons identifiedherewere retinorecipient.We identifiedPvalb+,Spp1+,Ecel1+, andPenk+ cells in

vLGNe that contained tdT, indicating that these populations of GABAergic neurons are capable of receiving

monosynapticinputfromtheretina(Figure6c-f).

Wenextsought to functionallyconfirmthatsomeof these transcriptomicallydistinctcell typesreceive

direct retinal input and to characterize their electrophysiological response properties. We utilized available

transgenicreporterlinestorecordfromandcharacterizeseveralGABAergicsubtypesinvLGN.Figure7a-cprovide

examplesofbiocytinfilledvLGNneuronsrecordedfromPvalb-Cre::Thy1-Stop-YFP(n=12),Sst-Cre::Rosa-Stop-tdT

(n=15)andGAD67-GFP(n=5)micealongwithrepresentativevoltageresponsestocurrentinjectionandsynaptic

responsesevokedbyoptic tract (OT)stimulation.Overall, theirdendriticarchitecturevariedwidely fromeach

other. Pvalb+ neurons had a hemispheric architecture with several elongated but sparsely branched primary

dendrites,whereasSst+neuronshadmuchsmallerbipolardendritic fieldsthatcontainedshortmulti-branched

processes.BiocytinfilledvLGNneuronsfromGAD67-GFPmicehadsimilarmorphologiestointrinsicinterneurons

indLGN,displayingexpansiveandcomplexarborsthatarisefromoppositepolesoffusiform-shapedsoma(Guillery

1966;Charalambakisetal.2019;Seabrooketal.2013).Thevoltageresponsestocurrentinjectionalsorevealed

differencesintheirintrinsicmembraneandfiringproperties.Forexample,therestingmembranepotential(Figure

7j)ofSst+(-57.5±1.6mV;n=22)andPvalb+(-59±1.2mV;n=18)neuronswassignificantly(P<0.05,One-Way

ANOVA)hyperpolarizedcomparedtoGAD67-GFP+neurons(-50±2.2mV;n=6).Theinputresistance(Figure7k)

ofPvalb+(140±18MΩ;n=18)wasalsosignificantly(P<0.05,One-WayANOVA)lowercomparedtoSst+(575±46

MΩ;n=22)andGAD67-GFP+neurons(278±56MΩ;n=6).Responsestohyperpolarizingcurrentpulsesfailedto

evokealargetriangularshaped,reboundlowthresholdCa2+spike(Figure7b,e,h).However,bothSst+andGAD67-

GFP+neuronsshowedastrongdepolarizingsagwhich is likelymediatedbyhyperpolarizationactivatedcation

conductance(Ih),whilePvalb+neuronsshowedlittleornosuchinwardrectification.Forallthreecelltypes,small

depolarizingcurrentsteps(~80pA)evokedatrainofspikesthatdisplayedspikefrequencyadaptation.However,

when larger steps were employed (>100 pA), Pvalb+ neurons displayed high frequency firing (84 ± 18 Hz).

However,Sst+andGAD67-GFP+neuronshaddifficultymaintainingfiringthroughoutthedurationofthecurrent

step,showingaprogressiveinactivationofNa2+spikes.


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Finally, we examined the synaptic responses that were evoked by OT stimulation (Figure 7c,f,i). As

expected, OT stimulation evoked EPSC activity in neurons (38/46, 82.6%) locatedwithin vLGNe, while those

recorded(n=14)invLGNifailedtorespond.ThehighprevalenceofsynapticresponsesinthevLGNewasobserved

amongPvalb+ (18/19),Sst+ (13/15)andGAD67-GFP+ (4/7)neurons.Thepeakamplitudeofexcitatorycurrents

(Figure7l)forPvalb+(613±79pA;n=15)andGAD67-GFP+(486±95pA;n=4)neuronswassignificantly(P<

0.05,One-WayANOVA)highercomparedtoSst+neurons(237±31;n=13).Synapticresponsesevokedbya10Hz

stimulustrainshowedamoderatedepression(75-85%)aftertheinitialresponse.Measurementsofpairedpulse

depression (Figure 7g) revealed no significant (p>0.1, One-way ANOVA) differences between Pvalb+ (0.76±

0.03;n=15),Sst+(0.84±0.04;n=13)andGAD67-GFP+(0.7±0.02;n=4)neurons.


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DISCUSSION

In this study,we identified novel vLGN cell typemarkerswhich label GABAergic cells throughout the

nucleusanddistinguish it fromitsdorsalcounterpart– thedLGN.Byperformingaseriesofmultiplex labeling

experimentsusingthesenewlyidentifiedmarkers,werevealedaremarkablyorganizedlaminararchitectureof

vLGNe,composedofatleastfouradjacent,transcriptionallydistinctsublaminae.Usinganterogradetrans-synaptic

viral tracing and patch-clamp electrophysiology, we determined that many of these regionally and

transcriptomicallydistinctsubtypesofGABAergicneuronsreceivedirectretinalinput.Thus,thesedatareveala

novelcellularorganizationofthevLGNandsuggestsuchorganizationmayhaveimportantimplicationsforhow

differentstreamsofretina-derivedvisualinformationareprocessedinthispartofvisualthalamus.

DifferenttypesofhiddenlaminaeexistinvLGNanddLGN

Incontrasttotheclearlaminationoftheprimateandcarnivorelateralgeniculatenucleus,therodentdLGN

andvLGNhavenoobviouscytoarchitectoniclamination–saveforthedivisionofthevLGNintoretinorecipient

vLGNeandnon-retinorecipientvLGNi(Niimietal.1963;Hickey&Spear1976;Gabbott&Bacon1994;Harrington

1997;Sabbaghetal.2018).TheneuronalcytoarchitectureoftherodentdLGNiscomposedofthreewell-defined

classes of glutamatergic thalamocortical relay cells and just one or two classes of GABAergic inhibitory

interneurons(roughly10%ofitsneuronalpopulation)(Arcellietal.1997;Jaubert-Miazzaetal.2005;Evangelioet

al.2018).Whileinterneuronsarepresentthroughoutthenucleus,relaycellsofeachclassappeartoexhibitregional

preferences intheirdistributionwithinthedLGN(Kraheetal.2011).Theseregionalpreferences intherodent

dLGN, however, do not alone capture the level of organization seen in primate and carnivore LGN. Instead, it

appearsthatretinalafferentsimpartorderintherodentdLGNbysegregatingtheirarborsinto“hiddenlaminae”

bothintermsofeye-specificdomainsandsubtype-specificlamina(Martin1986;Reese1988;Hong&Chen2011).

RecentadvancesintransgeniclabelingofindividualRGCsubtypeshasrevealedtheprecisearchitectureofthese

hidden laminae of subtype-specific retinal arbors in dLGN (Cruz-Martín et al. 2014; Huberman et al. 2008;

Hubermanetal.2009;Kayetal.2011;Kimetal.2010;Kimetal.2008;Marterstecketal.2017;Rivlin-Etzionetal.

2011;Kerschensteiner&Guido2017;Monavarfeshanietal.2017).Inthisstudy,however,weidentifiedadifferent

kindof‘hiddenlaminae’withinvLGN.Notably,thefewidentifiedsubtypesofvLGN-projectingRGCsdonotappear

tosegregate their terminalarbors into laminae invLGN(withthenotableexceptionthat theyarerestrictedto

vLGNe)(Hattaretal.2006;Osterhoutetal.2011;Monavarfeshanietal.2017).Thediffuseterminalarborizationof

thesenon-imageformingsubtypesofRGCsraisesthequestionofwhethervisualinformationis,infact,parsedinto

parallelchannelsinvLGN(Hattaretal.2006;Osterhoutetal.2011).Thestratificationoftranscriptomicallydistinct

neuronspresentedinthisstudyintoadjacentsublaminaeinvLGNemaycontributetotheparallelprocessingof

sensoryinformationinthisbrainregion.Justastheorganizationofretinalinputsimposesorderontheotherwise

lessorganizedcytoarchitectureofdLGN,thediversityandorganizationofintrinsiccellsinvLGNmayimposeorder

ontheunorganizedinputitreceivesfromtheretina.


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DolaminatedretinorecipientcircuitsorganizevisualpathwaysthroughthevLGN?

Is the quantifiable segregation of distinct GABAergic subtypes into sublaminae a potential means of

organizingvisual informationarrivingfromtheretina?Toaddressthis,wesoughttodeterminewhetherthese

subtypesweredirectlyinnervatedbyRGCs.Usinganterogradetrans-synaptictracing,weidentifiedEcel1+,Pvalb+,

Spp1+,andPenk+cellsasretinorecipient,togetherrepresentingatleastthreesublaminaeofvLGNe.Whilewefailed

toobserveanyretinorecipientcellsinvLGNiusingthismethod,itcertainlyremainspossiblethatthedendritesof

cellsinvLGNiextendintovLGNeandreceivemonosynapticinputfromtheretina.Nevertheless,ourviraltracing

results suggest that the cell type-specific organizationof the vLGN is relevant to organizing visual input. Such

organization of retinorecipient cells hints at a potentially novel role for vLGN in visual processing, by which

incomingvisual input issampledbyspecificGABAergiccell typesandparsed intoparallelchannelsofsensory

informationtobetransmittedtodownstreamtargets.

The specificity of these organized cell types in vLGN raises questions of whether they are projection

neuronsor local interneurons.Ourelectrophysiologicalandmorphologicalanalysesofsomeof thesecell types

suggeststhatcellslabeledinGAD67-GFPmicearelikelyvLGNinterneurons.Basedontheirpreponderanceinthe

vLGN,itislikelythatatleastsomeoftheothersubtypesofGABAergicneuronshereareprojectionneurons.Unlike

thedLGN,whichhasafferentprojectionstojustvisualcortexandthethalamicreticularnucleus,neuronsinvLGN

projecttoadiversesetofoveradozendownstreamsubcorticalregionsincludingtheSC,thenucleusoftheoptic

tract and other pretectal nuclei (Cadusseau& Roger 1991; Swanson et al. 1974; Trejo & Cicerone 1984), the

suprachiasmaticnucleus(atleastinhamsters)(Harrington1997),thehabenula(Ohetal.2014;Huangetal.2019),

andzona incerta(Ribak&Peters1975),allcontributing tovisuomotor,ocular,vestibular,circadian,andother

innatebehaviors(Monavarfeshanietal.2017).ItisknownthatsomeGABAergiccellsinvLGNreceiveretinalinput

andprojecttothelateralhabenula,althoughitremainsunclearwhichGABAergicsubtypesthisincludes(Huanget

al.2019).

Might the different transcriptionally distinct GABAergic cell types in vLGN each project to different

downstreamnucleiandcontributetouniquefunctionsandbehaviours?First,wenoteherethatitisconceivablethat

vLGNnotonlyorganizesandtransmitsvisualinformationinseparatechannels,butalsosamplesspecificfeatures

ofretinalinputinsublamina-specificmanner–consistentwithlabeled-linetheory.Unfortunately,wedonotyet

havesubtypespecificresolutionofvLGNprojectionneuronsbuthopethatthedatapresentedherewillhelpto

create a molecular toolkit for such circuit tracing in future studies. Such experiments will be crucial in a)

determiningwhetherparsingvisualinformationintothesehiddenlaminaeisimportantforparallelprocessingand

b)whetherthereisafunctionaland/orprojection-specificlogictothelaminationofvLGNcelltypes.


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TranscriptomicheterogeneityunderlyingcellulardiversityinvLGN

Thereisacurrentpushtouseunbiasedapproachestoidentifyallofthecelltypesinthebrain–essentially

tocreatea‘parts’list.ThesestudieshavetypicallyemployedsinglecellRNAsequencing(scRNAseq)tounderstand

thedevelopment,structure,andevolutionofthebrain(Krienenetal.2019;Saundersetal.2018;Pengetal.2019).

In aneuroscience community that consists of ‘lumpers’ and ‘splitters’, it is clearweare currently in an eraof

‘splitting’ – as our technology to detect transcriptomic heterogeneity of cell types evolves, so too does the

granularityoftheirdiscretization.Here,wehavebegunto‘split’therodentvLGNintomanydistinctGABAergiccell

types.

Rather than performing scRNAseq, we tackled the heterogeneity question by using bulk RNAseq and

generatingriboprobeswithnoaprioriknowledgetoperformabatteryofinsituhybridizationsfortranscriptional

heterogeneityinvLGN.Bygeneratingriboprobesagainstsinglemolecules,wedelineateddistinctpopulationsof

transcriptomicallyandspatiallydistinctcells,anadvantageofthisapproachoverscRNAseq.Conservatively,our

resultsdemonstratetheexistenceofatleastahalfdozendiscreteandseparablesubtypesofGABAergicneuronsin

vLGN,(althoughthenumberislikelymuchhigherthanthis)andfourdistinct,adjacentsublaminaeofGABAergic

neuronsinvLGNe.

WhethertherearesixGABAergiccelltypesinvLGNormanymorehasmadeuspondertheoldquestion

(Cajal1893)ofhowdoesonedefineacelltype?Traditionally,classesandtypesofneuronshavebeencharacterized

onthebasisofmorphological,electrophysiological,neurochemical,connectomic,orgeneticinformation(Sanes&

Masland2015).Unfortunately,rarelyarealltheseaspectsofneuronalidentityaccountedforinacomprehensive

waytogleanamoreaccurateunderstandingaboutthestructureofthenervoussystem.Thishasledtodiscrepant

subtype classification across technical methodologies and challenges to comparing results between research

groups.Here,weusedspatialdistributionandtranscriptionalprofilestoclassifyneuronsintodistinctsubtypes.

Molecularmarkersremainatpresentaleadingcharacteristicforsuchclassifications,thoughtheyarenotwithout

theirlimitations.ItmaywellbethatvLGNneuronscanbefurthersubdividedifclassifiedby2-3molecularmarkers

ratherthanone(asweobservedinSpp1+andPvalb+neurons).However,theveryfactthatdifferentialexpression

ofonemoleculewassufficienttodifferentiatetwovLGNpopulationsisastrongindicatorthatthenucleusasa

wholeismorediverselypopulatedthanpreviouslyappreciated.Nevertheless,weacknowledgeheretheeffortsto

createamorecomprehensiveframeworktoclassifyingcelltypesinthefield.Asetofrecentstudiesrepresenta

major step towards this goalbyutilizingPatch-seq to simultaneously characterize corticalGABAergicneurons

electrophysiologically, morphologically, and transcriptomically (Scala et al. 2020; Gouwens et al. 2020). Our

approach here did not take into account these additional functional aspects of neuronal identity for all of the

GABAergiccelltypesidentified.

Ourdata,whentakentogether,suggestthepossibilitythatfunctionalorganizationofnon-image-forming

informationfromretinatovLGNisextractedfromthesegregationoftranscriptionallydistinctretinorecipientcells.


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Weviewtheseresultsasaframeworkforfurtherdissectingthestructure,circuitry,andfunctionsofthevLGNata

cell-typespecificlevel.Howthisheterogeneityandorganizationcontributestotheyet-to-bedeterminedfunctions

ofthevLGNremainstobedefined.


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Graphicalabstract.ThevLGNisorganizedintosubtype-specificsublaminaewhichreceivevisualinput.

Theventrallateralgeniculatenucleus(vLGN)ispartofthevisualthalamus.Itcanbroadlybeseparatedintotwo

structuraldomainsorlaminae,theexternalvLGNe(whichreceivesretinalinput)andtheinternalvLGNi(receives

no retinal input). In this study, we describe subtypes of transcriptomically distinct GABAergic neurons that

populatethevLGNandorganizeintodiscrete,adjacentsublaminaeinthevLGNe.Takentogether,ourresultsshow

foursubtype-specificsublaminaeofretinorecipientneuronsinvLGNe.


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Figure1.


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Figure1.Cellsinretinorecipientandnon-retinorecipientzonesofvLGNaremolecularlydistinct

(a)InsituhybridizationforGad1mRNAinP60dLGNandvLGN.(b,c)QuantificationofthepercentageofDAPI+cells

thatareGad1+indLGN(b)andvLGN(c).Datapointsrepresentbiologicalreplicates,barsrepresentmean±SD.(d)

TransgeniclabelingofGad2+GABAergicneuronsinP60LGNofGad2-Cre::Sun1-Stop-GFPmice.(e,f)Quantification

of the percentage of DAPI+ cells that are Gad2+ in dLGN (e) and vLGN (f) of Gad2-Cre::Sun1-Stop-GFP. Data

presentedasin(c).(g)TransgeniclabelingofGFP+cellsinP60LGNofGAD67-GFPmice.(h,i)Quantificationofthe

percentageofDAPI+cellsthatareGFP+indLGN(h)andvLGN(i)ofGAD67-GFPmice.Datapresentedasin(c).(j)

RawtranscriptreadsofNxph1mRNAinvLGNanddLGNobtainedbyRNAseq. Individualdatapointsplottedas

whitecircles,min/maxvaluesareconfinedtotheredbars,andverticalblacklinewithbarsdepictsmean.(k-k’’)

DoubleISHforNxph1andGad1mRNAsinP10vLGN.(l)RawtranscriptreadsofArxmRNAinvLGNanddLGN

obtainedbyRNAseq,presentedasin(j).(m-m’’)DoubleISHinvLGNusingriboprobesgeneratedagainstArxand

Gad1mRNAsinP10vLGN.(n,o)QuantificationofthepercentofNxph1+(n)orArx+(o)cellsthatco-expressGad1

mRNA.Datapresentedasin(c).(p)IntravitrealinjectionofAlexa-conjugatedCholeraToxinsubunitB(CTB)labels

retinothalamicprojections.(q,r)ISHforNxph1(q)andArx(r)mRNAsinP10CTB-labeledvLGN. Allscalebars=

100µm.


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Figure2.


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Figure2.CanonicalmarkersofGABAergicneuronslabelsubsetsofneuronsinvLGN

(a-c)RawtranscriptreadsSst(a),Calb1(b),andPvalb(c)mRNAinvLGNanddLGNobtainedbyRNAseq.Individual

datapointsplottedaswhitecircles,min/maxvaluesareconfinedtotheredbars,andverticalblacklinewithbars

depictsmean.(d)TransgeniclabelingofSst+neuronsinvLGNofP60Sst-Cre::Rosa-Stop-tdTmice.(e)ISHforGad1

mRNAinSst-Cre::Rosa-Stop-tdTvLGN.(f)QuantificationofthepercentageofSst+cellsthatco-expressGad1mRNA.

Datapointsrepresentbiologicalreplicates,barsrepresentmean±SD.(g)TransgeniclabelingofSst+neuronsin

vLGNe and vLGNi of Sst-Cre:Rosa-Stop-tdT mice following intravitreal CTB injection. (h) Quantification of the

densityoftransgenicallylabeledSst+cellsinvLGNeandvLGNi.Dataplottedasin(f).(i)ImmunolabelingofCalb+

cells in P60 vLGN. (j) IHC-ISH for Calb protein andGad1 mRNA. (k) Quantification of Calb+ andGad1+ signal

colocalization as seen in (j). Data plotted as in (f). (l) Calb+ neurons in vLGNe and vLGNi visualized by Calb-

immunolabelingandintravitrealCTBinjection.(m)QuantificationofthedensityofCalb-immunoreactivecellsin

vLGNe or vLGNi. Data plotted as in (f). (n) Transgenic labeling ofPvalb+ neurons in P60 vLGN of P60Pvalb-

Cre::Thy1-Stop-YFP mice. (o) ISH for Gad1 mRNA in Pvalb-Cre::Thy1-Stop-YFP vLGN. (p) Quantification of the

percentageofPvalb+cellsthatco-expressGad1mRNA.Dataplottedasin(f).(q)ImmunolabelingofPvalb+neurons

invLGNeandvLGNiofwildtypemouse following intravitrealCTBinjection.(r)Quantificationof thedensityof

Pvalb+cellsdensityinvLGNeandvLGNi.Dataplottedasin(f).Allscalebars=100µm.


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Figure3.


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Figure3.NovelgeneticmarkerslabelsubtypesofGABAergicneurons

(a-d)Raw transcript readsSpp1 (a), Penk (b), Lypd1 (c), andEcel1 (d)mRNA in vLGN anddLGNobtained by

RNAseq.Individualdatapointsplottedaswhitecircles,min/maxvaluesareconfinedtotheredbars,andvertical

blacklinewithbarsdepictsmean.(e)ISHforSpp1mRNAinP60vLGN.(f)DoubleISHforSpp1andGad1mRNAin

vLGN. (g) Quantification of the percentage of Spp1+ cells that co-express Gad1 mRNA. Data points represent

biological replicates, bars representmean± SD. (h) ISH-labelingSpp1+ neurons in vLGNeandvLGNi following

intravitrealCTBinjection.(i)QuantificationofthedensityofSpp1+neuronsinvLGNeandvLGNi.Dataplottedasin

(g).(j)ISHforPenkmRNAinP60vLGN.(k)DoubleISHforPenkandGad1mRNAinvLGN.(l)Quantificationofthe

percentageofPenk+cellsthatco-expressGad1mRNA.Dataplottedasin(g).(m)ISH-labelingPenk+neuronsin

vLGNeandvLGNifollowingintravitrealCTBinjection.(n)QuantificationofthedensityofPenk+neuronsinvLGNe

andvLGNi.Dataplottedasin(g).(o)ISHforLypd1mRNAinP10vLGN.(p)DoubleISHforLypd1andGad1mRNA

invLGN.(q)QuantificationofthepercentageofLypd1+cellsthatco-expressGad1mRNA.Dataplottedasin(g).(r)

ISH-labelingLypd1+ neurons in vLGNeandvLGNi following intravitrealCTB injection. (s)Quantificationof the

densityofLypd1+neuronsinvLGNeandvLGNi.Dataplottedasin(g).(t)ISHforEcel1mRNAinP60vLGN.(u)

DoubleISHforEcel1andGad1mRNAinvLGN.(v)QuantificationofthepercentageofEcel1+cellsthatco-express

Gad1mRNA.Dataplottedasin(g).(w)ISH-labelingEcel1+neuronsinvLGNeandvLGNifollowingintravitrealCTB

injection.(x)QuantificationofthedensityofEcel1+neuronsinvLGNeandvLGNi.Dataplottedasin(g).Allscale

bars=100µm.


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Figure4.


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Figure4.TranscriptomicallydistinctGABAergicneuronsorganizeintodiscretesublaminaeofvLGNe

(a-c)ISH-labelingforSpp1+neuronsinvLGNofP60Pvalb-Cre::Thy1-Stop-YFPtransgenicreportermice.Insetin(c)

showssinglechannelandmergedhi-magnificationimagesandillustratesautomatedlinescanapproachusedto

quantify spatial expression. (d)Quantificationof vLGNneuronswhich generate eitherorbothSpp1 andPvalb

mRNA.Datapoints representbiological replicates, bars representmean± SD. (e) Line scan analysis of spatial

distributionofSpp1+ andPvalb+ cells.Arbitrary fluorescenceunits (a.f.i.) areplottedagainstdistance from the

lateral-mostpartofthevLGNtothemostmedial.SolidlinerepresentsmeanandshadedarearepresentsSEM(n=3).

(f-h) Double ISH for Spp1 and Penk mRNA in P60 vLGN. Inset in (h) shows single channel and merged hi-

magnification imagesandillustratesautomated linescanapproachusedtoquantifyspatialexpression.(i)Line

scananalysisofspatialdistributionofSpp1+andPenk+cellsinvLGNplottedasin(e).(j-l)DoubleISHforEcel1and

PenkmRNAinvLGNofP60Pvalb-Cre::Thy1-Stop-YFPtransgenicreportermice.(m)Linescananalysisofspatial

distributionofEcel1+,Penk+,andPvalb+cellsinvLGN,plottedasin(e),revealingfourdiscretespatialdomainsof

vLGN.TheblackarrowispointingatapotentialfifthlayerbetweenEcel1+andPvalb+layers.(n-p)DoubleISHfor

Ecel1andSpp1mRNAinvLGNofP60Gad2-Cre::Sun1-Stop-GFPtransgenicreportermice.(q)Schematicofsubtype

expressionobservedin(p),whereeachdotcorrespondstoacell(magenta=Ecel1+,cyan=Spp1+,black=Ecel1-Spp1-

Gad2+).OnlyGad2+neuronsbetweenthetwolabeledlayersareidentifiedbyblackdots.(r)Linescananalysisof

spatialdistributionofEcel1+,Spp1+,andGad2+cellsinvLGN,plottedasdescribedin(e).Blackarrowpointsatsame

region as arrow in (m). (s-u) Double ISH forEcel1 andGad1mRNA in vLGN of P60Pvalb-Cre::Thy1-Stop-YFP

transgenicreportermice.(v)Schematicofsubtypeexpressionobservedin(u),whereeachdotcorrespondstoa

cell (magenta=Ecel1+, cyan=Pvalb+,black=Ecel1-Pvalb-Gad1+neurons),as in(q). (w)Linescananalysisofspatial

distributionofEcel1+,Pvalb+,andGad1+cellsinvLGN,plottedasin(e).Blackarrowpointsatsameregionasarrow

in(m,r).Allscalebars=100µm.


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Figure5.


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Figure5.SubtypespecificlaminarorganizationofvLGNneuronsalongtherostro-caudalaxis

(a)SchematicofhorizontalmousebrainsectioningusedtofindvLGN.(b)IdentificationofthemousevLGNand

thalamicreticularnucleus(TRN)inthehorizontalplaneusingthePvalb-Cre::Thy1-Stop-YFPtransgenicreporter

andDAPIcounter-staining.(c-e)ISH-labelingforSpp1+neuronsinhorizontalvLGNofP60Pvalb-Cre::Thy1-Stop-

YFP transgenic reportermice. (f) Line scan analysis of spatial distribution ofSpp1+ andPvalb+ cells. Arbitrary

fluorescenceunits(a.f.i.)areplottedagainstdistancefromthelateral-mostpartofthevLGNtothemostmedial.(g-

j)DoubleISHforEcel1andPenkmRNAinhorizontalvLGNofP60Pvalb-Cre::Thy1-Stop-YFPtransgenicreporter

mice. (k) Line scan analysis of spatial distribution ofEcel1+,Penk+, andPvalb+ cells in vLGN, plotted as in (f),

revealingfourdiscretespatialdomainsofvLGN.Allscalebars=100µm.


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Figure6.


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Figure6.SeveralsubtypesofGABAergicneuronsinvLGNereceivedirectretinalinput

(a) Schematic of trans-synaptic viral tracing strategy to label retinorecipient vLGN neurons, where AAV1-Cre

induces recombination (and therefore expression of tdT) in transfected cells. (b) Image of an LGN with

retinorecipientcells labeledusingstrategy in (a). (c-c’) low-magnificationandhigh-magnificationmicrographs,

respectively,oflabeledretinorecipientneuroninvLGN.(c’’-c’’’)ImmunolabelingofPvalb+neuronsinvLGN(c’’)

and colocalization with tdT+ signal (c’’’). (d-d’) low-magnification and high-magnification micrographs,

respectively,oflabeledretinorecipientneuroninvLGN.(d’’-d’’’)ISHlabelingofSpp1+neuronsinvLGN(d’’)and

colocalizationwithtdT+signal(d’’’).(e-e’)low-magnificationandhigh-magnificationmicrographs,respectively,of

labeledretinorecipientneuroninvLGN.(e’’-e’’’)ISHlabelingofEcel1+neuronsinvLGN(e’’)andcolocalizationwith

tdT+ signal (e’’’). (f-f’) low-magnification and high-magnification micrographs, respectively, of labeled

retinorecipientneuron invLGN.(f’’-f’’’) ISHlabelingofPenk+neurons invLGN(f’’)andcolocalizationwithtdT+

signal(f’’’).Insetsaresingleplaneconfocalimages.Scalebars(c,d,e,f)=100µm,(c’,d’,e’,f’)=25µm.


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Figure7.


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Figure7.Morphology,synapticresponses,andmembranepropertiesofGABAergicsubtypesinvLGNe

(a-i)RepresentativeconfocalreconstructionsofPvalb+(a),GAD67+(d),andSst+(g)neuronsalongwithexamples

oftheirvoltageresponsestohyperpolarizinganddepolarizingcurrentpulses(top),andsynapticresponsestooptic

tract(OT)stimulation(bottom).(j)PlotdepictingrestingmembranepotentialofPvalb+,Sst+,andGAD67+neurons.

(k)PlotdepictingmembraneinputresistanceofPvalb+,Sst+,andGAD67+neurons.(l)Plotdepictingpeakexcitatory

postsynaptic currents (EPSC) amplitude of Pvalb+, Sst+, and GAD67+ neurons. (m) Plot depicting paired pulse

depression ration (PPD) during repeated stimulation of Pvalb+, Sst+, and GAD67+ neurons. Each data point

representsanindividualvalueandbarsreflectmean±SEM.Allscalebars=50µm.


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Table S1.Riboprobe screen of genes enriched in vLGN.Symbols are qualitative indicators of

expressionineachregion.TheminussymbolindicatesnocellsexpressingthismRNAobservedand

plus symbols indicate that cells were observed, ranging from some (+) to many (+++). SCN –

suprachiasmaticnucleus;dLGN–dorsallateralgeniculatenucleus;vLGN–ventrallateralgeniculate

nucleus;SC–superiorcolliculus;n.d.–notdone.

Gene SCN dLGN vLGN SC Gene SCN dLGN vLGN SC1 Adcyap1r1 + + +++ + 36 Sh3bgrl2 – + + +2 Ankrd34b – – ++ - 37 Slc39a14 – – ++ +3 Anxa3 – – + – 38 Spp1 – – ++ +4 Arx – – ++ – 39 Sst n.d. n.d. ++ ++5 Asic4 – + +++ +++ 40 Steap2 – – + –6 Atpaf1 – ++ + + 41 Tcf7l2 – +++ + +7 Cabp7 – – ++ + 42 Unc5d – – + –8 Calb1 + – ++ ++ 43 Zfp804a – +++ + –9 Cbln4 – – + + 10 Cd24a +++ – + + 11 Chrm2 – + ++ ++ 12 Chst2 – + + ++ 13 Cntn4 – + + – 14 Col15a1 + – + – 15 Coro6 – +++ + + 16 Ecel1 – – ++ + 17 Gad1 +++ + +++ +++ 18 Gad2 +++ + +++ +++ 19 Grik1 ++ ++ + + 20 Islr2 + – +++ n.d. 21 Lmo3 – – + + 22 Loc433436 – – + – 23 Lypd1 – – ++ – 24 Nacc2 – + + + 25 Nos1 – – + – 26 Nos1ap ++ + + + 27 Nxph1 – – ++ – 28 Pvalb – – + + 29 Pcdh11x + – + + 30 Penk ++ – ++ + 31 Prkcd – +++ + – 32 Ptprk – – + – 33 Rab37 – +++ + – 34 Sdk1 n.d. – + n.d. 35 Sdk2 n.d. – + n.d.


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References

Arcelli, P., Frassoni, C., Regondi, M., Biasi, S. and Spreafico, R. (1997) GABAergic neurons in

mammalianthalamus:amarkerofthalamiccomplexity?Brainresearchbulletin42,27-37.

Berson, D. (2008) Retinal ganglion cell types and their central projections. The senses: a

comprehensivereference1,491-520.

Cadusseau,J.andRoger,M.(1991)Corticalandsubcorticalconnectionsoftheparscompactaofthe

anteriorpretectalnucleusintherat.Neuroscienceresearch12,83-100.

Cajal,S.R.(1893)Laretinedesvertebres.Cellule9,119-255.

Charalambakis, N. E., Govindaiah, G., Campbell, P. W. and Guido, W. (2019) Developmental

remodelingofthalamicinterneuronsrequiresretinalsignaling.JournalofNeuroscience39,

3856-3866.

Cruz-Martín,A.,El-Danaf,R.N.,Osakada,F.,Sriram,B.,Dhande,O.S.,Nguyen,P.L.,Callaway,E.M.,

Ghosh,A. andHuberman,A.D. (2014)Adedicated circuit linksdirection-selective retinal

ganglioncellstotheprimaryvisualcortex.Nature507,358-361.

Dhande,O.S.,Hua,E.W.,Guh,E.,Yeh,J.,Bhatt,S.,Zhang,Y.,Ruthazer,E.S.,Feller,M.B.andCrair,M.

C.(2011)Developmentofsingleretinofugalaxonarborsinnormalandβ2knock-outmice.

TheJournalofNeuroscience31,3384-3399.

Dhande,O.S.andHuberman,A.D.(2014)Retinalganglioncellmapsinthebrain:implicationsfor

visualprocessing.Currentopinioninneurobiology24,133-142.

Dhande, O. S., Stafford, B. K., Lim, J.-H. A. and Huberman, A. D. (2015) Contributions of Retinal

GanglionCellstoSubcorticalVisualProcessingandBehaviors.AnnualReviewofVisionScience

1,291-328.

El-Danaf,R.N.,Krahe,T.E.,Dilger,E.K.,Bickford,M.E.,Fox,M.A.andGuido,W.(2015)Developmental

remodelingof relay cells in thedorsal lateral geniculatenucleus in theabsenceof retinal

input.Neuraldevelopment10,19.

Evangelio, M., García-Amado, M. and Clascá, F. (2018) Thalamocortical projection neuron and

interneuronnumbersinthevisualthalamicnucleioftheadultC57BL/6mouse.Frontiersin

neuroanatomy12,27.

Fleming,M.D.,Benca,R.M.andBehan,M.(2006)Retinalprojectionstothesubcorticalvisualsystem

incongenicalbinoandpigmentedrats.Neuroscience143,895-904.

Gabbott,P.andBacon,S.(1994)Anorientedframeworkofneuronalprocessesintheventrallateral

geniculatenucleusoftheratdemonstratedbyNADPHdiaphorasehistochemistryandGABA

immunocytochemistry.Neuroscience60,417-440.


https://doi.org/10.1101/2020.05.03.073197


36

Gaillard,F.,Karten,H. J. andSauvé,Y. (2013)Retinorecipientareas in thediurnalmurine rodent

Arvicanthisniloticus: adisproportionally large superior colliculus. Journal of Comparative

Neurology521,1699-1726.

Gale,S.D.andMurphy,G.J.(2014)Distinctrepresentationanddistributionofvisualinformationby

specificcelltypesinmousesuperficialsuperiorcolliculus.JournalofNeuroscience34,13458-

13471.

Gale, S.D. andMurphy,G. J. (2016)Active dendritic properties and local inhibitory input enable

selectivity forobjectmotioninmousesuperiorcolliculusneurons. JournalofNeuroscience

36,9111-9123.

Godement, P., Salaün, J. and Imbert, M. (1984) Prenatal and postnatal development of

retinogeniculate and retinocollicular projections in the mouse. Journal of Comparative

Neurology230,552-575.

Gouwens,N.W.,Sorensen,S.A.,Baftizadeh,F.etal. (2020)Towardan integratedclassificationof

neuronal cell types: morphoelectric and transcriptomic characterization of individual

GABAergiccorticalneurons.BioRxiv.

Guillery,R.(1966)AstudyofGolgipreparations fromthedorsal lateralgeniculatenucleusof the

adultcat.JournalofcomparativeNeurology128,21-49.

Hammer,S.,Carrillo,G.L.,Govindaiah,G.,Monavarfeshani,A.,Bircher,J.S.,Su,J.,Guido,W.andFox,

M. A. (2014) Nuclei-specific differences in nerve terminal distribution, morphology, and

developmentinmousevisualthalamus.Neuraldevelopment9,1.

Harrington, M. E. (1997) The ventral lateral geniculate nucleus and the intergeniculate leaflet:

interrelated structures in the visual and circadian systems.Neuroscience & Biobehavioral

Reviews21,705-727.

Hattar,S.,Kumar,M.,Park,A.,Tong,P.,Tung,J.,Yau,K.W.andBerson,D.M.(2006)Centralprojections

of melanopsin-expressing retinal ganglion cells in the mouse. Journal of Comparative


He, J.,Xu,X.,Monavarfeshani,A.,Banerjee, S., Fox,M.A. andXie,H. (2019)Retinal-input-induced

epigeneticdynamicsinthedevelopingmousedorsallateralgeniculatenucleus.Epigenetics&

chromatin12,13.

Hickey, T. and Spear, P. (1976) Retinogeniculate projections in hooded and albino rats: an

autoradiographicstudy.ExperimentalBrainResearch24,523-529.

Hong, Y. K. and Chen, C. (2011)Wiring and rewiring of the retinogeniculate synapse.Curr Opin

Neurobiol21,228-237.


https://doi.org/10.1101/2020.05.03.073197


37

Hoy,J.L.,Bishop,H.I.andNiell,C.M.(2019)Definedcelltypesinsuperiorcolliculusmakedistinct

contributionstopreycapturebehaviorinthemouse.CurrentBiology29,4130-4138.e4135.

Huang,L.,Xi,Y.,Peng,Y.etal.(2019)Avisualcircuitrelatedtohabenulaunderliestheantidepressive

effectsoflighttherapy.Neuron102,128-142.e128.

Huberman,A.D.,Manu,M.,Koch,S.M.,Susman,M.W.,Lutz,A.B.,Ullian,E.M.,Baccus,S.A.andBarres,

B. A. (2008) Architecture and activity-mediated refinement of axonal projections from a

mosaicofgeneticallyidentifiedretinalganglioncells.Neuron59,425-438.

Huberman,A.D.,Wei,W.,Elstrott, J., Stafford,B.K.,Feller,M.B.andBarres,B.A. (2009)Genetic

identificationofanOn-Offdirection-selectiveretinalganglioncellsubtyperevealsa layer-

specificsubcorticalmapofposteriormotion.Neuron62,327-334.

Inamura,N.,Ono,K.,Takebayashi,H., Zalc,B. and Ikenaka,K. (2011)Olig2 lineage cells generate

GABAergic neurons in the prethalamic nuclei, including the zona incerta, ventral lateral

geniculatenucleusandreticularthalamicnucleus.Developmentalneuroscience33,118-129.

Jager, P., Ye, Z., Yu, X. et al. (2016) Tectal-derived interneurons contribute to phasic and tonic

inhibitioninthevisualthalamus.NatureCommunications7.

Jaubert-Miazza,L.,Green,E.,Lo,F.-s.,Bui,K.,Mills,J.andGuido,W.(2005)Structuralandfunctional

compositionofthedevelopingretinogeniculatepathwayinthemouse.Visualneuroscience

22,661-676.

Kalish, B. T., Cheadle, L., Hrvatin, S., Nagy, M. A., Rivera, S., Crow, M., Gillis, J., Kirchner, R. and

Greenberg, M. E. (2018) Single-cell transcriptomics of the developing lateral geniculate

nucleus reveals insights into circuit assemblyandrefinement.Proceedingsof theNational

AcademyofSciences,201717871.

Kay,J.N.,DelaHuerta,I.,Kim,I.-J.,Zhang,Y.,Yamagata,M.,Chu,M.W.,Meister,M.andSanes,J.R.

(2011) Retinal ganglion cells with distinct directional preferences differ in molecular

identity,structure,andcentralprojections.TheJournalofNeuroscience31,7753-7762.

Kerschensteiner,D.andGuido,W.(2017)Organizationofthedorsallateralgeniculatenucleusinthe

mouse.VisualNeuroscience34.

Kim, I.-J.,Zhang,Y.,Meister,M.andSanes, J.R. (2010)Laminarrestrictionofretinalganglioncell

dendrites and axons: subtype-specific developmental patterns revealed with transgenic

markers.TheJournalofNeuroscience30,1452-1462.

Kim, I.-J.,Zhang,Y.,Yamagata,M.,Meister,M.andSanes, J.R. (2008)Molecular identificationofa

retinalcelltypethatrespondstoupwardmotion.Nature452,478-482.


https://doi.org/10.1101/2020.05.03.073197


38

Krahe, T. E., El-Danaf, R.N., Dilger, E. K., Henderson, S. C. andGuido,W. (2011)Morphologically

distinct classes of relay cells exhibit regional preferences in the dorsal lateral geniculate

nucleusofthemouse.TheJournalofNeuroscience31,17437-17448.

Krienen,F.M.,Goldman,M.,Zhang,Q.etal.(2019)Innovationsinprimateinterneuronrepertoire.

bioRxiv,709501.

Langel, J., Ikeno, T., Yan, L., Nunez, A. A. and Smale, L. (2018) Distributions of GABAergic and

glutamatergicneuronsinthebrainsofadiurnalandnocturnalrodent.Brainresearch1700,

152-159.

Leist,M.,Datunashvilli,M.,Kanyshkova,T.,Zobeiri,M.,Aissaoui,A.,Cerina,M.,Romanelli,M.N.,Pape,

H.-C.andBudde,T.(2016)Twotypesofinterneuronsinthemouselateralgeniculatenucleus

arecharacterizedbydifferenth-currentdensity.Scientificreports6.

Lim,L.,Mi,D.,Llorca,A.andMarín,O.(2018)Developmentandfunctionaldiversificationofcortical

interneurons.Neuron100,294-313.

Ling,C.,Hendrickson,M.L.andKalil,R.E.(2012)Morphology,classification,anddistributionofthe

projectionneuronsinthedorsallateralgeniculatenucleusoftherat.PloSone7,e49161.

Martersteck,E.M.,Hirokawa,K.E.,Evarts,M.etal. (2017)DiverseCentralProjectionPatternsof

RetinalGanglionCells.CellReports18,2058-2072.

Martin, P. (1986) The projection of different retinal ganglion cell classes to the dorsal lateral

geniculatenucleusinthehoodedrat.Experimentalbrainresearch62,77-88.

Mo,A.,Mukamel,E.A.,Davis,F.P.etal.(2015)Epigenomicsignaturesofneuronaldiversityinthe

mammalianbrain.Neuron86,1369-1384.

Monavarfeshani,A.,Sabbagh,U.andFox,M.A.(2017)Notaone-trickpony:Diverseconnectivityand

functionsoftherodentlateralgeniculatecomplex.VisualNeuroscience34.

Monavarfeshani,A.,Stanton,G.,Su,K.,MillsIII,W.A.,Swilling,K.,Kerr,A.,Huebschman,N.A.,Su,J.

andFox,M.A.(2018)LRRTM1underliessynapticconvergenceinvisualthalamus.eLife7,

e33498.

Morin,L.P.andStudholme,K.M.(2014)Retinofugalprojectionsinthemouse.JournalofComparative

Neurology522,3733-3753.

Muscat, L., Huberman, A. D., Jordan, C. L. andMorin, L. P. (2003) Crossed and uncrossed retinal

projectionstothehamstercircadiansystem.JournalofComparativeNeurology466,513-524.

Niimi,K.,Kanaseki,T.andTakimoto,T.(1963)Thecomparativeanatomyoftheventralnucleusof

thelateralgeniculatebodyinmammals.JournalofComparativeNeurology121,313-323.


https://doi.org/10.1101/2020.05.03.073197


39

Oh,S.W.,Harris,J.A.,Ng,L.etal.(2014)Amesoscaleconnectomeofthemousebrain.Nature508,

207-214.

Oliveira, A. F. and Yonehara, K. (2018) The mouse superior colliculus as a model system for

investigatingcelltype-basedmechanismsofvisualmotortransformation.Frontiersinneural

circuits12,59.

Osterhout, J.A., Josten,N.,Yamada, J.etal.(2011)Cadherin-6mediatesaxon-targetmatchingina

non-image-formingvisualcircuit.Neuron71,632-639.

Peng,Y.-R.,Shekhar,K.,Yan,W.etal.(2019)Molecularclassificationandcomparativetaxonomicsof

fovealandperipheralcellsinprimateretina.Cell176,1222-1237.e1222.

Piscopo,D.M.,El-Danaf,R.N.,Huberman,A.D.andNiell,C.M.(2013)Diversevisualfeaturesencoded

inmouselateralgeniculatenucleus.TheJournalofNeuroscience33,4642-4656.

Reese, B. (1988) ‘Hidden lamination’in the dorsal lateral geniculate nucleus: the functional

organizationofthisthalamicregionintherat.Brainresearchreviews13,119-137.

Reinhard,K.,Li,C.,Do,Q.,Burke,E.G.,Heynderickx,S.andFarrow,K.(2019)Aprojectionspecific

logictosamplingvisualinputsinmousesuperiorcolliculus.Elife8.

Ribak, C. E. and Peters, A. (1975) An autoradiographic study of the projections from the lateral

geniculatebodyoftherat.Brainresearch92,341-368.

Rivlin-Etzion,M.,Zhou,K.,Wei,W.,Elstrott,J.,Nguyen,P.L.,Barres,B.A.,Huberman,A.D.andFeller,

M.B.(2011)Transgenicmicerevealunexpecteddiversityofon-offdirection-selectiveretinal

ganglion cell subtypesandbrain structures involved inmotionprocessing.The Journal of

Neuroscience31,8760-8769.

Sabbagh,U.,Monavarfeshani,A., Su,K. etal. (2018)Distributionanddevelopmentofmolecularly

distinctperineuronalnetsinvisualthalamus.Journalofneurochemistry.

Sanes, J. R. and Masland, R. H. (2015) The types of retinal ganglion cells: current status and

implicationsforneuronalclassification.Annualreviewofneuroscience38,221-246.

Saunders,A.,Macosko,E.Z.,Wysoker,A.etal.(2018)Moleculardiversityandspecializationsamong

thecellsoftheadultmousebrain.Cell174,1015-1030.e1016.

Scala, F., Kobak, D., Bernabucci, M. et al. (2020) Phenotypic variation within and across

transcriptomiccelltypesinmousemotorcortex.bioRxiv.

Seabrook,T.,Burbridge,T.,Crair,M.andHuberman,A.(2017)Architecture,Function,andAssembly

oftheMouseVisualSystem.Annualreviewofneuroscience40,499.


https://doi.org/10.1101/2020.05.03.073197


40

Seabrook,T.A.,Krahe,T.E.,Govindaiah,G.andGuido,W.(2013)Interneuronsinthemousevisual

thalamusmaintainahighdegreeofretinalconvergencethroughoutpostnataldevelopment.

Neuraldevelopment8,1.

Shang,C.,Chen,Z.,Liu,A.etal.(2018)Divergentmidbraincircuitsorchestrateescapeandfreezing

responsestoloomingstimuliinmice.Naturecommunications9,1-17.

Shang,C.,Liu,Z.,Chen,Z.,Shi,Y.,Wang,Q.,Liu,S.,Li,D.andCao,P.(2015)Aparvalbumin-positive

excitatoryvisualpathwaytotriggerfearresponsesinmice.Science348,1472-1477.

Su,J.,Charalambakis,N.E.,Sabbagh,U.,Somaiya,R.D.,Monavarfeshani,A.,Guido,W.andFox,M.A.

(2020) Retinal inputs signal astrocytes to recruit interneurons into visual thalamus.

ProceedingsoftheNationalAcademyofSciences117,2671-2682.

Su, J.,Gorse,K.,Ramirez,F. andFox,M.A. (2010)CollagenXIX isexpressedby interneuronsand

contributes to the formation of hippocampal synapses. Journal of Comparative Neurology

518,229-253.

Su,J.,Haner,C.V.,Imbery,T.E.,Brooks,J.M.,Morhardt,D.R.,Gorse,K.,Guido,W.andFox,M.A.(2011)

Reelin isrequiredforclass-specificretinogeniculatetargeting.TheJournalofNeuroscience

31,575-586.

Swanson,L.,Cowan,W.andJones,E.(1974)Anautoradiographicstudyoftheefferentconnections

oftheventrallateralgeniculatenucleusinthealbinoratandthecat.JournalofComparative


Trejo,L.J.andCicerone,C.M.(1984)Cellsinthepretectalolivarynucleusareinthepathwayforthe

directlightreflexofthepupilintherat.Brainresearch300,49-62.

Tremblay,R.,Lee,S. andRudy,B. (2016)GABAergic interneurons in theneocortex: fromcellular

propertiestocircuits.Neuron91,260-292.

Wang,Q.,Marcucci,F.,Cerullo,I.andMason,C.(2016)Ipsilateralandcontralateralretinalganglion

cellsexpressdistinctgenesduringdecussationattheopticchiasm.eneuro3.

Yonehara,K.,Ishikane,H.,Sakuta,H.,Shintani,T.,Nakamura-Yonehara,K.,Kamiji,N.L.,Usui,S.and

Noda, M. (2009) Identification of retinal ganglion cells and their projections involved in

centraltransmissionofinformationaboutupwardanddownwardimagemotion.PloSone4.

Yuge,K.,Kataoka,A.,Yoshida,A.C.,Itoh,D.,Aggarwal,M.,Mori,S.,Blackshaw,S.andShimogori,T.

(2011) Region-specific gene expression in early postnatal mouse thalamus. Journal of

ComparativeNeurology519,544-561.


https://doi.org/10.1101/2020.05.03.073197


41

Zingg,B.,Chou,X.-l.,Zhang,Z.-g.,Mesik,L.,Liang,F.,Tao,H.W.andZhang,L.I.(2017)AAV-mediated

anterogradetranssynaptictagging:mappingcorticocollicularinput-definedneuralpathways

fordefensebehaviors.Neuron93,33-47.


https://doi.org/10.1101/2020.05.03.073197


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