1
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.CC-BY-NC-ND 4.0 International licensewas not certified by peer review) is the author/funder.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
2
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.
.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
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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;
.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
4
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.
.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
5
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),
.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
6
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.
.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
7
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).
.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
8
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.
.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
9
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.
.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
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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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12
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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14
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.
.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
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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.
.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
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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.
.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
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