Combined GABA-immunocytochemistry and TMB-HRP histochemistry of pretectal nuclei projecting to the

inferior olive in rats, cats and monkeys

A K E Horn * and K -P Hoffmann Abttlhmg!ur Vergleichende Neuroblologu'. Uruverslfat U/m, V/rn (F R G)

(Acccpt.cd 2\ OClober 1986)

Key words Nucleu$ uf thc OpllC trael, Infe n or ohve, y-Ammobulync aC id, Horserad lsh perOXidase . Tct ramelhylbe nZldmc, Monkey, Cat, Rat

Cells 10 the pretcctal noclcus of thc optlC trae! (NOT) of rats calS and monkeys wen: ren ograde ly labeled wllh horseradlsh perox­Idase (HRP) stereotaxtcally rnJcclcd mto thc inferior ohve (JO) A procedure for stablll1:mg thc letramClhyJbenzldme (TMB)-HRP rcacuon product wa5 used to vlsuahze oombmed TMB - HRP and ImmunohlSlochemtca lly locahzed y-ammobutync aCid (GABA) In

the same seChQns POSI\lVC GABAcqpc rcachon product was fOllod to be restncted 10 sma lJer-slze InlnnSlc neurons Larger NOT cclIs proJech ng 10 Ihe 10 were oonslslently frec of GABA reaCll on producI and, m addlUon , appcared to be contacted by rclauvely few GABAerglc tennmals

A speclfH: popul atIon of neurons In the nucleus of the 0rtlc tract (NOT) and the dorsal te rminal nucleus of the accessory optlC tract (DTN) In the pretectum of mammals I~ mvolved 10 the cont rol of hOTlzontal optokmeuc nys tagmus~ f> I() t~ 13 If> 23 30 These neu­rons recetve a vIsual proJeetloll malnly from the tOIl­tralateral eye1 26 as well as from the lpstlate ral visual cortex1734 and they 1Il turn prOJect to Ihe tpsllatera l dorsal cap of the Infenor o live (10)142833 The out­standmg prope rt y of these NOT and DTN neurons tS thelr dtrecltonall y speclflc response 10 lemporal 10 nasal movement of fu ll-ft e ld vtsual sttmuh presentcd to the contralateral eye4 ~ tO t: 13 The resuhs of phar­macologlca l experimen ts "> uggesl that y-amtnobutyr­te aCId (GA BA) plays an Im portalll role tn the mllla­tton 01 dlreclton speclflctly tn cells of the re lma and vlsua l cortex1~"111 Inhl bltory GA BAergic ce lls may lIbo shape the prete rred dlrectlOn of dlrecllOnal ce lls tn the NOT or med iale Ihelr tnhl blllOn caused by mo"ement tn Ihe non-prcferred dtrcctlon Rece nt lmmunoeylochcm tcal stud les havc shown Ihe pres­enee uf neurons wlth1l1 Ihe NOT and DTN tn a nu m-

ber of mammals thaI use GA BA as a ncurolransmlt­terY l~ ~1 1: 35 In Ihe preselll study we Invesugaled the

pOSSlbJe structural basIs for a dlrecllOn se lectlve mecha msm III Ihe NOT and DTN by deie rmmmg whelher neurons pTOJeCIlllg 10 the 10 are contacled by GA BAcrgic lermmals We used thc ret rograde axonal transport ot horserad lsh peroXidase (HRP) to label NOT and DTN neurons. and combmed HRP hlstochemlslry wlth subsequent Immullocylocheml­cdl me thods for Ihe localtzatlon of GABA

Four pIgmented rats, IwO normally plgmenled cats and two mon keys (Macoca mululla) were used FOT Ihe stereolaxlC tnJectlO1I of HRP each anlm al was deepl} ane~thet lzed Rats were glvcn a smgle dose of Nembuta l (5 mg/WO g) and cal~ as weil as monkeys were glven an tnlt ml subcutaneou~ tnJect ton of ket ­amme (20 mg/kg) tn combmll tlon wll h Rompun (0 05 ml/kg), fo llowed by subsequent dosts of ketamme ( 10 mg/kg) I v glven as needed dunng surgc ry The cats and monkeys were Ifltubated and aTllflclally ven­lil ated wlth a mixt ure of N20/0~ (3 1) 10 mamtam ad­eq uate ane., thesl3

" Present 3d"re,~ Phv~ , ologl"che~ InstItut, PellenJ"olcr.>tr I.! ROOO Muenchcn 2 F R G (oTTr~polldellce K ·1' Hoffmann AbI f VergleIchende Neurob,ologle Un,verS 'lat Ulm Oberer Esdsberg D-7'XJ(J Ulm FR G

Each ammal was subsequcntly placed m an appro­pnate stereotaxlc mstrument To approach Ihe 10 10 rats and cals. the foramen magnum was enlarged anel thc durd exclsed 10 cxpose thc medulla oblonga'a The caudal part of the cerebellum was gently elc­

valed to visua hze the forth ventnele and a Hamllton mlcrohter synnge was advanced towards the 10 FOT ralS. Ihe mlcrohter synnge was mserted to a dcpth ot 2 7 mm through the mldhne 01 the open mg 01 thc obex angled fo rward at .In angle 01 5~ A slml ldr .tp­proach was used tor the first cat Thc ~ynnge w.t\ In­serted 1 mm lateral to the obex. anglcd 45° forwdTd dnd advanced 7 mm bc10w the bram~tem surfacc A second cat recelved an HRP InJectlOn mto thc latem l gcmculatc nueleus The syrmge was ;, tercotaxlc.tlly gUided wlth referencc 10 the atlas of Berman ' .• md It" fmal posit ion was determllled by elcctrophyslOloglcd l recordmg of visually evoked dCtlVlty In tbe monkc y. the 10 was approached stereotaxlcally accordmg to the atlas of Snyder and Lee31 wlth a synnge angled forward 45° Tbe rats recelved 0 I 111. thc Colt<; dnt! monkeys recelved 0 2 p i of d 30% H RP solullon In 2% dlmethylsulfoxlde (DMSO) m dlSlllled wdter AI[ IßJectlOns were made ;,lowly ovcr a penod of20 mm

Followmg a sUTVlval penad of 72 h after an mJcc­tlOn mto the 10, and 45 h after the mJeelion mto thc gemculate body, the amrnals were deeply ane<;thc­tlzed and perfused transcard13lly wlth the followmg solutlons (I) 1 hter Rmger (0 9% NaCI) wlth 0 1% Novocam at 37°C, (2) 3 liters of a fixative eonslstlng of 1% paraformaldehyde and 1 % glutaraldehyde In

OlM phosphate buffer (pH 7 4) at 4 oe and (3) I h­ter of a second fixative conslsllng of 2% paraformal­dehyde and 0 1 % glutaraldehyde at 4 oe Thc brams were Immedlately blocked and stored overmght m OlM phosphate buffer The next day, 20-40 ~m se-

nal sectlons of the mldbram were cut on a vlbratome and collected mOl M phosphate buffer All sect!ons wer\! lir .. t processed fo r HRP-dctl \> lt} wlth h::trdmC'­Ihylbenzldme (TMB) a~ a chromogt: n In order tn demonstrate lTansported HRP <lnd GABA-lmmullO~ hlstochemlstry III the samt: .,('CIIOII thc tollowmg. modlflcatlOn of the Me~ul;Jml ~ prol\l{ol for rMB-hl'o­tochem l~ITY W<J\ u<,ed

H R P prolocol

(I ) Rm~e "ectlons 6 tlmes m Icc-cold dlsti lled Wd­Icr

(2) Premcubale m a mixture 01 solution A ll.H) mg sodlUm mtroferncyamde. 92 5 ml dlslllled water and 5 ml 0 2 M citrate buffer (pH 3 3), <lnd B 5 mg TMB dlssolved In 2 5 ml of reagent grade absolute dlcohol

(:\) Incubate by addmg 200 mg ß-o.glucose. 40 mg ammoß\urn chlonde and 0 5 mg glucose OXidase (GOD) (m~tead of HZ02)1 ~, 10 100 ml of premcuba­tlOn balh for about 40 mm untll the demed degrec 01 ~ldmlßg IS obtamed

(4) Slabll!ze TMß m an Ice-coolcd 5% ammonIUm heplamolybdate (A HM) solutlOnll .2<1 for 20 mm (nole wlthoullhlS Ißltl3l stablhzatlon . TMB reactlon product mdY be losl Iß the second S!ablhzal1on step.)

(5) Rmse sectlons 30 S mOl M phosphate buffer (rH 7 4)

(6) Furthet stabllize TMB for 5- H mm wlth DAß­Co as descnbed by Rye et al ~ prt:pare <I sol~tlon of 50 mg DAB m 100 ml 0 I M phosphate buffer (pH 74), and whde sttrnng slowly add 2 5 ml of a I % co­balt acetate solution dropWlse , followed by addJllOn of H20 Z (for a fmal dilution ofO 01 % HP2)

(7) Rmse 5 tlmes m cold 001 M phosphate-buf­fered sa hne (PBS). pH 73

Fig 1 Mlcrographs showmg the blaek tetramethylberwdlOe (TMB) reaetlon produel of relJogradely transporied horseradlSh perox­Idase (HRP) and the brown label of y-ammobulyne aCid (GABA) bom an Immuooreacuon W1lh GABA"anubody (Immuno Nudear) m the same seeIIon Sohd blaek arrowheads poml to GABAcrgle termInals (puncta) on somata and proximal dendntcsof retrogradely labeled cells Open blaek arrows pOInt 10 eclls 001 retrogradely labeled bul outhned by dense dustenog of GABAer8le termmals Whlte arrows POint to GABAergie neurons A GABAergtc neuron In the pengenlCUlate nudellS of a eat The brown soma aod den­dote are dearly YlSlble due to thelr high contenl of GABA In additlon, the soma cootalnS the granular blaek 'QofB reactlon product due 10 Ibe retrogradely Iransported HRP from an InJeenon 10tO Ihe lateral g1:rueuJate nucleus B cell in the lotermedtate layen of Ihe: supeoor cothculus In Ihe eat The eell was retrognulely labcled by an HRP UlJe/;IIOD IDIO the Inferior olive and GABAergtc puncta (marke<! by blaek arrows) oulhne soma and dendnles These tennlna.ls probably stern from the rugroteclal prOJeetlOnl7 C-F cells m Ihe nude:us of the opue Irael retrogradely labeled by HRP lDJecbOßS mto the lofenor OllYe are not GABAersae C monkey (Maellc/i mu/aull) D plgmented ral E,F eal In contrast to the oolhcular neuron (B) the pretectal neurons are c:ontacted by only a few GABA­er&Jc termmals Bars = 25l1m

After completion of the HRP reaction , thc free· floating sections werc processed for GABA·immu· nocytochemistry using the unlabeled antibody pcrox­idasc.antiperoxidase (PA P) methodl2• Sections were first incubated in 10% methanoll3% H20 Z 10 deslroy the remaining activity of HRP and endogenous per­oxidases. Non·specific background slaining was sup' prcssed incubating the scctions for 30 min at room temperalUre in 10% normal swine serum (NSS) with 0.5% Trilon X-IOO in PBS. Then the sections were incubated for 12- 16 h in a solution at4 oe containing rabbit anli·GABA serum (Immuno Nuclear Corp) diluted 1:3000 in PßS containing 5% NSS and 0.5% Triton X·IOO. The specific antigen was labeled by subsequently incubat ing the sections in swine anti· rabbil IgG (Da kopaus) diluted I: 100 in the same so· lution fo r 1 h at room temperature (RT) , and in rab· bit PAP (Dakopaus) diluted 1:50 in PBS for 1 h at RT. The P AP complexcs were visualized by a 5- IO min incubation in the fresh ly prepared solution con· sisting of 0.05% DA.B in 0.05 M Tris·buffered saline (TßS) , pH 7.6 and 0.01% H20 2 0r by 20-30 min in· cubat ion using the DAß·glucose oxidase method of hoh et aL 15 without cobalt. As a eontrol , same sec· tions were incubated in a solution in which the pTi· mary antibody was replaced with normal rabbit se· rum. After rinsing , mounting and drying, alternate sections were counterstained wilh 0.1 % Cresyl violet before dehydrating and coverslippi ng.

The double· label method resulted in homogeneous brown labeled GAßA·positive cell struclures and granu lar black·labeled retrogradely transported IIRP conlained in neurons. The two labels were clearly distinguishable from one anOlher by color and appearance of the reaction product (Fig. 1). GABA· immunoreactivity was fOllnd primarily in cell bodies and punctate profiles determined from elcctron mi· croscopic studies to be axon terminals24

. Due 10 lim· ited abilily of the antibody to penetratc the lissue GABA·positive labcJing is restricted to lhe outer 8 .um of the sect ion's surface. As a control , we firsl ver· ified two critical points: ( 1) both markers can bc inde· pendently demonstratcd in the same cell, and (2) GAßAergic lerminals can be distinguished on retro· gradely labeled cells. For the first point we per· formed the conlrol experiment described by Fitzpa· trick et a1.7 . The neurons of the perigeniculate ou· deus are known to be GABAergic and thcy project

A

Fig. 2. Camera lucida drawings showing ctose proximity and size ditrerencc between t eils in the nucleus of the optic tract projetling 10 the inferior olive (black) and GABAergie somata (openoutlines) . A: nt. B: eal. Bars - lOO.um.

to Ihc hileral geniculaIe body. Following injections of HRP inlO the lateral genicu late body of a cat, we were ablc to demonstrate GAßA·immunoreactivity in rctrogradely labeled cclls of the perigeniculate nu· deus (Fig. JA). To provc the second point, we exam. incd the large cells in thc intermediate layers of the superior colliculus which were relrogradc1y filled with HRP after 10 injections. These neurons receive a substantial input from the substantia nigra pars reti· culaiaul . Lu et al. 17 have demonstrated Ihat these terminations Are glutamate decarboxylase (GAD) im· munoreactive. Fig. IB shows a rel rogradely labeled neuron in the intermediate layer of the cat's superior colliculus out lined by numerous GABA·immuno­reactive tcrminals. These Icchnical conlrols vcrify the validity of our observations concerning cells in the prelectum. tn our material, in ral , cat and mon· key the distribution or GABA·immunoreac!ivc neu· ronal somata and terminals in the vicinity of retro· gradc1y labeled preteetal cells resemblcs that

observed In prevlous studles usmg antibodlcS to GABA21 or to the syntheslzmg enzyme glutamate de­carboxylase (G AD)9 19 J~ (Flg ID-F) In no case dld we fmd any NOT or DTN neuron labeled for both the prescnce of Iransp0rlcd HRP and GABA-lmmuno­reactlvlly Measu rements 01 ce ll body areas In thc NOT rcvcaled tha! m rat as weil as cat, GAßA-posl­live neurons are ~lgOl flcan!ly srnaller than retro­gradcly labeled cells In the same SCC! lon (V-lest . P < 00]) Thc Ime drawlngs shown 10 Fig 2 Illustrate the relative slze and pO~ ltlon~ of the GA ßAergic mtrm­~ IC ,md non-GAßAcrglc prOJecl1on neurons m the NOT 01 ral and cat SlIllllar relauons were fo und In the mon key By count lng ever)' GABA-lm muno­redctlve :md NIssl-stamed neuron In sampies taken trom the NOT. wc e~tlm ,l ted thc re lative proporllo n of GAßA-posltlve neurons In IhlS nucleus In bolh r,11 .lnd ca l, GABA-posll lve neurons made up about 30q· of Ihe IOIal neu ronal popu lallon Iß the NOT We hdvt' aha eXdm llled thc relatIve proportion of GABA-Immunorcacllve neurons In the superflClal Idyer (SGS) 01 the supe nor colhculu~. a ~ t ruc ture Ihat also recelve~ .md procc<;\c<; vlsual mform.1I10n fro m Ihe rellll.l ,:IOd \' I ~ua l cortex Thc propOri lon 01

GABA-p0<;lllve neurons In the SGS of the cal (47t'i)

IS ~ l gOlflc,lßtly hlgher Ihan 111 the correspondmg area 01 the ral (3OCi )

U.,mg the double-label method. It wa~ posslble 10

demomtrdtc In <;ome casc<; rctrogr.adely 1,lbeled ncu­rons wll h thclr somata .md/or proximal dendntes do~cI}' a~soe 1<l ted wll h GA BA-posl\lve pu nela rc­~em [1lmg pre~umed nerve lermmals (FIg IC- F) The~c proJectlon neurons . howcvcr. were contacled b} ont}' ,I few GAßA-p0<;lll\C te rm mals. suggcstmg thm GABAergic mtcractlOnS .I re weak or re~t Tlcted

to the dl ... tal pMt of Ihe dendnles Smcc Ihe dppca r-

I Balla .. I Hoflmann K -p ~nd Wagner, ,I J Rellnal pro· J<!won 10 Ihe nuc\eu, 01 Ihe OpllC tracI In Ilte eat a~ re\'eatcd b) rClrogr,\de tt.m~porl of Ito~er"dl~h perO~lda~ Nl'u,u, l(/ L<,II ~tl(I'lRI) 1')7- ::!IC

2 B .. IIJS J and BuHmann K -P A corrclJIIOO bCllleen rc· eepll»e f,eld properlles Jod morphoJoglcJI Sl ruclurcs In Ihe prCleclum 01 Ilte cal J Comp Nl'urol 23R (19RS) 41 7-421-1

1 l3ermJIl '\ L TI,.. 8ralll~I<'I" O{llrt (m. "fhc \lol\cr\ll~ uf WI:>con'ln pte~, J%R

4 (dll" L PReh .. W dnd Lmnou J I'alhway~ mcd13llog oplOt..IIlCIIC ti.·SPOIl":~ of ,e~l l h ul .lr neurons 10 the t.,1 I'{lll~er' 4.1(.11 J K4 (1')80) 1'1- 1')

anee 01 stalOed GABA-poSll1ve te rm mals I:' re­st neted 10 the ~urface 01 the scellon and therefore the exammallon , 100. Tl O attc mpl has been made 10 sys­Icmallcall y qUdnllfy the num be r of GABA-pmlllve term mals assoclated wl!h NOT ne uron~ proJect lOg 10

the 10 These rcsulls were crHlcally dcpendenr upon the

10rmuldt l01l of thc new Iwo-step procedure for stabl­hzmg thc TMB- HRP reactlon product Smce thc TMB-reacllOn prod uct IS normally unsldble Iß neu­Iral butfers. subsequent Immunocytochcmlcal ~tam­

mg usua lly Cduses a marked 10'>5 of rcaClIon product Thls dltflculty w .. s overcome uSll1g the ~ tablhza tl OIl

prolocot of Rye CI .. I .!5 . comblOlIlg the ~cnslll vlly 01

TMB wlt h the s.tabll ll} of the DAß-rCdctl on pmduct Furthcrmore. wc tound that the preced mg mcuba-1I0n ~ tep wlth 5C,( AHM~ ~II prevcnted the los<; ot TMB-redellon produci prevlously ob~en'eJ dunng the short DAß rcact lon

Dur resulls spcclflca lly dl~prove thc hypothe~ I ~I~

Ihat thc NüT· DTN cclls <;end a GA ßAergic proJec­tlOn to thc 10 Smlll,lT double label lOg ~Iud les wllh IIRP mJccllon ... 1010 Ihe MTN or cont r"la leral NOT ~h ould bc pertormcd to pOint out whelher the reclp­rocdl connectlOm bctween the NOT :111<1 olher prc­tcctal ,md dccc<,~ory optlC nudel <irc GABAergic Such mhlbllory mlcraCllom are d<;'umed to contn b­ute to the dlSt lllCt dlrc('tlon ,e lectlvll\ 01 ncuronal populations m the~c nuckl

Thl <; work wa5 ~upported by Grdnts DFG Ho 450117 and For~ch ung~schwerpun kt 24 01 Ihe ~ta tc uf ß ddc n-Wucrnemberg We Ihank the membcr~ ol lhc Departme nt of CllOlcal Morphology lor theJT J...md tcchmcal help. Dr R Enckson tor cntlcally readlOg the O1a nu~eT1 pt and C DIst ier fo r typmg 1\

5 Chcva!JeT Ci Th,ert\, A M Sh,b.t:l:l,t..1 T Jnd FeJ!er J E\lueoec fOT d GABAergic mhlbuory nrgrotcelal p;lIhIlJ\ 'nlhcrJI Vellrp" I LI'II 21(191l1Jn7- 70

tl CUI/CIII}11 1-1 DlreCIIOI1-sc\ew\'c uni" 111 llte r~hhu ~ nu­cleu~ uf Ihe optle Irdel Bram R~\"lmh IUO 11'175) 4l'<)-50~

7 FjlZpdlnü, 0 Pel1l1':~ G R JO(] ~chmcchcJ D E Glu· IdllllC .Icld dccarnoxvld":-umnul1orc.lct,,c I1CurOI1~ 311d ler. IIllllal~ 10 Ihe 1.11I,·ral geoltuldle Il ucleus "r Ihe c.tl J Nellro' IU . '" (I'JR4) IRI)'} - 1l'29

R FUJII M .lOd KU~dOld T. FI\,1I100 01 Itl)t,<:tddl~h PCTO~ ­Iddse react,un products \\ rlh ,\mmOOlliOl molvhdale "'euro­fli Re) 1(I<.)H4)19- I'\n

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IS h oh. K . KOOlSh .. A ,Nomura, S . Mlzuno, N , Nakamura. Y and Suglmoto. T Appllcallon of coupled OXidation reactlon to electron mlcroscaplc demonstration of horse­radlsh perQJudase cobalt -glucose OXIdase method Bram Research , 175 (1979) 341-346

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25 Rye. D B ,Sapcr C B and Wamel B li , Stdblhzmg of the le tramethylbenztdille (TMB) teaC I1OI1 producl apphed-1I0n for ret rograde and ante rogr"de traelOg and comblna· tlon w/th ImmunohlSlochernlstrv.] Hlwx:hem l ytIXhem, 32 (1984) 1145- 1153

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