niarB hcroe$cR ,nitelluB .loV ,31 pp. .MBC87 %51 o ohknA Intematbnal Inc. Printed in the .A.S.U 48wm!~1630 00.3$ + .oo Proopiomelanocortin Peptide Immunocytochemistry in Rhesus Monkey Brain HENRY KHACHATURIAN, MICHAEL E. LEWIS, SUZANNE N. HABER,* QUDA AKIL AND STANLEY J. WATSON Mental htlaeH Research Institute, University fo ,nagihciM nnA Arbor, MI 48109 *Department fo Anatomy, University fo Rochester, Rochester, NY 14442 Received 14 rebmeceD 1984 ,NURUTAHCAHK ,.H .fh E. LEWIS, S. N. HABER, H. AKIL DNA .S J. .NOSTAW Proopiomclanocorrin peptide immunocytochemistry in susehr monkey brain. BRAIN RES BULL 13(6) 78MW, l!%t.-The lacimehcotyctcnummi noitubirtsid of proopiomeIanocortin (POMC) seditpep ,nihprodne-@ ACTS, ,HSM-a 16K )tnemgarf saw deiduts ni the niarb of the rhesus yeknom (Muccrco .)ottalum Some slamina erew deretsinimda enicihkoc mbmecmtni yitkucirtnev prior to sacri6ce to ecnahne the noitaxilausiv of layrahirep .ytivitcaeronummi evitcaetonummI eyrakitep are dexhack to -opyb kmalaht rkuiidnufni ,eukcun givinR rise to several tcnitsid projections. Rosrraf projections dnetxe thf0@ midline cbshpecneid dna preoptic ,saera dna enter the .nolahpecnket ~ttolA siht course, evitrerwummi mebIf are seen ni cnhdim kmkah&pyh dna ktpoerp ,i&ua sukcun of the lanogaid ,dnab olfactory ,kcrebut sukcun ,rnebmucca deb sukcun of airts ,silanimret ,mutpes dna other cibmil structures ni .nolahpecnelct laduaC to the anterior commissure, enas nebIf dnecsa yllasrod to enter the enildim ,sumalaht hcihw yeht .etavrenni laretaL projections of the iiimufni ayrakirep course through the lasab-laidem ,sumalahtopyh dorsal to the optic tracts, dna enter the k&yma no&m erehw yeht innervate more yllaidem detautis dioladgyma .ielcun lasuaC projections of the POMC neurons also dnetxe @mht enildim ,nolahpecnkd some coursing gnola a perivcntricular htap to innervate enhdim cim&htopyh dna cimkaht .ielcun sihT projection sdnetxe into the cilahpecnesem aitnatsbus @ea centralis dna yam also contribute to the noitavrenni of more yllastod detautis ikcun ni the ports dna .alludem hcus sa the laihcarbmap ikcun dna su&m tractus solitarhrs. Other laduac projections originating ni the sumkahtopyh course through the ventral mutnemget of nolahpecnesem dna posts dna yam contribute to the noitavrenni of enildim ehpar dna other ventrrdly detautis ikcun ni the snap dna .kludem ebT noitubirtsid of evitcaeronummi ayrahitep dna nebIf ni the niarb of rhesus yeknom si y-s rkimis to taht dnuof ni the tar .niarb However, eltbus differences raeppa to tsixe ni the inoervation snrettap of ralucitrap niarb regions. Pmopiomelanocortin &Endoqhin ACTH 16K fragment Rhesus yeknom niarB yrtsimehcotyconummI PROOPIOMELANOCORTIN (POMC) dna sti evitavired devired seditpep nihtiw the emas etaucra )ralubidtutfni( seditpep evah yltnecer neeb the tcejbus of hcum evisnetni snoruen [S, 37, 42, 491. nI rat, the etaucra FOMC snoruen noitagitsevni (see [3, 4, 23, 34, 481). ehT tseretni ni CMOF tcejorp ot ynam ,klahpecnelet ,cilahpecneid dna metaniarb sevired morf the fact that ti sniatnoc lareves nwonk evitcaoib ;setis these snoitcejorp evah neeb deppam’ylevisnetxe nj the seditpep ,.e.i( ,nihprodnc-lf ACTH, a-M%), that ti si tnedor lartnec suovren metsys [6,7,16, 19,23,32,49,51,52, decudorp ni htob yratiutip dna niarb ecneh( the laitnetop for 551. emoS yranimilerp snoitavresbo evah osla neeb edam ni htob ekil-enomroh dna ekil-rettimsnart snoitca of sti -dorp the liatgip ,yeknom acacuM anirrsemen .]l[ ehT rettal -ni ,)stcu dna osla that sti dne stcudorp evah elbissop -goloisyhp srotagitsev evah debircsed HTCA ytivitcaeronummi ni -ni laci selor ni the noitaludom of ,niap stress, ,enircodneoruen iidnuf ayrakitcp dna ni deta coslrebif ni the citpoerp ,ralucsavoidrac ,yrotaripser dna lareves other cimonotua area, ,sumalahtopyh ralucibnevarap ,rumthaht aitnatsbus processes [23]. aesirg silartnec latcudeuqairep( ,)yarg dna enildim ralukter tsoM of ruo tnerruc egdelwonk of CMOP dna sti -ssecorp .noitamrof gni otni evitcaoib seditpep sevired morf work no the yratiutip tsoM of ruo egdelwonk of the ymotana of CMOF smetsys dnalg [ 14, 17.541. ehT gnissecorp of CMOP ni niarb snoruen sevired morf work no the rat lartnec suovren metsys [3]. si ssel llew ,dootsrednu tub appears ot eb ralimissid ot that ,revewoH there are nwonk seicepsretni sewereffid ni the -nec gnirrucco ni the yratiutip (see [So]). snorueN that ezisehtnys lart suovren metsys noitubirtsid of rettimsnattoruen -bus POMC are detacol ni the etaucra suelcun of rat -alahtopyh secnats [18, 40, 431. nA elpmaxe hcihw si tsom tnenitrep ot .sum sihT suelcun si the tnelaviuqe of the etamirp -ubidnufni the tneserp yduts si the work yb rebaH dna edlE [ 181 no the ral .suelcun Immunocytochemica seidutsl ni htob rat dna noitubirtsid of nilahpekne-teM ytivitcaeronummi ni the niarb namuh evah detartsnomed the noitazilacoloc of -CMQF of the nacirfA neerg yeknom (Cercopithocus ,)spoihtea ni 587 NAIRJITAHCAHK 1:“/ ./A. LIST OF ABBREVIATIONS 3 rotomolllco .n me laidem etalucineg .n v3 driht elcirtnev flm laidem lanidutignol sulucicsaf v4 htruof elcirtnev frm cilahpecnesem raluciter noitamrof aa roiretna aladgyma sm laidem mutpes ba lasab .n fo aladgyma ntm rotom lanimegirt .n ca roiretna erussinunoc vm lartnev-laidem .n fo sumalaht bca .n snebmucca scn .n silartnec roirepus )ehpar( eca lartnec .n fo aladgyma ucn .n sutaenuc eca lacitroc .n fo aladgyma bdn .n fo lanogaid dnab aha roiretna cimalahtopyh area rgn .n silicarg al laretal .n fo aladgyma mrn .n siraluciter siralullecongam ma laidem .n fo aladgyma prn .n siraluciter siralullecovrap bma .n suugibma stn .n sutcart suiratilos qa larberec tcudeuqa co citpo msaihc roiretna .n fo sumalaht no rotomoluco evren 2 muihcarb muvitcnujqoc 01 citpo tcart pb muihcarb sitnop uto yrotcaflo elcrebut tsb deb .n fo airts silanimret nbp laihcarbarap .n cau etaduac CP roiretsop erussimmoc lbc mulleberec fP ralucicsafarap .n fo sumalaht cc suproc musollac ahp roiretsop cimalahtopyh area ec lartnec lanac rip mrofirip xetroc ic lanretni eluspac IP laretal-roiretsop .n fo sunukaht lC murtsualc np snap mc murtnec munaidem .n fo sumalaht aep citpoerp area larberec elcnudep atp latceterp area CZ noitassuced fo nru&arb muvitcnujqoc luP ranivlup gd etatned suryg tuP nematup ahd lasrod cimalahtopyh area vP ralucirtnevirep .n fo sumalaht md dridemosrod .n fo sumalahtopyh nvP ralucirtnevarap .n ypd noitassuced fo ladimaryp tcart ryp ladimaryp tcart sd lasrod mutpes dr ehpar silasrod nf laicaf .n er sneinuer .n fo sumalaht xf ximof lm ehpar sungam Pg subolg sudillap nr der .n lh laretal alunebah tr raluciter .n fo sumalaht mh laidem alunebah CS roirepus sulucilloc cph supmacoppih cgs aitnatsbus aesirg silartnec ci roirefni sulucilloc is aitnatsbus atanimonni fni ralubidnufni .n fo sumalahtopyh ns aitnatsbus argin oi roirefni yravilo .n nos citpoarpus .n IC sucol suelureoc tS cimalahtbus .n ngl laretal etalucineg .n nts lanips lanimegirt .n ahI laretal cimalahtopyh area av roiretna-lartnev .n fo sumalaht mI laretal raluciter .n laretal-lartnev .n fo sumalaht VI sI laretal mutpes mv laidemortnev .n fo sumalahtopyh IV laretal elcirtnev lpv laretaloretsop-lartnev .n fo sumalaht am yrallimmam .n mpv laidemoretsop-lartnev .n fo sumalaht dm lasrod-laidem .n fo sumalaht tsv ralubitsev .n em naidem ecnenime atv lartnev latnemget area tem cilahpecnesem lanimegirt .n iz anoz atrecni hcihw lareves dekram secnereffid erew deton neewteb -nom lamina was dezitehtsena htiw muidos latibrabotnep (30 yek dna rat. nI the tneserp ,yduts we ebircsed the CMOP )gk/gm detcejni otni the suonehpas .niev ecnO yletelpmoc metsys of the susehr yeknom niarb gnisu aresitna desiar ,dezitehtsena the lamina was decalp no a lacigrus ,elbat na tsniaga ,HTCA ,nihprodne-@ ,HSM-a dna K61 ,tnemgarf laehcartodne ebut was detresni dna attached ot a ,rotaripser dna erapmoc these sgnidmf ot those deniatbo gnisu the emas dna the chest ytivac was exposed. ehT muidracirep was -xe aresitna ni rat. desic dna the apex of the selcirtnev was tuc .nepo A citsalp ebut detcennoc( ot the noisufrep )sutarappa was detresni hguorht the tfel elcirtnev otni the aorta dna was deit ni ecalp METHOD htiw a .erutus retfA gnittuc the thgir ,muirta the -ucsavoidrac ral metsys was dehsulf htiw 2 sretil of lamron enilas hguorht tludA elam dna elamef susehr syeknom (Macaca mulatto) the detresni .ebut gniwolloF the noitelpmoc of the enilas were deniatbo for the tneserp .yduts ehT syeknom were ,hsulf 16 sretil of dereffub-lartuen 4% edyhedlamrof were rehtie kept rednu lamron snoitidnoc )eno( or were treated depmup hguorht the erutalucsav for 30 .setunim ehT ebut htiw enicihcloc ,ruof( see .)woleb roirP ot ,ecifircas each was neht devomer dna the head was dereves morf the knurt 787 POMC PEPTIDES IN RHESUS MONKEY BRAIN FIG. 1. (Part 1) FIG. 1. (part 2) FIG. 1 parts 3-9 no gniwollof pages. FIG. 1. latnorF salta of eht susehr yeknom niarb hguurht 21 .slevel siidrooC are ni ecnerefer to eht laruaretni .cthl ~mtcurtS WitMdi@ra no eht ,tfel elihw POMC srebit trohs( devruc senil dna dots) dna ayrakirep ni( eht ralub~dnufni )suelcun are nwohs no eht thgir .edis See fptsil .snoitaiverbba and placed in a stereotaxic apparatus. The skull was ex- .erut latnorF dna parat&M snrktces were used in order to posed, the top of the calvarium removed, and the brain was cut into 1 cm thick slices in the frontal or paras&ttal planes using a Kopf stereotaxic device with blades attached. The skull was then removed from the stereotaxic apparatus and for 10 minutes, f&lowed by primary‘rabbit At&rum raised the brain slices were removed and postfixed in buffered 4% against ACTH, a-MSH, /3-endorphin, or 16K finSme@. The formaldehyde for 2-4 hours. The tissue was transferred into antisera were diluted with 0.62 M PBS containing 0.3% T& 15% sucrose in phosphate-b&&red saline (PBS) and stored ton X-100. Incubation was continued for an additional hour overnight at 4°C. The tissue was then &oxen in -WC after which the slides were tgu&red onto wet foam rubber isopentane or liquid nitrogen, and stored at -80°C. in a moist box and stored at 4% ovmn&ht. The next day, the For peroxidase-antiperoxidase immunocytochemistry tissue was washed thorou$@ in PBS for 30 minutes with [24], the tissue was sectioned at -20°C in a Bright cryostat several sohrtion chaqges. The slides wem a@n in and the sections were mounted onto subbed slides and were 37°C with NOS for 10 minutes, followed by @.mt either stored at -80°C or allowed to dry at room tempera- serum (Amei) at l/100 dilution for an additional 30 min&s. KHACHA’TURIAN t 7’ iii i, FIG. 1. (Part 3) .GIF .1 (Part 4) yehT erew neht derrefsnart otni eht tsiom xob and derots )ralubutoruen ,noitca enicihcloc noitartsi~da stluser ni a thginrevo at 4°C. On eht driht ,yad eht eussit was washed ni ssol fo tropsnart fo dezisehtnys slairetam ,snietorp( )seditpep SBP rof 30 ,setunim detabucni at 37°C htiw SGN rof 10 -nim ot lanoxa ,slanimret and an noita~mu~ca fo this lairetam ni ,setu and yltneuqesbus htiw tibbar hsi~resroh-it~ -ixorep eht lanoruen ayrakirep and lacnurt .snoxa esad )PRH( mures at 002/l noitulid rof 40 .setunim ehT sedils roF enicihcloc ,noitartsinimda eht animal was erew neht washed rof 30 setunim ni SBP and detabucni -id dezitehtsena htiw muidos latibrabotnep detcejni otni eht yltcer htiw 4 lm/g@ PRH emyzne ,amgiS( epyt )IV at 37°C rof suonehpas ,niev and eht daeh was decalp ni a cixatoerets 40 .setunim retfA noitelpmoc fo this pets and SBP wash, eht apparatus. ehT skull was desopxe and a small eloh was sedils erew desremmi ni a noitulos fo 0.125 l&n -id dellird otni eht skuh at a cixatoerets etanidrooc gmytrevo eht en~~n~o~ ed~o~co~y~tet )amgiS( and %30.0 ,O,H tsediw noiger fo eht thgir laretal elcirtnev 0.8( mm ,roiretna rof 15 setunim htiw tnatsnoc .gnirrits ehT eussit was neht 4.0 mm ,iaretal htiw ecnerefer ot .)amgerb A alunnac -ta( washed rof 30 setunim ni dellitsid ,retaw ylfeirb detacimso dehcat ot a detarbilac-emulov citsalp )ebut was decudortni htiw %10.0 OsO,, detardyhed hguorht dedarg slohocla and hguorht eht eloh and detresni otni eht elcirtnev 6.91( mm ,senelyx and detnuom ni .tnuomreP snoitavresbO and .)peed ehT lufsseccus noitresni otni eht ralucirtnev nemul sh~or~~otohp erew edam using a ztieL nalpohtrO was degduj yb a neddus pord ni eht nmuloc fo diulf -loc( .epocsorcim enicihc )noitulos ni eht citsalp ebut hcihw was dehcatta -rev yllacit evoba eht .alunnac enicihcloC ,57( 150, 300, 600 pg rep kg ydob )thgiew was thus dewolla ot esuffid otni eht Colckicine Treatment lanipsorberec .diulf erusserP was dewolla ot hcaer an -iuqe oT ecnahne eht noitazilausiv fo editpep ytivitcaeronummi ,muirbil retfa hcihw eht alunnac was ,deveirter eht eloh was ni lanoruen ,ayrakirep ruof animals erew detaert htiw -loc dekcolb htiw enob xaw and eht placs was .derutus ehT enicihc roirp ot eclhrcas .]U[ eniciheloC is a ,nixotoruen eno animal was dewolla ot revocer morf eht aisehtsena and was noitca fo hcihw is eht noitibihni fo elubutorcim noitamrof tpek rednu lamron snoitidnoc rof 24 ot 48 sruoh roirp ot .]33[ ecniS lanoxa tropsnart seriuqer ~~but~cinr ro( ecifircas and noitaif as debircsed .evoba POMC PEPTIDES IN RHESUS MONKEY BRAIN 789 FIG. 1. (Part 5) FIG. 1. @traP( RESULTS stainiug was noted with successively higher doses of col- chicine. Neuronal perikarya cove to POMC pep- The aresitna desu ni the present study have been charac- tides~l~~~eh~~ * f IiUCkUS terized extensively (see [50]). All immuuoreactivity seen tbrom its rost&ca&l extent, as well as scattered in usiq each antiserum was blockable by the preabsotptiou of theme&al-basalhypothalamas(Fa. 1: +14.5to+ll.%Figs. the Primary antibody by 1 gM contention of peptide 2,3 and 8). These parvocellular perikarya ag@ear to be mul- agaiust which the serum was raised. Extensive cross- tipolar, exhibiting numerous processes (Fig. 3C,D). Peri- blocking studies also confirmed the speci6city of each karyal and fiber immunomtivity was best demoru+trated by antiserum. Peptides with amino acid sequences homol~us ACTH and 16K antisera, with decreasiq ir&ensity of stain- to the peptide antigen did not block the immunoreactivity ~~~~~~~~~~~~~. when added to the primary antiserwn iu up to 50 phi concen- mmreacGve POMC pqde-co 5bersarelo- tration. The specificity ofthe ACTH a&serum is toward the calzto many brain regiom (Fm. 1 aad +-7), inctm the midregion of ACTH, i.e., ACTH(ll-24). The immuaoreac- dien&&naswellastheli&icf~aadthebrain raeppa dioved tivity observed using this ant&ram could be blocked by the stem (see F@. 8 for summary). Areas which of preabsorption of the pzimuy aetibody by 1 FM concentra- immanarsgctve Bbers include the cerebml cortex, hip- tions of ACI?H(l-24), ACTH{l-39), ACTH(l l-24) and ~,~tof~,~~~*~~~ ACI’H(ll-39), but not by up to SO a conceatrations of ~~~~~~~Of~~,~~- &lipo&opin, ,&eado@n or a_MSH. &Endoxphin im- dibul5rPOMCper&uyaofthemonkeybrainappeartocon- munorea&vity could not be blocked by Met- or Leu- tribute to several m&w projectional systems. enkephalin, ACI’H, or wMSH. The a-MSH immuaoreac- 1. larrsoR projections of the posvlc &a caa be tivity was not blocked by ACTH( l-24), ACTH(l-39), or traced tbpolllh much of the anterior hypotbplsmic and @endorphin. preoptic areas (Fig. 1: +~l~~~* ?D,.5A and 7), No perikaqal ~~~ti~ty couki be detected in the ~h~~~ . p~sjactto~ extend normal animal; however, increasiugly intense pexikaryal into the tele*phalon, innervating several m&r limbic- .GIF .I traP( 7) .GIF .I I traP 81 detaicossa .ielcun emoS srebif eunitnoc ni a lartsor noitcerid pears ot retne the nolahpecnesem raen the roiretsop -moc ot retne suelcun snebmucca dna suelcun of the lanogaid dnab ,erussim dna suht yam etubirtnoc ot the noitavrenni of the .giF( :1 5.12+ ot ;5.71+ .giF 4B). morF the l+nogaid ,dnab aitnatsbus aesirg silartnec .giF( :1 5.7+ ot ;5.0- .giF A6 dna scattered srebif are nees ot retne detaicossa-yrotcaflo areas. B) dna other enildim metsniarb .snoiger esehT snoitcejorp esehT srebif are yllareneg sparse, dna are yllanoisacco nees are rehtruf debircsed .woleb ni the yrotcaflo elcrebut .giF( :1 + 19.5). Other srebif ni the nI the citpoerp area dna the ,sumalahtopyh a lareneg lanogaid dnab wollof a lasrod esruoc dna appear ot retne the laretal-ot-laidem tneidarg of evitcaeronummi rebif ytisned si latpes ielcun .giF( :1 5.91+ ot .)5.61+ Scattered srebif are .nees ehT erom enildim ,serutcurts hcus as the laidem osla nees ni the head of the etaduac dna nematup at erom ,citpoerp roiretna cimalahtopyh dna ~lubidnufni ,ielcun -xe lartnev dna laidem aspects .giF( :1 5.52+ ot .)S-71+ tibih a ylevitaler esned noitalumucca of srebif .sgiF( 4D, A5 A tcnitsid eldnub of evitcaeronummi srebif stnevmucric dna 7), whereas the laretal citpoerp dna laretal cimalahtopyh the roiretna ,erussimmoc ylniam morf sti lartsor aspect, dna areas appear ot eb detavrenni ylthgil .giF( .)BS Other -opyh sretne otni the mutpes .giF( 8). Other srebif etavrenni the cimalaht ielcun hcihw appear ot eb ylesned detavrenni -ni deb suelcun of the roiretna erussimmoc .giF( 4C). -mI edulc ralucirtnevirep dna ralucirtnevarap ielcun dn& the -rod evit~eronum srebif hcihw esruoc laduac ot the roiretna laidemos suelcun .sgiF( C5 dna 7). Scatter&d -caeronummi erussimmoc etubirtnoc ot the noitavrenni of the deb suelcun evit srebif are osla nees ni the citpoarpus suelcun as lfew as of airts silanimret .giF( :1 5.61+ ot .)5.4i+ More ,ylladuac ni the laidemortnev suelcun .sgiF( CS dna 7). nI ,noitidda srebif are nees ot retne the ralucirtnevirep cimalaht suelcun there appears ot eb a yvaeh ytisned of srebif ni the lanretxe hcihw stibihxe ytivitcaeronummi tuohguorht sti -lartsor reyal of the naidem ecnenime .giF( 7). laduac tnetxe .giF( :1 5.21+ ot ;5.3+ .giF 5D). ,eroferehT 2. Lateraf snoitcejorp morf the ralubidnufni CMOP siht tnenopmoc of the lartsor noitcejorp metsys semoceb a ayrakirep nac eb nees tuohguorht the eritne lasab-laidem ylladuac gnitcejorp eldnub nihtiw the ~~c~nev~~ suelcun .su~ht~yh ehT ytisned of these processes sehsinim~ of the .sumalaht A noitaunitnoc of these enildim srebif -pa yllaudarg ni a laretal-ot-laidem tneidarg .giF( :I + 15.5 ot POMC SEDITPEP NI SUSEHR YEKNOM NIARB 791 eht noitavrenni fo eht subset aesirg silartnec .giF( 6A and .)B rehtO srebif ’tcejorp yllaretal and ylladuac hguorht eht lartnev latnemget ,aera gnisruoc lasrod ot eht substantia mgin ot retne eht strop .giF( :1 +9.5 ot +3.5). derettacS srebif era nees ot etartenep eht substantia argin pars -moc .atcap rehtO cilahpecnesem saera hcihw tibihxe rebif -mi ytivitcaeronum edulcni eht roirepus and roirefni ilucilloc ..giF( :1 ,)5.0- as llew as a tcnitsid band fo srebif gnidnetxe yllaretal morf eht substantia aesirg silartnec otni eht cilahpecnesem raluciter noitamrof .giF( :1 + .)5.1 esehT -rod yllaretalos gnitcejorp srebif era denioj yb eht laretalortnev snoitcejorp at eht level fo eht muihcarb ~~~ti~rn, and etubirtnoc ot eht noitavrenni fo eht laihcarbarap suelcun .giF( :1 ;5.2- .giF .)!C6 nI eht snop and ,alludem rebif -mi ytivitcaeronum is erom sparse, hcus that on tcnitsid -cejorp snoit nac eb denrecsid ees( .iiF .)8 ,revewoH srebif era nees ni such diverse braiustem nuclei as the raphe dorsalis and ehpar magnus, all yrosnes and rotom lanimegirt ,ielcun -un ielc siraluciter siratullecovrap and ,siralullecongnm suelcun ambiguus, suelcun sutcart ,suiratilos and laretal raluciter suelcun giF( :1 +1.5 ot ;5.8- .giF .)D6 NOISSUCSID ehT tneserp yduts se~snomed POMC editpep -mi ytivit~e~~m ot eb detuiirtsid ylediw tuolf eht susehr yeknom lartnec suovren ,metsys gnidulcni -id ,cilahpecne cibmil c~p~nelet and enildim metsniarb -er .snoig sihT ytivitcaeronummi stneserper ,nihptodneB ,HTCA HSM-a and K61 tnemgarf ,noitubirtsid tnetsisnoc htiw evisnetxe seiduts hcihw evah yllacoviuqenu -nomed detarts eht sisehtnys-oc fo eseht seditpep morf a elgnis -erp ,rosruc POMC 130, 31, 391 as llew as rieht noitazilacoloc nihtiw eht emas etaucra )ralubidnufnif aytakirep ni htob eht tar and eht human ,5[ 32, 42, 46, 47, 491. oT ,etad eno -mi lacimehcotyconum yduts has neeb deirrac tuo ni eht yeknom niarb gnitartsnomed eht noitubirtsid fo HTCA sllec and sessecorp .]l[ esehT srohtua evah osla debircsed -mi .GIF 1. traP( )9 evit~aeronum ayrakirep ni eht ~~~b~ suelcun fo eht ,sumalahtopyh and evah yllareneg deton rebif -caeronummi ytivit gnidnuorrus eht roiretna erussimmoc ,yllartnev -poerp cit ,aera suelcun ,snebmucca laduac ,ralucirtnevarap + .)5.11 A tcnitsid band fo evitcaeronummi srebif is nees ot ,ralucirtnevirep ,laidemosrod ,ralubidnufni roiretsop and -tal esruoc yllaretal revo eht citpo stcart and otni eht .aladgyma lare cimalahtopyh ielcun and ,snoiger ralucirtnevirep -un At eht level fo eht roiretna cimalahtopyh ,aera emos fo eseht suelc fo eht thalamus, substantia aesirg silartnec -neuqairep( srebif esruoc lasrod ot eht citp~pus ,suelcun ne et&ur ot eht latcud ,)yarg and .aladgyma ,ero~e~uF Abrams te al. ]l[ aladgyma .giF( :1 + .)5.41 esehT srebif spahrep etubirtnoc ot did ton evresbo HTCA rebif ytivitcaeronummi ni eht -erec eht noitavrenni fo eht citpoarpus .suelcun eroM ,ylladuac at larb ,xetroc ,supmacoppih substantia n&pa, ,mulleberec ro eht level fo eht laidemortnev ,suelcun eht yllaretal gnitcejorp .mutairts roF eht tsom ,trap eht stluser fo eht tneserp yduts srebif era dehciwdnas neewteb eht citpo tcart yllartnev and era ni doog tnemeerga htiw eht .rettal eW evah rehtruf -ed eht subolg pallidus yllasrod ,giF( :1 f .)5.31 nI ,aladgyma eht debircs lanoitidda rebif ytivitcaeronummi ni ynam -ecnelet evit~~onummi srebit era nees ot etavrenni eht erom laidem ,cilahp cilahpecneid and metsniarb .saera rehtO -mi ,ielcun yllacificeps eht laidem and lartnec ielcun .giF( :1 ~c~ehcoty~num ,seiduts ylralucitrap ni eht human ,niarb f 17.5 ot f ;5.21 .giF .)A4 derettacS srebif era osla nees ni evah tlaed htiw eht noitazilacol fo POMC editpep -mi eht lacitroc ,suelcun tub eht basal aud laretal ielcun raeppa ot ytivitcaeronum ylno ni eht h~~~~us .]83,8,5[ suhT eht eb dioved fo rebif .ytivitcaeronummi rehtonA elbaborp tneserp yduts sedivorp eht tsril lluf evitpircsed tnuocca fo ecruos fo dioladgyma srebif yam eb eht stria ,silanimret eht lacimotana noitubirtsid fo devired-CMOP seditpep ni a hcihw stibihxe evitcaeronummi .srebif etamirp .niarb 3. Cuudal projections of eht POMC snoruen dnetxe ehT noitub~tsid fo editpep-!CMOP ytivit~aero~mmi ni hguorht eht roiretsop and lasrod cimalahtopyh ,saera and lanoruen serutcurts fo susehr yeknom niarb is ralimis ni eunitnoc ylladuac ot retne eht nolahpecnesem .iiF( :1 + 11.5 ynam stcepser ot that nees ni tar niarb ,7-5[ 16, 23, 49, 51, ot +7.5). sihT noitcejorp setacrufib otni owt tcnitsid -nub 52,551. ehT rojam syawhtap ni tar osla raeppa ot eb tneserp .seld emoS srebif eunitnoc ni a erom laduacosrod noitcerid ni yeknom .niarb nI htob ,seiceps a tnenimorp yllartsor -orp and nioj eht ralucirtnevirep cimalaht srebif ni eht -ecnesem gnitcej eldnub sesruoc hguorht enildim cilahpecneid and -let nolahp .giF( :1 +5.5, +3.5; .giF .)8 esehT srebif etubirtnoc ot cilahpecne ,serutcurts gnitavrenni suoirav ,cimalahtopyh .GIF 2. A si a egatnomotohp fo eht susehr yeknom ralubidnufni suelcun gniwohs eht esned noitalumucca fo 16K evitcaeronummi ayrakirep ni a 23 nr& kciht .noitces nI ,B nihprodne-p evitcaeronummi ayrakirep era nees ni a noitces 23 mp laduac ot .A etoN taht elihw eseht owt seitivitcaeronummi era detubirtsid yrev ,ylralimis eht nihprodne-@ gniniats si ssel .decnuonorp A noiger fo lenap A deniltuo yb eht xob si deifingam ni .C Bar S.O=)B,A( .mm Bar (C)= 100 .mp POMC PEPTIDES IN RHESUS MONKEY BRAIN FIG. 3. A and B are adjacent 32 Frn thick horizontal sections bugh the infundi&lar nuckus showing the identical’disWwtio~ of 16K (A) and ACTH (8) cove perikaqa Note that both antisera stain the PGMC neurons with similar intensity. In C and D, numerous ACTH-containing perikarya and beaded varicosities are magnified. Bar (A,B)=ZOOp m. Bar (C,D)=25 ,&i. tunoreacbve tibers era shown in darkfield photomicrograpns rnrougn rour sueA accumhens .)B( bed nucleus of the anterior commissure (0. and the mc