- Email: [email protected]
A direct projection from the ventromedial nucleus and retrochiasmatic area of the hypothalamus to the medulla and spinal cord of the rat
807
Ne ~ ~ , ~ I7 (I980) $07--S.L? © N~iNo~oN~&md ~n~e Pub~er~ L~.
AD I ~
PIIO~ION
FNO~g ' ~ A OF ~ OF
VNNT~ObEEDIA.L ~ C L N U S ~-~D NYPOT~LikL-~NUS T O ~T
D e ~ e n ~ of Anatomy ~ Neurobfology, Wezhing~on U n i ~ i ~ School of Medicine, St. Lou.~, MO (U~_4o) ~ Deper~enf of A~,~tomy, E ~ r n ~ Unfuer~ii'y Medica~ School, RoOfer,,am (The NetherNmd~) ( ~ i v e ~ Deeem~r 3rd, 1979)
(Reviged vemion received January iTCh, !980) (A~p~,d January 31~, 1980 )
BD~A~Y
Ad~r flujec~io~ of the fluorescent ~ o ~ a d e tracers b~benzLmide, %rue blue', ' g r a a u ~ r blue ~aad E ~ blue in the medulla o b l o n g a ~ (cen@red in the ~ . o ~ ~ucleus) or m upper thoracic spinal segment~, a continuous g~oup of labeled neurons was found s t a r . r e d t h r o u ~ o u t the retro zhiasmatic area ~r~d v e n ~ p a ~ of ~he ven~r nucleus of %he hypo~h~amus, predominantly on, the i p ~ i a ~ r ~ ~ ~he inNction si~s, Finding~ afte mject~on~ of' two d t. in the animai (bisbenzimide and %rue blue ~, E blue and ' ~ u l a r blue') ~t tha$ a ~ 15% of ~he iabeled ne~rons in. ~he above a~e~ d i ~ b u ~ e divei°gen~ axon coilateral~ to both the medulla ~ d the ~NnaI cord°
ndiig cu~ IIi
ila
Inl @of I
Y! . . . . . p , eoniisimlly found m the. of a num~longaga
~#e~e made
I en% nzimide
~obIok~y~
a{;8
7 ra~), '~pa.e blue' (5% aqueo,~ ~ ; s ~ s i o n ; 0.t #l Le m e d ~ , 0o6--1o2 #l cord; 4 rats)~ d °granular blue' (2% a q u e o ~ suspension; 0o! #i in medulla, [email protected] ul '.incord; 8 mLs)° Evans blue (I0% a q u e o ~ suspen~on; 0.3 ~.~in m e d u ~ ; 1.0--2.4 ~i L~ cord; 4 ra%) w ~ mj.ee~d ~ e c t i y ~om the n e r v e of a 10-#| ~ r ~ t o n ~-rL~ge. ~ repo~+~ed earlier [1,7,8], e ~ h of t h ~ yahstances ~ Sranspor~d retrogr~Ae~y ~ o ~ to ~he~ parent ceR bodies, and each apF~aZ~ to ~ taken up by tennin~ a~ well as by ~o~o In an a d d i t i o ~ ~ries of double l a ~ g expeff~ents, indiv~dued rats re~ived one injection in zhe med-d~a ( b ~ z i m i d e , granular blue or Evans blue} and another iu the spk~al co~d (true blue, Evans blue or ~ ~ blue, ~espectively). Four t~ nine days after the injec~ons of Evans blue, grenuiar blue and true blue and 14--27 h after ~he injection of bi~be~z~de, the ra~s were perfused with 10% formalin (following a brief rir~e with 0.9% ~ u e ) . The brains were soaked overnigh~ in 80% buffered sucrose, and a 1-in-3 series of 2,5 ~ m frontal sec$ions was cut and mounted from water. The dried ~ctions were observed with Leitz P l o ~ o p a c k fluorescence microscope {see Refs. I, 7
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Fig. I. A ~eriesof line dra~:g~ to ahow the location of retro~adeb~ fil~edr~_euron~in ~he retrochia~matic a~'ea, v e n t r o m e d i g nucleus, and arcuate nucleus after a ~arge bi~benzirnide injection in upper ~horacic segmen~ o f the spinal cord and an injection of t ~ e b~ue in :the dorsomedial ~ e d u l l a of the ~ame a n i m a l The ~oca~ioa of labeted ce~& wa~ p~otted on an X-Y reco~der~ and confirmed by ~ubsequen~ N~l~coun~ers~a~ning of tbe ~ : n e ~ectionso S~ng~e labeled ~-ae bh~e c e ~ are shown b y open etrc}e$ (o), ~ n g ~ labeled b ~ n z ~ m ~ d e celh~ by ~Aangles (~ L and double | ~be~ed ceH~ by filled c~rc~es (~ }° L a d l e d eelL~ ~n ~he paraven~icu~ar n u c l ~ s are no~ s h o ~ ° The drawings are arranged f~om ~o~rat (A) to c a u d ~ (F)o A~A0 ~a°~}or hypot~,~1~ic area; A ~ H , ~xcua~e n; LHA, ~te~A h y l a ~ t h ~ c are~; ~ 0 ~ med~1 p~opt~c a~ea; ~ VH, ~aventricular n°; ~Ch~ re~roch~az~a~e ~xea; 8Ch,
supra~hi~ma~c n0 ~V~{H, ventromed}~ n4 VS~ ~ d soe~ supr~opt[c com~vt~ssures, x 250.
ventr~cle~fx, fo~a~x; o~0 opGe ~a~t~
309 ~ d 8 for d e ~ s ) . T h e su~.-iv~ t i m e for animals i n j e c ~ d ~dth bisbe~zimide w ~ k e p t short, t.o m L ~ z e g i ~ labeling arotu~d r e t ~ o ~ a d e l y labeled n e u r o n s {see below). ~ the d o u b ! ~ a c e r experiments, injections of Wae blue were made 3 - - 8 days before h~jections o f bisbenzimide. The m e d u ! t a ~ , injections were m a d e b y exposing t h e o ~ x and l o w e ~ n g the micropipe%e h a l f w a y b e t w e e n t h e o b e x ~ d t h e i ~ e r i o r cerebetlar veto; thus, the ~ p o f t h e injection p i p e t t e u s u r y ~ e t o rest just ventral to t h e solitary tract. T h e spinal bujec~io~ were ~ n t ~ r ~ in ~ e dorsolat~raI ~ n i c ~ u s , which c o n t a i n s the bulk o f paraventriculosph~ai fibers [ 1 2 , 1 4 ] . ~ ~ rats, 1 gl o f a 80% solution o f h o r s e r a d ~ h ~ r o x i d a s e (HRP; Sigma, t y p e VI) was injected into u p p e r thoracic levels o f the s p i n ~ cord; 48 h later t h e anhmals were perfused and 30 p m t h i c k frozen sections t h r o u g h the h y p o t h a l a m u s were r e a c t e d with the s u b s ~ t e ~ ~ e t h y l o b e n z i d i n e , according to a m e t h o d previously described [ i 8 ] . FoHow-ing hujections o f each of the 4 tracers into either the dorsomediai medu~a or u p p e r thoracic spinal segments, retrogradeiy labeled n e u r o n s were a p p a r e n t m t h e para-¢entricular nucleus~ the lateral h y p o t h a i a m i c area, and
Fig. 2, A k~w*pawer pho~orai ~h t~ show the den~siWof retrogradely labeled aeuro~ in ti~e ~etmchia~atic a~ea after a true blue injectio~ in upper thor~cic segmer~ts of the ~'at spinal
the zor~ i~cer~ as p~e~io~y ~ were founa !yLng of the ~ were generally o w~ a~
~
~ d w ~ not ~ ;
[~9~12~ o In a d ~ however~ ou~ the area~ often .cc o m m ~ ~ m (F~go I)o They neurons° ~ e d e ~ of ~ . ~ e ~ ~
~ 30 Jabeh~ ~
with nv ~oii (from
1 c e ~ continued i n ~ ~he ~4ma of the yen n u c i ~ . The n u m b ~ of ~ c h c e ~ gmd~/ly f~om 5--7 to 2--87 h~ m o t e c a ~ !~ of the . ~ e y w~.~ o ~Lzed m~/~ipo~ ,~ d ten be ~ o : ~ t of as for~/~..g~ thin cup of ce!~ in ~he ventr~d sspec~; of the ~ p e d nuc~~.. In addi~on~ a few mediumsized ~ebeled cells were found in ~he ~rc.ualenucleus; they appe~ed to be c o n ~ u o ~ with the ~ o u p of h~be~ed cells in ~ e e n t ~ s of the re~-oe~da~ marie area and veT~tromedial nucleus and were most numerous after medullary,~nject~ons.~ each region~ a ~ar~e ~ o d t y of the ~abeled ¢e~s were fecund on the side ip~i~e~al to ~;he[n~ec~ion site in ~he ~edulla or ~ p ' ~ cord. Skn~o ~ar ~esults were found in the H K P mater~A., ~d~hou~ m gene~a|, fewer cells
one qu i r to oae-t ) ~n double ~be~in~ exp~i~en~ w ~ h the %~acer corn on~ men~oned abo.ve, a small p on {about 15%) of ~he ~abeled ce~b ~ bo~h ~he re~o.chi~matie s2ea m~.d @ ~ vent~o nucle'~ con÷m~.r~ed both (Fig. 2~ in.~;tL ~g that, at ~ some he,Irons ~ the ventromed~ nu~leus ~.d rei~och~matie ~ea d u~ d axon c o ~ h ~ ~} both the ~ph~i cord and the vicgn~.tyof the solitary nuc]e~. How~er0 ~nce the pd~hway taken by ~ vent~_~ h y p o ~ m . ~ : ~ f/ibg~ to the.cord ~ not known~ pa~.~ or al~ of %he double ladling might be due to the.up~a~ of %'be ~ace~ by damaged fi[~-of.pa~s~ge ~ ~he do~me.d.~ medutta. However, ~be fact that in ~hes.eexperh~enV~ m~ neuro~ were s ~ e l a ~ k ~ ~ th~n~c ~be~g do~ m~u t~vns, was not due merely t< fi des for ~he spins/cordo and ~ch a patnwa~ was not ~dea.~_fi~ m. pre~ou~ ~n~ro ~ expert. men~s [6,13fl5~ and in reL~ogr~e H ~ P w i ~ d~Lnoobenz (DAB} as the sub,irate [9,12]. In view of the rather low density of ce~s in the ventra~ hypo~a~ar.lus~ it seems likely that in the autor~d~o~phic expeNments~ not.enough cells were labeled by the sma~l injections m th~ ~ea. At the sense ~n.~e~ ~he present results show bha~ at [e~t m some systems, ~he ~racer~ bish~enzhn~de, g r a n ~ blue, tr~e b~ue, ~ d Evans blue may be more senAtive than HRP (pa~ieular~y as demons by DAB}. In our experience bi~nzimJ.de is the mos~ sensitive of %.ae used he~. Hewer.cA ~2~:dse of the ~uo~escent ~belh~g of g [ ~ nuclei aro.umd retro~deiy
811
ne~orm ~hat m found in m ~ y exFefmaents ~ t h bisbenzimide, it b c!e~ ~hat the ~aeer can ~ g r a t e ou~ of r e V r o ~ d e ! y labeled neurons. Therefore, approp~.a~!y low d~ses and shor~ sur~va! Nines were determined and used. Under ~ h ~ c ~ a m s ~ c e s ~@Ie or no tabeling of gliaI nuclei occurs, a condition which s ~ e s ~ tbm~ no s~gnifi~% migration of b i s ~ n z ~ i d e ou% of remotely n e u ¢ o ~ has occurred. This b s u b s ~ a ~ d by the fact that the p o p ~ t i o n of n e u m n ~ nueiei retrogradely labeled with bisbenzimide at short s ~ 4 v a ! ~mes has ~ e s ~ , e di~t-~bu~on ~ neuronal perikarya labeled ~ t h ~ e blue, g r a n ~ blue and Evans blue ~ r 4--5~ay survival times (at a t ~ e when g l ~ Iabeling m barely d e ~ c ~ b l e wi~h these ~-.,acers). ~.It is unlikely that ~he p a t ~ m of cells described here is due to vascu!ar or cerebrospinN fluid tcanspo~ of ~he tracers, since most of the cells are ,~n ~:he s ~ e side of the b r ~ ~ the ~ e c t i o n , and their distribution does not correspond to that described by Broadwelt and Brightman [ 2 ]. The p o s s i b ~ t y that the ven~omediaI nucleus of the hy~ othalamus projects directly ~ the i c i n i W of ~he dorsal vagal complex and go the spinal cord is of m~res~ in view of evidence that the nucleus is involved in ingestive [ 8 ], aggre~ive [ I t ] ~ d sexual [ t 0 ] ~haviors, atl of which have autonomic componen~. The prec'~e sites of ~rmination of the fibe~ from the ver~ai hypoth~us remain to ~ d e s t i n i n g , N~hough it is ~mpging ~o speculate that they p r o j e ~ at least in par~ ~ the dorsal vagal complex and ~ thoracic levels of the i n ~ e d i o l a ~ r a I column, as do nearby cells in the p ~ a v e n ~ c u l a r nucleus [ I 2 , ! 4 , 1 7 ] . The present resulb do sugges~ that iimbic ~ystem inforo marion from the ~ y g d a l a [5] ~ d bippocampN formation [ i ~ ] may be relayed d~ec~ty ~o the medutN arid sp:;~al eo~d by the ve~tromec~ial r~ucleus. ACKNOWLEDGEMENTS
Thb work -~:'a.ssu~porb:d in pm~ by USPHS G r a ~ NS~!37~7 and a Gra~t from ¢he Sloa~ FeuadaNoh and by Gmn¢ 18°4gol 5 from the FUNGO/ZNO (D~ch O~a~o~ for damea~ R h m Medicine). We es~ciaily ~haak DL M. ~m~voglio for helpful discussions of ~his work ~ d for cm~zing ou~ a n u m ~ of con~oi ~pe~m~enN w i n bbbe~z~lideo REFERENCES I ~stfvog!io~ M. K u ~ ' ~ , H.GA.M., Ca~man-Be~revoets, C.E. and Dan~, O°, Fbore:scene re~;~og~ade neuronal l.a.b,eli~g in ~he rag by means of substances binding specifi o catly ¢o a d e n ~ e - ~ i n e rich DNA, Neuro~i. ~ [ ¢ . , 12 (i979) 235--240° 2 BroadweH, R°D, and Bdgh~:n~an, M.W., Entry of peroxid~e ia~,o rieurons of the cea~¢at and gmHpher~ aer~]ou~ ~y~~em~ fl0om e~-racerebr~J a~d eerebra~ b~ood~ J° cevap, Ne~ol,°, 166 (197g) 257--~284. 8 S,Po~ of ~he u,~ in ~he ~:e~la~ion of ~ d and water i~tak~:.0 o
: S~ (7975)
[~f~o,a (!97e} !79--i~o
224.
12
@~e b ~ e ~
f
c
p~n~ 8 Kuy~
~ d he~r~e~m in ~
t~f ~he v e n = o m e ~
za* ~
ca~ J~ e ~ p . Neu~i°o !7~ (1978)
nuc~e~ of ~be hypo~h~amu~ J° e~mpo
~79) 1--7~ C~E~ ~ P~d~, R,E.,
e axor~
• 2' --13a°
9 K u y ~ , H.G~Mo ~ ~ i ~ y ~ V~A~.,R e ~ axoaat ~a.~lmr~ .of h ~ , ~ r ~ ~r~ d u e e from ~p~M c¢~rd t~ brain ~ v a ¢e~i i ~ t ~ ~ ~ ca~, Ne~c~cL L ~ L , t (I975~ 9--14~ 1~ Ma~thew$, D. and E d w ~ . D.A. ~ e venix~,me~l nuc~ew of the hy~o~h#~nu~ and h~r~ona] around of ~exual beha~o~ ~ *~e remade ra~. Horra. ~h~v.~ 8 (1977) 4¢~51. II P ~ p , J . E f f ~ $ of hyp ic Mui,~ ~w,m ~ - k ~ and ~ h ~ k indu~ed f~.~httngin r a ~ Fhy~ioL Behav., 8 (1971 ) 311--31~. 12 Saper~ C.B., L~mw~ A.D. Swan~n, LW~ and Cowa~, W~h~.~Direc~ bypo~alamo~ aui~n~mi¢ c o n n e c ~ , Brain l%e$.0i17 (!97~i} 305--312. 13 ~a ~r, C.Bo, Swau~on~ L.W. and C~w~n, W,~L, The efferen~ e.~m~eCd~z of ~be w n ~ o media~ n ~ c ~ ~f the hy~a,o~hal~v~u~of ~he ra,, J, c~mp. NeuroL, 169 (I~7~) 4()9~442. l,i Sw~a~n~ L,W. ~ m u n ~ m ~ c h c ~ m ~ l evidence for a ~ e u r ~ h y ~ a ~ n L u ~ n i ~ a u ~ nomic pathway a~fi~ingin $he paravee~v~cu~arnucleus af ~he y p o ~ h a ~ u ~ ~a~n R~., 28 (i!+77) 345~353. 15 S:waa~on~L.W~ and Cowan, W.M., ~Fae ~fferen~ ¢vnnec~.~on~of ~h~ ~u~w~cht~a~t¢ nuci~uu .af the h~o~bala~u~ J. caa~p. N~amL, 160 (i975i 1--t,2. !.*~ g~'~so~% L.W° and Cowan, W,M,, An ,u~'ad~og~apbh.~,i:~dy of ~.heovganlza~i~n of •,he efferen~connec~.~onaof the hipp~e~pM fo'~aL~on i~ the ~aL J, ~-mp. Neu¢~i o, 172 (1977) 49~84. ! 7 Swan~n~ L.W° and Har~aa~ B,K.0 ~ h ~ c h ~ i e ~ ~ ¢ ~ ~e~ty it~ ce-ntra~ ~a~hway~ t ~ i ~ d 18 Wy~, J.M., ~wanson, L.W. ~nd Cowaa, W.M.~A aCudy of ~ a ~ o ~ ~ffe~en~ t~ ~ hippo~ompai forma~i~)n in ~he ra** N e ~ e ~ e v ~ e , 4 (1979) 4~3--476.
Ne ~ ~ , ~ I7 (I980) $07--S.L? © N~iNo~oN~&md ~n~e Pub~er~ L~.
AD I ~
PIIO~ION
FNO~g ' ~ A OF ~ OF
VNNT~ObEEDIA.L ~ C L N U S ~-~D NYPOT~LikL-~NUS T O ~T
D e ~ e n ~ of Anatomy ~ Neurobfology, Wezhing~on U n i ~ i ~ School of Medicine, St. Lou.~, MO (U~_4o) ~ Deper~enf of A~,~tomy, E ~ r n ~ Unfuer~ii'y Medica~ School, RoOfer,,am (The NetherNmd~) ( ~ i v e ~ Deeem~r 3rd, 1979)
(Reviged vemion received January iTCh, !980) (A~p~,d January 31~, 1980 )
BD~A~Y
Ad~r flujec~io~ of the fluorescent ~ o ~ a d e tracers b~benzLmide, %rue blue', ' g r a a u ~ r blue ~aad E ~ blue in the medulla o b l o n g a ~ (cen@red in the ~ . o ~ ~ucleus) or m upper thoracic spinal segment~, a continuous g~oup of labeled neurons was found s t a r . r e d t h r o u ~ o u t the retro zhiasmatic area ~r~d v e n ~ p a ~ of ~he ven~r nucleus of %he hypo~h~amus, predominantly on, the i p ~ i a ~ r ~ ~ ~he inNction si~s, Finding~ afte mject~on~ of' two d t. in the animai (bisbenzimide and %rue blue ~, E blue and ' ~ u l a r blue') ~t tha$ a ~ 15% of ~he iabeled ne~rons in. ~he above a~e~ d i ~ b u ~ e divei°gen~ axon coilateral~ to both the medulla ~ d the ~NnaI cord°
ndiig cu~ IIi
ila
Inl @of I
Y! . . . . . p , eoniisimlly found m the. of a num~longaga
~#e~e made
I en% nzimide
~obIok~y~
a{;8
7 ra~), '~pa.e blue' (5% aqueo,~ ~ ; s ~ s i o n ; 0.t #l Le m e d ~ , 0o6--1o2 #l cord; 4 rats)~ d °granular blue' (2% a q u e o ~ suspension; 0o! #i in medulla, [email protected] ul '.incord; 8 mLs)° Evans blue (I0% a q u e o ~ suspen~on; 0.3 ~.~in m e d u ~ ; 1.0--2.4 ~i L~ cord; 4 ra%) w ~ mj.ee~d ~ e c t i y ~om the n e r v e of a 10-#| ~ r ~ t o n ~-rL~ge. ~ repo~+~ed earlier [1,7,8], e ~ h of t h ~ yahstances ~ Sranspor~d retrogr~Ae~y ~ o ~ to ~he~ parent ceR bodies, and each apF~aZ~ to ~ taken up by tennin~ a~ well as by ~o~o In an a d d i t i o ~ ~ries of double l a ~ g expeff~ents, indiv~dued rats re~ived one injection in zhe med-d~a ( b ~ z i m i d e , granular blue or Evans blue} and another iu the spk~al co~d (true blue, Evans blue or ~ ~ blue, ~espectively). Four t~ nine days after the injec~ons of Evans blue, grenuiar blue and true blue and 14--27 h after ~he injection of bi~be~z~de, the ra~s were perfused with 10% formalin (following a brief rir~e with 0.9% ~ u e ) . The brains were soaked overnigh~ in 80% buffered sucrose, and a 1-in-3 series of 2,5 ~ m frontal sec$ions was cut and mounted from water. The dried ~ctions were observed with Leitz P l o ~ o p a c k fluorescence microscope {see Refs. I, 7
../-
t b<.
]-+
)>j ......
-'. ' 2~<
~i~J
L C~?,D
Fig. I. A ~eriesof line dra~:g~ to ahow the location of retro~adeb~ fil~edr~_euron~in ~he retrochia~matic a~'ea, v e n t r o m e d i g nucleus, and arcuate nucleus after a ~arge bi~benzirnide injection in upper ~horacic segmen~ o f the spinal cord and an injection of t ~ e b~ue in :the dorsomedial ~ e d u l l a of the ~ame a n i m a l The ~oca~ioa of labeted ce~& wa~ p~otted on an X-Y reco~der~ and confirmed by ~ubsequen~ N~l~coun~ers~a~ning of tbe ~ : n e ~ectionso S~ng~e labeled ~-ae bh~e c e ~ are shown b y open etrc}e$ (o), ~ n g ~ labeled b ~ n z ~ m ~ d e celh~ by ~Aangles (~ L and double | ~be~ed ceH~ by filled c~rc~es (~ }° L a d l e d eelL~ ~n ~he paraven~icu~ar n u c l ~ s are no~ s h o ~ ° The drawings are arranged f~om ~o~rat (A) to c a u d ~ (F)o A~A0 ~a°~}or hypot~,~1~ic area; A ~ H , ~xcua~e n; LHA, ~te~A h y l a ~ t h ~ c are~; ~ 0 ~ med~1 p~opt~c a~ea; ~ VH, ~aventricular n°; ~Ch~ re~roch~az~a~e ~xea; 8Ch,
supra~hi~ma~c n0 ~V~{H, ventromed}~ n4 VS~ ~ d soe~ supr~opt[c com~vt~ssures, x 250.
ventr~cle~fx, fo~a~x; o~0 opGe ~a~t~
309 ~ d 8 for d e ~ s ) . T h e su~.-iv~ t i m e for animals i n j e c ~ d ~dth bisbe~zimide w ~ k e p t short, t.o m L ~ z e g i ~ labeling arotu~d r e t ~ o ~ a d e l y labeled n e u r o n s {see below). ~ the d o u b ! ~ a c e r experiments, injections of Wae blue were made 3 - - 8 days before h~jections o f bisbenzimide. The m e d u ! t a ~ , injections were m a d e b y exposing t h e o ~ x and l o w e ~ n g the micropipe%e h a l f w a y b e t w e e n t h e o b e x ~ d t h e i ~ e r i o r cerebetlar veto; thus, the ~ p o f t h e injection p i p e t t e u s u r y ~ e t o rest just ventral to t h e solitary tract. T h e spinal bujec~io~ were ~ n t ~ r ~ in ~ e dorsolat~raI ~ n i c ~ u s , which c o n t a i n s the bulk o f paraventriculosph~ai fibers [ 1 2 , 1 4 ] . ~ ~ rats, 1 gl o f a 80% solution o f h o r s e r a d ~ h ~ r o x i d a s e (HRP; Sigma, t y p e VI) was injected into u p p e r thoracic levels o f the s p i n ~ cord; 48 h later t h e anhmals were perfused and 30 p m t h i c k frozen sections t h r o u g h the h y p o t h a l a m u s were r e a c t e d with the s u b s ~ t e ~ ~ e t h y l o b e n z i d i n e , according to a m e t h o d previously described [ i 8 ] . FoHow-ing hujections o f each of the 4 tracers into either the dorsomediai medu~a or u p p e r thoracic spinal segments, retrogradeiy labeled n e u r o n s were a p p a r e n t m t h e para-¢entricular nucleus~ the lateral h y p o t h a i a m i c area, and
Fig. 2, A k~w*pawer pho~orai ~h t~ show the den~siWof retrogradely labeled aeuro~ in ti~e ~etmchia~atic a~ea after a true blue injectio~ in upper thor~cic segmer~ts of the ~'at spinal
the zor~ i~cer~ as p~e~io~y ~ were founa !yLng of the ~ were generally o w~ a~
~
~ d w ~ not ~ ;
[~9~12~ o In a d ~ however~ ou~ the area~ often .cc o m m ~ ~ m (F~go I)o They neurons° ~ e d e ~ of ~ . ~ e ~ ~
~ 30 Jabeh~ ~
with nv ~oii (from
1 c e ~ continued i n ~ ~he ~4ma of the yen n u c i ~ . The n u m b ~ of ~ c h c e ~ gmd~/ly f~om 5--7 to 2--87 h~ m o t e c a ~ !~ of the . ~ e y w~.~ o ~Lzed m~/~ipo~ ,~ d ten be ~ o : ~ t of as for~/~..g~ thin cup of ce!~ in ~he ventr~d sspec~; of the ~ p e d nuc~~.. In addi~on~ a few mediumsized ~ebeled cells were found in ~he ~rc.ualenucleus; they appe~ed to be c o n ~ u o ~ with the ~ o u p of h~be~ed cells in ~ e e n t ~ s of the re~-oe~da~ marie area and veT~tromedial nucleus and were most numerous after medullary,~nject~ons.~ each region~ a ~ar~e ~ o d t y of the ~abeled ¢e~s were fecund on the side ip~i~e~al to ~;he[n~ec~ion site in ~he ~edulla or ~ p ' ~ cord. Skn~o ~ar ~esults were found in the H K P mater~A., ~d~hou~ m gene~a|, fewer cells
one qu i r to oae-t ) ~n double ~be~in~ exp~i~en~ w ~ h the %~acer corn on~ men~oned abo.ve, a small p on {about 15%) of ~he ~abeled ce~b ~ bo~h ~he re~o.chi~matie s2ea m~.d @ ~ vent~o nucle'~ con÷m~.r~ed both (Fig. 2~ in.~;tL ~g that, at ~ some he,Irons ~ the ventromed~ nu~leus ~.d rei~och~matie ~ea d u~ d axon c o ~ h ~ ~} both the ~ph~i cord and the vicgn~.tyof the solitary nuc]e~. How~er0 ~nce the pd~hway taken by ~ vent~_~ h y p o ~ m . ~ : ~ f/ibg~ to the.cord ~ not known~ pa~.~ or al~ of %he double ladling might be due to the.up~a~ of %'be ~ace~ by damaged fi[~-of.pa~s~ge ~ ~he do~me.d.~ medutta. However, ~be fact that in ~hes.eexperh~enV~ m~ neuro~ were s ~ e l a ~ k ~ ~ th~n~c ~be~g do~ m~u t~vns, was not due merely t< fi des for ~he spins/cordo and ~ch a patnwa~ was not ~dea.~_fi~ m. pre~ou~ ~n~ro ~ expert. men~s [6,13fl5~ and in reL~ogr~e H ~ P w i ~ d~Lnoobenz (DAB} as the sub,irate [9,12]. In view of the rather low density of ce~s in the ventra~ hypo~a~ar.lus~ it seems likely that in the autor~d~o~phic expeNments~ not.enough cells were labeled by the sma~l injections m th~ ~ea. At the sense ~n.~e~ ~he present results show bha~ at [e~t m some systems, ~he ~racer~ bish~enzhn~de, g r a n ~ blue, tr~e b~ue, ~ d Evans blue may be more senAtive than HRP (pa~ieular~y as demons by DAB}. In our experience bi~nzimJ.de is the mos~ sensitive of %.ae used he~. Hewer.cA ~2~:dse of the ~uo~escent ~belh~g of g [ ~ nuclei aro.umd retro~deiy
811
ne~orm ~hat m found in m ~ y exFefmaents ~ t h bisbenzimide, it b c!e~ ~hat the ~aeer can ~ g r a t e ou~ of r e V r o ~ d e ! y labeled neurons. Therefore, approp~.a~!y low d~ses and shor~ sur~va! Nines were determined and used. Under ~ h ~ c ~ a m s ~ c e s ~@Ie or no tabeling of gliaI nuclei occurs, a condition which s ~ e s ~ tbm~ no s~gnifi~% migration of b i s ~ n z ~ i d e ou% of remotely n e u ¢ o ~ has occurred. This b s u b s ~ a ~ d by the fact that the p o p ~ t i o n of n e u m n ~ nueiei retrogradely labeled with bisbenzimide at short s ~ 4 v a ! ~mes has ~ e s ~ , e di~t-~bu~on ~ neuronal perikarya labeled ~ t h ~ e blue, g r a n ~ blue and Evans blue ~ r 4--5~ay survival times (at a t ~ e when g l ~ Iabeling m barely d e ~ c ~ b l e wi~h these ~-.,acers). ~.It is unlikely that ~he p a t ~ m of cells described here is due to vascu!ar or cerebrospinN fluid tcanspo~ of ~he tracers, since most of the cells are ,~n ~:he s ~ e side of the b r ~ ~ the ~ e c t i o n , and their distribution does not correspond to that described by Broadwelt and Brightman [ 2 ]. The p o s s i b ~ t y that the ven~omediaI nucleus of the hy~ othalamus projects directly ~ the i c i n i W of ~he dorsal vagal complex and go the spinal cord is of m~res~ in view of evidence that the nucleus is involved in ingestive [ 8 ], aggre~ive [ I t ] ~ d sexual [ t 0 ] ~haviors, atl of which have autonomic componen~. The prec'~e sites of ~rmination of the fibe~ from the ver~ai hypoth~us remain to ~ d e s t i n i n g , N~hough it is ~mpging ~o speculate that they p r o j e ~ at least in par~ ~ the dorsal vagal complex and ~ thoracic levels of the i n ~ e d i o l a ~ r a I column, as do nearby cells in the p ~ a v e n ~ c u l a r nucleus [ I 2 , ! 4 , 1 7 ] . The present resulb do sugges~ that iimbic ~ystem inforo marion from the ~ y g d a l a [5] ~ d bippocampN formation [ i ~ ] may be relayed d~ec~ty ~o the medutN arid sp:;~al eo~d by the ve~tromec~ial r~ucleus. ACKNOWLEDGEMENTS
Thb work -~:'a.ssu~porb:d in pm~ by USPHS G r a ~ NS~!37~7 and a Gra~t from ¢he Sloa~ FeuadaNoh and by Gmn¢ 18°4gol 5 from the FUNGO/ZNO (D~ch O~a~o~ for damea~ R h m Medicine). We es~ciaily ~haak DL M. ~m~voglio for helpful discussions of ~his work ~ d for cm~zing ou~ a n u m ~ of con~oi ~pe~m~enN w i n bbbe~z~lideo REFERENCES I ~stfvog!io~ M. K u ~ ' ~ , H.GA.M., Ca~man-Be~revoets, C.E. and Dan~, O°, Fbore:scene re~;~og~ade neuronal l.a.b,eli~g in ~he rag by means of substances binding specifi o catly ¢o a d e n ~ e - ~ i n e rich DNA, Neuro~i. ~ [ ¢ . , 12 (i979) 235--240° 2 BroadweH, R°D, and Bdgh~:n~an, M.W., Entry of peroxid~e ia~,o rieurons of the cea~¢at and gmHpher~ aer~]ou~ ~y~~em~ fl0om e~-racerebr~J a~d eerebra~ b~ood~ J° cevap, Ne~ol,°, 166 (197g) 257--~284. 8 S,Po~ of ~he u,~ in ~he ~:e~la~ion of ~ d and water i~tak~:.0 o
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