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Effects of intracerebral injections of 5,6-dihydroxytryptamine on central monoamine neurons: Evidence for selective degeneration of central 5-hydroxytryptamine neurons
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Effects of intracerebral injections of 5,6-dihydroxytryptamine on central monoamine neurons: Evidence for selective degeneration of central 5-hydroxytryptamine neurons
JOHN DALY, K JELL FUXE ~"~D GOSTA JONSSON Sectton o/Pharma~odvnamu,s, Laboratory oJ Chemtstry N I A M D , National In~ttttltea o/ Heallh, Betheada, M d 20014 (U S A ) and Department o/ Histology, Karohnaka htstttutet, S-104 Ol Nlo~khohn 60 (Sweden)
(Accepted September 27th, 1972)
It IS well-known that the neurotoxlc c o m p o u n d 6-hydroxydopamlne (6-OH-DAy causes a selective degeneration of peripheral and central catecholamme (CA) neuronsIS,t9, :1. Recently, it has been demonstrated that 5,6-dihydroxytryptamine (5,6HT) may cause a similar selective degeneration o f serotonin (5-HT) neurons (see refs. 2--4, 6, 8 and 13). The present paper gives evidence that stereotaxlc Injections ot 5,6-HT prowdes a powerful tool in the morphological and functional analysis of the central 5-HT neurons. Male Sprague-Dawtey rats (150-200 g, body wt.) have been used. The effects o f 5,6-HT injected either lntraventrlcularly or mtracerebrally were evaluated by follov~ing the m vm'o uptake of [aH]5-HT or [all]CA m slices from various brain regions(for techmcal details see ref. 17). In addition some of the 5,6-HT treated animals were taken for amine fluorescence hlstochemlstry according to Falck et al. 9 (see also refs 5. 10 and 12) hTtraventrtcular in/ection~" were made stereotaxically into the lateral ventricle under fluothane-oxygen anesthesia The doses given ranged from 25 to 100 /~g. 5,6-HT (creatmine sulfate-H~,O):' dissolved in Ringer solution containing0.2 mg/ml ascorbic acid All the doses of 5,6-HT given refer to the free base and the volume injected was 10 td. The animals taken for m v,tro uptake studies were killed 14 day's after the injection o f 5,6-HT. A reduction in [all]amine uptake mdmates a damage and degeneration o f amine nerve terminals (see refs. 14 and 17). Shces from the following brain regions were studied' the cerebral cortex, the hypothalamus, the neostrmtum and the spinal cord The m vitro uptake o f Jail]CA (10 -v M, 10 rain) and [aH]5-HT (10-7 M, 10 min) was studied and c o m p a r e d to saline treated controls. Fluorescence
* Samples of 5,6-HT (creatmme sulfate-H~O) were prepared as descr,bed" fiom 5,6-dJbene)loxytryptamme Additional samples, synthesized by Regis Chemical Co, Chmago, 111.under Research Contract No SA 43-ph-3021, were provided by Dr A A Maman, Psychopharmacology Service Center, National Institute of Mental Health, Bethesda, Md, U S.A.
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SHORT COMMUNICATIONS
histochemical analysis was performed 1, 7 and 14 days following the 5,6-HT injections. In these latter experiments the animals were injected with the monoamine oxldase inhibitor nialamide (500 mg/kg, 1.p.) 4-6 h before killing in order to ensure that as many as possible of the 5-HT nerve terminals remaining were visuahzed (see ref. 7). The extensor hmdhmb reflex of the acutely spinalized rat lS highly dependent on 5-HT receptor activity (see refs. 1 and 16). Therefore, this reflex was studied in some of the rats 10-14 days after an intraventricular injection of 5,6-HT. The animals were treated with nialamlde (300 mg/kg, i,p.) 3 h before testing and with chlorimipramine (10-20 mg/kg, ~.p.) 30 rain before testing. Thls treatment is known to markedly enhance extensor reflex activity (ref. 16). lntracerebral injections. In order to damage many of the ascending 5-HT pathways, 5,6-HT in doses of 5-25 #g in 10 #l of Ringer solution containing ascorblc acid were slowly refused bilaterally into the caudal raphe region (nucleus raphe dorsahs; nucleus raphe medianus; B7 and B8 according to Dahlstrom and Fuxe 7) of the mesencephalon. The coordinates were according to K6nig and Khppe115: anterior (A) = ~- 0.35 ram; lateral (L) = ~ 0.6 ram: vertical (V) =- - - 1.4. The operations were performed under fluothane-oxygen anesthesia and the flow was directed medially towards the 5-HT nerve cell containing nuclei and their axons. The brains were taken to histochemical fluorescence analysis and to [3H]amine uptake studies 10-14 days after the operation. Histochemical analysis was performed at various time-intervals (1-14 days) following the injection. The effects of the 5,6-HT injections on the nlalamlde (300 mg/kg, 4-6 h before testing, i.p ) and tryptophan (200 mg/kg, 1-2 h before testing, i.p.) induced changes in gross behavior (see ref. ! 1) and body temperature were also evaluated 10-14 days after the injection of the 5,6-HT. Unilateral stereotaxic injections of 5,6-HT (25 yg/8 yl) into the ascending CER CORTEX
HYPOTHAL
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Fig. 1. Effect of m t r a v e n t r i e u l a r rejection of 5,6-HT (25, 50 or 100 yg, 14 days) on the m vttro uptake of [3H]5-HT (10 7 M , 10 ram) in shces from cerebral cortex, hypothalamus and spinal cord (lumbar region). The results are presented as percentages of controls (I00 ± 4-8 %). Each column represents the mean ± S.E M. of 6-8 determinations.
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Fig 2 Effect ol mtrace~ebral mject~on ot 5,6-HT (10. 25 or 50 Hg, 14 days bilaterally into the caudal raphe region) on the m ~ t ~ o uptake o f [~H]5-HT (l(; - M , 10 m m ) m shoes f r o m cerebral c u r ( e \ , h y p o t b a l a m u s and cerebellar co)rex The results are presented as percentages of controls (ll}0 , 5-7°,,) Each column represents fine mean i S E.M of 0-8 d e t m m m a t i o n s
noradrenaline (NA) and dopamme (DA) bundles were also performed in a similar way as descr)bed above and by Ungerstedt 2(). The coordinates were for the dorsal NA bundle' A - -2.2 ram. L %0 8 ram: V - - 0 7 mm and for the nigro-neostrlatal DA bundle' A ~.2 mm: L , 1.4 ram, ~,' ---3 ram. The ammals were killed 10-14 days later for h~stochemlcal analysis and for [all]CA and [aH]5-HT uptake studies as described above The results from the mtraventricular rejections of 5,6-HT are summanzed m Fig. 1. A dose-dependent reduction of ['~H]5-HT uptake Is observed, which is panicularly pronounced m the spinal cord. The uptake of [aH]5-HT m the cortex cerebn was practmally unchanged, whlch was also the case for the uptake of [aH]NA m all regions investigated. After the largest dose, 100/,g 5,6-HT mtraventncularly, some 100"
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Fig 3. Effect of mtracerebral mjecnon of 5,6-HT (50 pg, 14 days as m Fig 2) on the it1 v t t m uptake of [aH]NA (10 7 A I , 10 ram) In slice~ from cerebral cortex, h y p o t h a l a m u s and cerebellar cortex Tile results are presented as percentages of controls (100 '- 5-7°o) Each column represents the mean L- S.E.M. o f 6-8 determinations
479
SHORT COMMUNICA~IIONS
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A Fig 4 Nucleus raphe medlanus (A) and adjacent medial reticular formation of the mesencephalon. The area of the Injection needle is seen to the right. 5,6-HT was infused medially (25/rg/10/11) 14 days before kllhng The strongly yellow fluorescent dots represent the damaged 5-HT axons in this area The 5-HT cell bodies (A) on the other hand appear to be relahvely unaffected " 120
of the animals did not survive, and in many cases a marked dllatatmn of the ventricles was observed indicating unspecific destrucnon of brain tissue by 5,6-HT. These results are m agreement with the results of previous workers (see refs 2 and 6). The fluorescence histochemlstry revealed accumulations of yellow fluorescence in swollen and distorted fibers close to the surface of the subarachnoldal space m the medulla oblongata and spinal cord. The accumulattons were most pronounced during the first days after the operation. A dose-dependent reduction of the yellow fluorescent nerve terminals was observed in the brain stem and particularly in the spinal cord The 5-HT cell bodies were not notably affected. The studies on the extensor hlndhmb reflex revealed a reduced enhancement of the reflex following nialamlde--chlorlmipramine treatment in the animals that had received 100 ktg 5,6-HT intraventricularly. These findings give functional evidence for a degeneration of the descending bulbospinal 5-HT neurons. The results from the uptake studies in the rats treated with local injections of 5,6-HT into raphe area are summarized in Fig 2 The data reveal a marked and selective decrease of [aH]5-HT uptake m the hypothalamus, an effect which is dose-dependent. The [aH]NA uptake m the hypothalamus was not significantly affected (Fig. 3), nor was the [JH]5-HT uptake in the cortex cerebri and in the spinal cord (Fig 2) The [aH]5-HT uptake in the cerebellar cortex was slightly reduced (Fig. 2). Fluorescence analysis revealed marked accumulations of yellow fluorescence in the medial ascending 5-HT fibers from the groups B7 and B8 (Fig. 4) In a preliminary microspectrofluorimetrical study the emission spectra of the yellow fluorescence accumulations have so far only showed emission maxima at 525 nm, typical of that of the 5-HT-fluorophor. Fourteen days after the 5,6-HT injection, signs of sprouting from the strongly fluorescent 5-HT axons were observed m the rejection area. Strongly
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I )g 5 Nuclcu~ laphc d~,~ahs ,,a~dal it) Ibc 'qtc of reJection Fhe 5,6-HT |njectlon (25 /¢g,20 ,I) was made ink) (he 5-HT d \ O l ) ~ o l l g l l l a t l l l g ~l'Om th~ nucleus at a level anterior to the 5-HT cctl bodies (about l nllll) ][[ t.dl] b c ~een that M)o)lg fluorescence appeals in the 5-HT axons ~ ~) tH' I(~ the 5-HT cell bod~ bill then abruptl~ e~)d, 200 fluorescent growth cones emerging fi-om weakly fluorescent slender axons were observed. T h e r e ~ e r e also signs o| mner~atlon or" nerve cell bodies near the site o f m j e c t l o n . These cell bodies are normall~ not innervated by 5-HT nerve terminals a n d do not contain m o n o a m m c s . The plhng up ol ~ellow fluorescence in the axons could be traced up into the 5-HT cell b o d y areas, which themselves had not been affected (Fig. 5). However, the cell bodies showed reduced a c c u m u l a t i o n s o f 5 - H T fluorescence following m a l a m i d e t r y p t o p h a n l r e a t m e n t Unspecific d a m a g e was only obser~,ed close to the site o f injection (200 fire) but with small ~olumes and high doses f a l r b large uHspectBc ~lecrosls call occur The 5-HT nerve t e r m i n a l s had c o m p l e t e l y d i s a p p e a r e d m the nucleus s u p r a c h i a s m a t l c u s 10-14 days following the 5,6-HT injection a n d m the globus palhdus N o fluorescence was observed, not even after n i a l a m l d e - t r y p t o p h a n , a t r e a t m e n t known to cause a m a r k e d increase m the yellow 5 - H T fluorescence. One d a y after the reJection, on the o t h e r hand, an increased fluorescence intensity in the s u p r a c h i a s m a t l c 5 - H T nerve terminals ~ a s observed InJections of the solvent into the raphe a r e a p r o d u c e d no effects on the 5-HT neurons. Studies on gross b e h a v i o r showed that following local injection o f 5,6-HT m the r a p h e area, the m a l a m ~ d e - t r y p t o p h a n s y n d r o m e o f i m m o N h z a t i o n , t r e m o r , a b d u c tion o f the h m b s and head m o v e m e n t s was r e d u c e d In addition, the h y p e r t h e r m m observed after n i a l a m ~ d e - t r y p t o p h a n t r e a t m e n t was less p r o n o u n c e d after this type o f 5,6-HT injection InJections o f 5,6-HT {25 fig) into the dorsal a n d ventral N A and the m g r o neostriatal D A bundles did not result m any sigmficant changes o f [ a H ] N A and [aH]D A u p t a k e m the cortex cerebrl and the neostriatum, respectively, or m the dlsappetuance o f N A nerve terminals m the teled~encephalon, as shown in the fluorescence microscopical analysis However, a few hours after mjecUon in the substantla m g r a a r e a yellowish a c c u m u l a t l o n s o f fluorescence were f o u n d m the D A tracts a n d in the
SHORT COMMUNICATIONS
481
D A cell bodies, p r o b a b l y representing u p t a k e o f 5,6-HT into these nerve cells an d their axons. Th~s u p t a k e was a p p a r e n t l y insufficient to cause any substantial d e g e n e r a tion o f the N A and D A neurons. These studies reveal that 5,6-HT provides a tool for the selective chemical d e g e n e r a t i o n o f central 5 - H T tracts when p r o p e r l y used. In addition, the results presented p r o v i d e evidence for the existence o f a specific medial 5 - H T tract from groups B7, B8 which is i n v o l v e d in the subcort~cal l n n e r v a t l o n o f the telediencephalon, especmlly the h y p o t h a l a m u s . P r e l i m i n a r y data suggest that m o r e laterally ascending 5 - H T fibers partly those f r o m g r o u p B9 (surrounds the dorsal surface to the lemniscus medialls (see ref. 7)) send fibers to the cortex cerebri as revealed w~th 5,6-HT injections laterally into the v e n t r o m e d m l t e g m e n t u m (the area o f g r o u p B9) Th e present studies also indicate that this t e c h n i q u e is helpful in the analysis o f the functional role o f the central 5 - H T neurons, a l t h o u g h it should be emphasized that 5,6-HT can cause unspecific d a m a g e o f brain t~ssue. T h e r e f o r e f u r t h er investigations are needed before ~t can be used routinely m studies on 5 - H T t r a n s m i t t e r mechanisms. This study has been s u p p o r t e d by G r a n t s B73-O4X-715 and B73-04X-2295-06B f r o m the Swedish M e d ic a l R e s e a r c h Council. T h e skilful technical assistance of Mr s
Agneta
Ehasson,
Mrs
Ulla-Br~tt
F l n n m a n , Miss Bodil F l o c k and Miss Elisabeth H a g m a n is gratefully a c k n o w l e d g e d
I ANDI~N, N -I~ , Discussion of serotonln and dopamme m the extrapyramldal system, ,4dvanc Pharmacol, 6A (1968) 347 349 2 BAUM3¢RTEN, H G , BJORKLUND, A , LACHE',IMAYER, L , NOBIb,, A , AND STENE\I, U , Longlasting selective depletion of brain serotonln by 5,6-dlhydroxytryptamme, Acta phystol scand, Suppl 373 (1971) 3 BAUM,~ARTEN, H G , BJORKLUND,A , HOLSTEIN, A F , AND NOBI~,, A , Chemical degeneration of lndolamlne axons m rat brain by 5,6-dlhydro×ytryptamme, Z ZelIJbt ~ch, 129 (1972) 256 271 4 B~UM~ARTEN, H. G , L&CHENMAYER, L , ~ND SCHEOSSBERGER, H G , E,,ldence for a degeneration of mdolam~ne containing nerve terminals ~n rat brain, induced by 5,6-dlhydroxytr~ptamme, Z Zell]o~ch , 125 (1972) 553-569. 5 CORRODI, H., AND JONSSON, G , The formaldehyde fluorescence method for the histochem~cal demonstration of b~ogenlc monoammes. A rewew on the methodology, J Htstochem Cyto~hem , 15 (1967) 65-78 6 COSTA, E , D,XL~, J , LEFEVRE, H . , MEEK, J , REVUELTA, A , SPARRO, F , AND STRADA, S , Serotomn and catecholamme concentrations m brain of rats reJected lntracerebrally with 5,6-dthydroxytryptamme, Brain Research, 44 (1972) 304-308 7 DAHLSTROM, A , AND FUXE, K , Evidence for the existence of monoamme nevrons m the central nervous system. I Demonstration of monoamlnes m the cell-bodies of brain stem neurons, A~taplo~tol. ~ca~td, 64, Suppl 232 (1964). 8 DCLY, J , FuxE, K , AND JONSSON, G , 5,6-D~hydroxytryptamme' a new tool m mapping out of central 5-HT neurons, Ab~tr 4th Int. Congr Ht~to~hem and Cytochem , K~oto, (1972) 487 488 9 FALCK, B , HILLARP, N.-/~, THIEME, G , A~,D TORP, A , Fluorescence of catecholamlnes and related comnounds condensed with formaldehyde, J Ht~tochem Cvto~hem , 10 (1962) 348 354 l0 F U \ E , K , HOKFELT, T , JONSSONr, G , A.ND UNGERSTEDT, U , Fluorescence m~croscopy m neuroanatomy In W J H N¢UTA ~'~D S O E EBBESSO'q(Eds), Contemporao' Re~ea~ch m Netnoanatomy, Springer, l~erhn, 1970, pp. 275 314 11 GR~HAME-SMIT~,D G , Studies m v~vo on the relationship between brain tryptophan, brain 5-HT synthes~s and hyperactivity m rats treated with a monoamme ox~dase inhibitor and Ltryptophan, J N e u t o ( h e m , 18 (1971) 1953.
]2 JONS~,()\. (~, Ou,llltltatto~ o[ b~ogem~ ii]onoanllocq denlgnstrated ~ttb .~h~ I o f l t h t l ~ . ' .[~ fluorescence method, P~,~,, t h w o c h e l ~ ( , t o c h e m , 2 ( 1 9 7 1 ) 2 9 9 334 13 JONSS'~'n, (; F,. \~, k . x'.t, D i n ; l Intracerebrat mje~tkm,, of 5d,-dllq_,,d~okSh3.,ta ~J,. (5,6-HF) ExJdel~c {oL ~,.le~_ll~,c dcgencrauon of t~cntla[ ~-h,,dro\3tlyptammc n c m o r ~, ~,, pllcIttlltll~o[ Abr! , 31. bdl~pl I (1972) 24 14 JONSSI)N (~ , A.ND Szk ~, I,5, Q H , Degenerative and non-degeneraU\e ettects ol I ~ - h > , d r o \ ) d t q ~ , ~ c on adrenerglc nez'c~ ,1 P h r . m ~ . ¢ , l exl) Ftte~ . 180 (1972) 625 635 15 KOF\t(,, J F R , x'~I) KI [L'I'I'L, R R , fl/te R a l B r o t h a ~Sleteotart( ,tt[as o l The [ o t ¢ l ) i t . ~ ,#ta Lol~et Pa~ls o]" 171e Btal,l Nletll. Wtlham~ and Wflklns, Baltunore, 1963 16 M u K. J , Fu-.~, k , ~-.t) A\Dz-~. N -h , Effects of antidepressant drugb of the ~mlprammc tSpc on centrdt 5-h)dro',3tt~ptamme neurotran,,mr,,,Ion. E m o p .I P h a r m a ( o l , ~) (1970) 325 332 17 Sa( us, ('H aN~) .IoNsso\ (_z , D,3gdner0.tlon of central and peripheral n o r a d r e n a h n c t~e~,H,,w, pzodttued by 6-h3d~o\~-DOPA, J ~ / e u ~ , ~ h e m , 19 (1972) 1561-1575 18 TI'.ANzIR 1 P , ~ \ D TH,~NI'~. H . t'hram.)rpholog~sche Velanderungcn der ,,)lnpathr,~.hen Nervenend~gungcn de~ K a t / e na~_h Vorbehandlung mlt 5- und 6 - H ~ d r o x y - D o p a m m , \'(zt/zt~ttS~hmzedeh.,e's t ~ h e t p P~th Pha~mal. . 257 (19671 343 19 U N ( , E R N I I D T , e. ~}-Hydroxs-dopanamc reduced degenerauon of central m o n o a n u n e neHon',, /;{ll~q~ [ PJt(lFllldtO/. 5 (1968)107 lit) 20 UNcd RSFI I)l, U , LI'~c ol Jnha~.e~eb~al nUc~.t~ons oi 6-h3 d r o x y d o p a m m e as a tool lot zno~pho!ogit.al and ltLnt.t~onal Mud~e,, on ¢e~atral uatecholamme netwon,, In T M A L M F ( ) R S A N D m Tttol-~Y (Kd', }. 6 - H v t h o x ieb'p(t~ll,~t' ,~/,¢[ f ,/h'* /lOhlltlllh' Nt('ttt()ll~, N o r t h - H o l l a n d Publ . AmMerdan~ t'171. pp 315 3~2 21 U r F T S k ~ N .I , x",t) lxl-rSt ",. L L . Etl.:~r., of o - h y d r o x y d o p a m m e on n o r a d r e n a h n e - c o n t d m m g n,:urone, ,~ the ~at b~a,n, .\tlllltt, ] ond .. 221 (1969) 557 559
Printed ~~ t,,c Nc,h~' .
Effects of intracerebral injections of 5,6-dihydroxytryptamine on central monoamine neurons: Evidence for selective degeneration of central 5-hydroxytryptamine neurons
JOHN DALY, K JELL FUXE ~"~D GOSTA JONSSON Sectton o/Pharma~odvnamu,s, Laboratory oJ Chemtstry N I A M D , National In~ttttltea o/ Heallh, Betheada, M d 20014 (U S A ) and Department o/ Histology, Karohnaka htstttutet, S-104 Ol Nlo~khohn 60 (Sweden)
(Accepted September 27th, 1972)
It IS well-known that the neurotoxlc c o m p o u n d 6-hydroxydopamlne (6-OH-DAy causes a selective degeneration of peripheral and central catecholamme (CA) neuronsIS,t9, :1. Recently, it has been demonstrated that 5,6-dihydroxytryptamine (5,6HT) may cause a similar selective degeneration o f serotonin (5-HT) neurons (see refs. 2--4, 6, 8 and 13). The present paper gives evidence that stereotaxlc Injections ot 5,6-HT prowdes a powerful tool in the morphological and functional analysis of the central 5-HT neurons. Male Sprague-Dawtey rats (150-200 g, body wt.) have been used. The effects o f 5,6-HT injected either lntraventrlcularly or mtracerebrally were evaluated by follov~ing the m vm'o uptake of [aH]5-HT or [all]CA m slices from various brain regions(for techmcal details see ref. 17). In addition some of the 5,6-HT treated animals were taken for amine fluorescence hlstochemlstry according to Falck et al. 9 (see also refs 5. 10 and 12) hTtraventrtcular in/ection~" were made stereotaxically into the lateral ventricle under fluothane-oxygen anesthesia The doses given ranged from 25 to 100 /~g. 5,6-HT (creatmine sulfate-H~,O):' dissolved in Ringer solution containing0.2 mg/ml ascorbic acid All the doses of 5,6-HT given refer to the free base and the volume injected was 10 td. The animals taken for m v,tro uptake studies were killed 14 day's after the injection o f 5,6-HT. A reduction in [all]amine uptake mdmates a damage and degeneration o f amine nerve terminals (see refs. 14 and 17). Shces from the following brain regions were studied' the cerebral cortex, the hypothalamus, the neostrmtum and the spinal cord The m vitro uptake o f Jail]CA (10 -v M, 10 rain) and [aH]5-HT (10-7 M, 10 min) was studied and c o m p a r e d to saline treated controls. Fluorescence
* Samples of 5,6-HT (creatmme sulfate-H~O) were prepared as descr,bed" fiom 5,6-dJbene)loxytryptamme Additional samples, synthesized by Regis Chemical Co, Chmago, 111.under Research Contract No SA 43-ph-3021, were provided by Dr A A Maman, Psychopharmacology Service Center, National Institute of Mental Health, Bethesda, Md, U S.A.
477
SHORT COMMUNICATIONS
histochemical analysis was performed 1, 7 and 14 days following the 5,6-HT injections. In these latter experiments the animals were injected with the monoamine oxldase inhibitor nialamide (500 mg/kg, 1.p.) 4-6 h before killing in order to ensure that as many as possible of the 5-HT nerve terminals remaining were visuahzed (see ref. 7). The extensor hmdhmb reflex of the acutely spinalized rat lS highly dependent on 5-HT receptor activity (see refs. 1 and 16). Therefore, this reflex was studied in some of the rats 10-14 days after an intraventricular injection of 5,6-HT. The animals were treated with nialamlde (300 mg/kg, i,p.) 3 h before testing and with chlorimipramine (10-20 mg/kg, ~.p.) 30 rain before testing. Thls treatment is known to markedly enhance extensor reflex activity (ref. 16). lntracerebral injections. In order to damage many of the ascending 5-HT pathways, 5,6-HT in doses of 5-25 #g in 10 #l of Ringer solution containing ascorblc acid were slowly refused bilaterally into the caudal raphe region (nucleus raphe dorsahs; nucleus raphe medianus; B7 and B8 according to Dahlstrom and Fuxe 7) of the mesencephalon. The coordinates were according to K6nig and Khppe115: anterior (A) = ~- 0.35 ram; lateral (L) = ~ 0.6 ram: vertical (V) =- - - 1.4. The operations were performed under fluothane-oxygen anesthesia and the flow was directed medially towards the 5-HT nerve cell containing nuclei and their axons. The brains were taken to histochemical fluorescence analysis and to [3H]amine uptake studies 10-14 days after the operation. Histochemical analysis was performed at various time-intervals (1-14 days) following the injection. The effects of the 5,6-HT injections on the nlalamlde (300 mg/kg, 4-6 h before testing, i.p ) and tryptophan (200 mg/kg, 1-2 h before testing, i.p.) induced changes in gross behavior (see ref. ! 1) and body temperature were also evaluated 10-14 days after the injection of the 5,6-HT. Unilateral stereotaxic injections of 5,6-HT (25 yg/8 yl) into the ascending CER CORTEX
HYPOTHAL
SPINAL CORD
100-
% m
80.
~ 60-
&
,///~
40-
//M
L6 20-
,///,
25
50
100
25
50
100
25
50
tO0 p c /
Fig. 1. Effect of m t r a v e n t r i e u l a r rejection of 5,6-HT (25, 50 or 100 yg, 14 days) on the m vttro uptake of [3H]5-HT (10 7 M , 10 ram) in shces from cerebral cortex, hypothalamus and spinal cord (lumbar region). The results are presented as percentages of controls (I00 ± 4-8 %). Each column represents the mean ± S.E M. of 6-8 determinations.
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Fig 2 Effect ol mtrace~ebral mject~on ot 5,6-HT (10. 25 or 50 Hg, 14 days bilaterally into the caudal raphe region) on the m ~ t ~ o uptake o f [~H]5-HT (l(; - M , 10 m m ) m shoes f r o m cerebral c u r ( e \ , h y p o t b a l a m u s and cerebellar co)rex The results are presented as percentages of controls (ll}0 , 5-7°,,) Each column represents fine mean i S E.M of 0-8 d e t m m m a t i o n s
noradrenaline (NA) and dopamme (DA) bundles were also performed in a similar way as descr)bed above and by Ungerstedt 2(). The coordinates were for the dorsal NA bundle' A - -2.2 ram. L %0 8 ram: V - - 0 7 mm and for the nigro-neostrlatal DA bundle' A ~.2 mm: L , 1.4 ram, ~,' ---3 ram. The ammals were killed 10-14 days later for h~stochemlcal analysis and for [all]CA and [aH]5-HT uptake studies as described above The results from the mtraventricular rejections of 5,6-HT are summanzed m Fig. 1. A dose-dependent reduction of ['~H]5-HT uptake Is observed, which is panicularly pronounced m the spinal cord. The uptake of [aH]5-HT m the cortex cerebn was practmally unchanged, whlch was also the case for the uptake of [aH]NA m all regions investigated. After the largest dose, 100/,g 5,6-HT mtraventncularly, some 100"
%
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40-
~
20'
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Fig 3. Effect of mtracerebral mjecnon of 5,6-HT (50 pg, 14 days as m Fig 2) on the it1 v t t m uptake of [aH]NA (10 7 A I , 10 ram) In slice~ from cerebral cortex, h y p o t h a l a m u s and cerebellar cortex Tile results are presented as percentages of controls (100 '- 5-7°o) Each column represents the mean L- S.E.M. o f 6-8 determinations
479
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4 O
•
j
A Fig 4 Nucleus raphe medlanus (A) and adjacent medial reticular formation of the mesencephalon. The area of the Injection needle is seen to the right. 5,6-HT was infused medially (25/rg/10/11) 14 days before kllhng The strongly yellow fluorescent dots represent the damaged 5-HT axons in this area The 5-HT cell bodies (A) on the other hand appear to be relahvely unaffected " 120
of the animals did not survive, and in many cases a marked dllatatmn of the ventricles was observed indicating unspecific destrucnon of brain tissue by 5,6-HT. These results are m agreement with the results of previous workers (see refs 2 and 6). The fluorescence histochemlstry revealed accumulations of yellow fluorescence in swollen and distorted fibers close to the surface of the subarachnoldal space m the medulla oblongata and spinal cord. The accumulattons were most pronounced during the first days after the operation. A dose-dependent reduction of the yellow fluorescent nerve terminals was observed in the brain stem and particularly in the spinal cord The 5-HT cell bodies were not notably affected. The studies on the extensor hlndhmb reflex revealed a reduced enhancement of the reflex following nialamlde--chlorlmipramine treatment in the animals that had received 100 ktg 5,6-HT intraventricularly. These findings give functional evidence for a degeneration of the descending bulbospinal 5-HT neurons. The results from the uptake studies in the rats treated with local injections of 5,6-HT into raphe area are summarized in Fig 2 The data reveal a marked and selective decrease of [aH]5-HT uptake m the hypothalamus, an effect which is dose-dependent. The [aH]NA uptake m the hypothalamus was not significantly affected (Fig. 3), nor was the [JH]5-HT uptake in the cortex cerebri and in the spinal cord (Fig 2) The [aH]5-HT uptake in the cerebellar cortex was slightly reduced (Fig. 2). Fluorescence analysis revealed marked accumulations of yellow fluorescence in the medial ascending 5-HT fibers from the groups B7 and B8 (Fig. 4) In a preliminary microspectrofluorimetrical study the emission spectra of the yellow fluorescence accumulations have so far only showed emission maxima at 525 nm, typical of that of the 5-HT-fluorophor. Fourteen days after the 5,6-HT injection, signs of sprouting from the strongly fluorescent 5-HT axons were observed m the rejection area. Strongly
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I )g 5 Nuclcu~ laphc d~,~ahs ,,a~dal it) Ibc 'qtc of reJection Fhe 5,6-HT |njectlon (25 /¢g,20 ,I) was made ink) (he 5-HT d \ O l ) ~ o l l g l l l a t l l l g ~l'Om th~ nucleus at a level anterior to the 5-HT cctl bodies (about l nllll) ][[ t.dl] b c ~een that M)o)lg fluorescence appeals in the 5-HT axons ~ ~) tH' I(~ the 5-HT cell bod~ bill then abruptl~ e~)d, 200 fluorescent growth cones emerging fi-om weakly fluorescent slender axons were observed. T h e r e ~ e r e also signs o| mner~atlon or" nerve cell bodies near the site o f m j e c t l o n . These cell bodies are normall~ not innervated by 5-HT nerve terminals a n d do not contain m o n o a m m c s . The plhng up ol ~ellow fluorescence in the axons could be traced up into the 5-HT cell b o d y areas, which themselves had not been affected (Fig. 5). However, the cell bodies showed reduced a c c u m u l a t i o n s o f 5 - H T fluorescence following m a l a m i d e t r y p t o p h a n l r e a t m e n t Unspecific d a m a g e was only obser~,ed close to the site o f injection (200 fire) but with small ~olumes and high doses f a l r b large uHspectBc ~lecrosls call occur The 5-HT nerve t e r m i n a l s had c o m p l e t e l y d i s a p p e a r e d m the nucleus s u p r a c h i a s m a t l c u s 10-14 days following the 5,6-HT injection a n d m the globus palhdus N o fluorescence was observed, not even after n i a l a m l d e - t r y p t o p h a n , a t r e a t m e n t known to cause a m a r k e d increase m the yellow 5 - H T fluorescence. One d a y after the reJection, on the o t h e r hand, an increased fluorescence intensity in the s u p r a c h i a s m a t l c 5 - H T nerve terminals ~ a s observed InJections of the solvent into the raphe a r e a p r o d u c e d no effects on the 5-HT neurons. Studies on gross b e h a v i o r showed that following local injection o f 5,6-HT m the r a p h e area, the m a l a m ~ d e - t r y p t o p h a n s y n d r o m e o f i m m o N h z a t i o n , t r e m o r , a b d u c tion o f the h m b s and head m o v e m e n t s was r e d u c e d In addition, the h y p e r t h e r m m observed after n i a l a m ~ d e - t r y p t o p h a n t r e a t m e n t was less p r o n o u n c e d after this type o f 5,6-HT injection InJections o f 5,6-HT {25 fig) into the dorsal a n d ventral N A and the m g r o neostriatal D A bundles did not result m any sigmficant changes o f [ a H ] N A and [aH]D A u p t a k e m the cortex cerebrl and the neostriatum, respectively, or m the dlsappetuance o f N A nerve terminals m the teled~encephalon, as shown in the fluorescence microscopical analysis However, a few hours after mjecUon in the substantla m g r a a r e a yellowish a c c u m u l a t l o n s o f fluorescence were f o u n d m the D A tracts a n d in the
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D A cell bodies, p r o b a b l y representing u p t a k e o f 5,6-HT into these nerve cells an d their axons. Th~s u p t a k e was a p p a r e n t l y insufficient to cause any substantial d e g e n e r a tion o f the N A and D A neurons. These studies reveal that 5,6-HT provides a tool for the selective chemical d e g e n e r a t i o n o f central 5 - H T tracts when p r o p e r l y used. In addition, the results presented p r o v i d e evidence for the existence o f a specific medial 5 - H T tract from groups B7, B8 which is i n v o l v e d in the subcort~cal l n n e r v a t l o n o f the telediencephalon, especmlly the h y p o t h a l a m u s . P r e l i m i n a r y data suggest that m o r e laterally ascending 5 - H T fibers partly those f r o m g r o u p B9 (surrounds the dorsal surface to the lemniscus medialls (see ref. 7)) send fibers to the cortex cerebri as revealed w~th 5,6-HT injections laterally into the v e n t r o m e d m l t e g m e n t u m (the area o f g r o u p B9) Th e present studies also indicate that this t e c h n i q u e is helpful in the analysis o f the functional role o f the central 5 - H T neurons, a l t h o u g h it should be emphasized that 5,6-HT can cause unspecific d a m a g e o f brain t~ssue. T h e r e f o r e f u r t h er investigations are needed before ~t can be used routinely m studies on 5 - H T t r a n s m i t t e r mechanisms. This study has been s u p p o r t e d by G r a n t s B73-O4X-715 and B73-04X-2295-06B f r o m the Swedish M e d ic a l R e s e a r c h Council. T h e skilful technical assistance of Mr s
Agneta
Ehasson,
Mrs
Ulla-Br~tt
F l n n m a n , Miss Bodil F l o c k and Miss Elisabeth H a g m a n is gratefully a c k n o w l e d g e d
I ANDI~N, N -I~ , Discussion of serotonln and dopamme m the extrapyramldal system, ,4dvanc Pharmacol, 6A (1968) 347 349 2 BAUM3¢RTEN, H G , BJORKLUND, A , LACHE',IMAYER, L , NOBIb,, A , AND STENE\I, U , Longlasting selective depletion of brain serotonln by 5,6-dlhydroxytryptamme, Acta phystol scand, Suppl 373 (1971) 3 BAUM,~ARTEN, H G , BJORKLUND,A , HOLSTEIN, A F , AND NOBI~,, A , Chemical degeneration of lndolamlne axons m rat brain by 5,6-dlhydro×ytryptamme, Z ZelIJbt ~ch, 129 (1972) 256 271 4 B~UM~ARTEN, H. G , L&CHENMAYER, L , ~ND SCHEOSSBERGER, H G , E,,ldence for a degeneration of mdolam~ne containing nerve terminals ~n rat brain, induced by 5,6-dlhydroxytr~ptamme, Z Zell]o~ch , 125 (1972) 553-569. 5 CORRODI, H., AND JONSSON, G , The formaldehyde fluorescence method for the histochem~cal demonstration of b~ogenlc monoammes. A rewew on the methodology, J Htstochem Cyto~hem , 15 (1967) 65-78 6 COSTA, E , D,XL~, J , LEFEVRE, H . , MEEK, J , REVUELTA, A , SPARRO, F , AND STRADA, S , Serotomn and catecholamme concentrations m brain of rats reJected lntracerebrally with 5,6-dthydroxytryptamme, Brain Research, 44 (1972) 304-308 7 DAHLSTROM, A , AND FUXE, K , Evidence for the existence of monoamme nevrons m the central nervous system. I Demonstration of monoamlnes m the cell-bodies of brain stem neurons, A~taplo~tol. ~ca~td, 64, Suppl 232 (1964). 8 DCLY, J , FuxE, K , AND JONSSON, G , 5,6-D~hydroxytryptamme' a new tool m mapping out of central 5-HT neurons, Ab~tr 4th Int. Congr Ht~to~hem and Cytochem , K~oto, (1972) 487 488 9 FALCK, B , HILLARP, N.-/~, THIEME, G , A~,D TORP, A , Fluorescence of catecholamlnes and related comnounds condensed with formaldehyde, J Ht~tochem Cvto~hem , 10 (1962) 348 354 l0 F U \ E , K , HOKFELT, T , JONSSONr, G , A.ND UNGERSTEDT, U , Fluorescence m~croscopy m neuroanatomy In W J H N¢UTA ~'~D S O E EBBESSO'q(Eds), Contemporao' Re~ea~ch m Netnoanatomy, Springer, l~erhn, 1970, pp. 275 314 11 GR~HAME-SMIT~,D G , Studies m v~vo on the relationship between brain tryptophan, brain 5-HT synthes~s and hyperactivity m rats treated with a monoamme ox~dase inhibitor and Ltryptophan, J N e u t o ( h e m , 18 (1971) 1953.
]2 JONS~,()\. (~, Ou,llltltatto~ o[ b~ogem~ ii]onoanllocq denlgnstrated ~ttb .~h~ I o f l t h t l ~ . ' .[~ fluorescence method, P~,~,, t h w o c h e l ~ ( , t o c h e m , 2 ( 1 9 7 1 ) 2 9 9 334 13 JONSS'~'n, (; F,. \~, k . x'.t, D i n ; l Intracerebrat mje~tkm,, of 5d,-dllq_,,d~okSh3.,ta ~J,. (5,6-HF) ExJdel~c {oL ~,.le~_ll~,c dcgencrauon of t~cntla[ ~-h,,dro\3tlyptammc n c m o r ~, ~,, pllcIttlltll~o[ Abr! , 31. bdl~pl I (1972) 24 14 JONSSI)N (~ , A.ND Szk ~, I,5, Q H , Degenerative and non-degeneraU\e ettects ol I ~ - h > , d r o \ ) d t q ~ , ~ c on adrenerglc nez'c~ ,1 P h r . m ~ . ¢ , l exl) Ftte~ . 180 (1972) 625 635 15 KOF\t(,, J F R , x'~I) KI [L'I'I'L, R R , fl/te R a l B r o t h a ~Sleteotart( ,tt[as o l The [ o t ¢ l ) i t . ~ ,#ta Lol~et Pa~ls o]" 171e Btal,l Nletll. Wtlham~ and Wflklns, Baltunore, 1963 16 M u K. J , Fu-.~, k , ~-.t) A\Dz-~. N -h , Effects of antidepressant drugb of the ~mlprammc tSpc on centrdt 5-h)dro',3tt~ptamme neurotran,,mr,,,Ion. E m o p .I P h a r m a ( o l , ~) (1970) 325 332 17 Sa( us, ('H aN~) .IoNsso\ (_z , D,3gdner0.tlon of central and peripheral n o r a d r e n a h n c t~e~,H,,w, pzodttued by 6-h3d~o\~-DOPA, J ~ / e u ~ , ~ h e m , 19 (1972) 1561-1575 18 TI'.ANzIR 1 P , ~ \ D TH,~NI'~. H . t'hram.)rpholog~sche Velanderungcn der ,,)lnpathr,~.hen Nervenend~gungcn de~ K a t / e na~_h Vorbehandlung mlt 5- und 6 - H ~ d r o x y - D o p a m m , \'(zt/zt~ttS~hmzedeh.,e's t ~ h e t p P~th Pha~mal. . 257 (19671 343 19 U N ( , E R N I I D T , e. ~}-Hydroxs-dopanamc reduced degenerauon of central m o n o a n u n e neHon',, /;{ll~q~ [ PJt(lFllldtO/. 5 (1968)107 lit) 20 UNcd RSFI I)l, U , LI'~c ol Jnha~.e~eb~al nUc~.t~ons oi 6-h3 d r o x y d o p a m m e as a tool lot zno~pho!ogit.al and ltLnt.t~onal Mud~e,, on ¢e~atral uatecholamme netwon,, In T M A L M F ( ) R S A N D m Tttol-~Y (Kd', }. 6 - H v t h o x ieb'p(t~ll,~t' ,~/,¢[ f ,/h'* /lOhlltlllh' Nt('ttt()ll~, N o r t h - H o l l a n d Publ . AmMerdan~ t'171. pp 315 3~2 21 U r F T S k ~ N .I , x",t) lxl-rSt ",. L L . Etl.:~r., of o - h y d r o x y d o p a m m e on n o r a d r e n a h n e - c o n t d m m g n,:urone, ,~ the ~at b~a,n, .\tlllltt, ] ond .. 221 (1969) 557 559