622
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v a r i e d f r o m n e r v e - l i k e t h r e a d s t o c o m p l e x p l e x u s e s in w h i c h i n d i v i d u a l fibres could b e seen (Figures 3 a n d 4). I n t h e 3 areas f r o m e a c h of t h e e x p e r i m e n t a l sections a t l e a s t 4 labelled s t r u c t u r e s were f o u n d , t y p i c a l p h o t o m e t e r r e a d i n g s b e i n g as s h o w n in t h e Table. No such s t r u c t u r e s were f o u n d in l u n g tissue f r o m t h e c o n t r a l a t e r a l side or t h e u n t r e a t e d c o n t r o l hen. Conclusion. W e c o n c l u d e t h a t t h i s t e c h n i q u e c a n b e used t o d e m o n s t r a t e t h e p e r i p h e r a l d i s t r i b u t i o n of v a g a l a f f e r e n t n e r v e fibres w h i c h h a v e t h e i r cell b o d i e s i n t h e nodose g a n g l i o n a n d t h i s w o r k is n o w in progress.
EXPERIENTIA 31/5
Rdsumd. P a r m a r q u a g e d u g a n g l i o n n o d o s u m avec la *H-leucine n o u s a v o n s 6tudi6 Ia r e p a r t i t i o n des aff6rences vagales a n n i v e a u de la p a t t i e inf6rieure du s y s t 6 m e r e s p i r a t o i r e d u p o u l e t p a r des t e c h n i q u e s d ' a u t o r a d i o graphic. A. J. BOWER, V. MOLONY a n d C. M. BROWN Department ol Anatomy and Department o] Veterinary d natomy, University of Liverpool, Brownlow Hill, P.O. Box 147, Liverpool L69 3 B X (England), 12 November 1974.
Samplin~ of the Rodent Dorsal Lateral Geniculate Nucleus for Electron Microscopy I n o u r l a b o r a t o r y we h a v e b e e n c o n c e r n e d w i t h t h e e l e c t r o n m i c r o s c o p y of r e t i n a l a x o n t e r m i n a t i o n a n d d i s t r i b u t i o n of t h e n e u r o n s of t h e dorsal l a t e r a l g e n i c u l a t e n u c l e u s ( D L G N ) in t h e r a t . I n o r d e r t h a t c o r r e l a t i o n c o u l d be m a d e w i t h o t h e r a n a t o m i c a l a n d physiological d a t a i t was n e c e s s a r y to d e v e l o p a n a c c u r a t e s a m p l i n g t e c h n i q u e t o i n s u r e precise localization w i t h i n t h e D L G N of i n f o r m a t i o n o b t a i n e d in u l t r a s t r u c t u r a l studies. T h e b r a i n s of l a b o r a t o r y r a t s were p e r f u s e d t h r o u g h t h e a o r t a w i t h a m i x t u r e of 4 % p a r a f o r m a l d e h y d e a n d 0.5% g l u t a r a l d e h y d e w i t h 4 % sucrose in 0.1 M p h o s p h a t e b u f f e r ( p H 7.3). T h e p e r f u s a t e was used a t r o o m t e m p e r a t u r e . Sufficient p r e s s u r e for g r a v i t y p e r f n s i o n was achieved by maintaining the perfusate reservoir at a h e i g h t of 3.5 feet (78.5 m m Hg) a b o v e t h e a n i m a l s . T h e p e r f u s i o n s c o n t i n u e d for a t o t a l of 20 min. I m m e d i a t e l y a f t e r p e r f u s i o n e a c h a n i m a l w a s dec a p i t a t e d b y first s e v e r i n g t h e soft tissue s t r u c t u r e s a r o u n d t h e n e c k region. T h i s w a s followed b y a l a m i n e c t o m y to expose t h e u p p e r cervical cord. T h e cervical spinal cord was t r a n s e c t e d w i t h a s h a r p r a z o r blade. T h i s step is p e r f o r m e d t o a v o i d s t r e t c h i n g a r t i f a c t of t h e b r a i n s t e m . T h e c a t v a r i u m was r e m o v e d w i t h a s m a l l p a i r of ' r o n g e u r s ' .
After removing the brain from the cranial cavity, the b r a i n was sliced s a g i t t a l y . E a c h half of t h e b r a i n was t r i m m e d so t h a t t h e e n t i r e desired n u c l e a r region was c o n t a i n e d in o n e t i s s u e block. T h e tissue b l o c k was imm e r s e d for t w o h o u r s in cold 4 % g l u t a r a l d e h y d e in t h e s a m e b u f f e r as t h e p e r f u s a t e . T h e b l o c k was t r a n s f e r r e d t o a rinse s o l u t i o n of 0.1 M p h o s p h a t e b u f f e r a n d 4 % sucrose. T h e b l o c k of b r a i n tissue was f u r t h e r t r i m m e d u n d e r a dissecting m i c r o s c o p e to t h e s m a l l e s t d i m e n s i o n s t h a t still c o n t a i n e d t h e e n t i r e n u c l e a r g r o u p of choice. W i t h t h e aid of a S m i t h - F a r q u h a r tissue s e c t i o n e r t h i s tissue b l o c k was c u t i n t o serial slices 250 [~m t h i c k . I n t e g r i t y of t h e slice series was m a i n t a i n e d b y t h e a g a r m e d i a used to s u p p o r t t h e block. T h e slices were p l a c e d serially in p a r t i t i o n e d p l a s t i c cases t h a t c o n t a i n e d t h e rinse solution. Sections were r i n s e d for a n a d d i t i o n a l 10 rain. P r i o r to p o s t - f i x a t i o n e a c h slice w a s t r a n s f e r r e d t o a s m a l l p e t r i d i s h t h a t c o n t a i n e d a s o l u t i o n of 0.5% o s m i u m - t e t r o x i d e a n d 4 % sucrose in 0.1 M p h o s p h a t e buffer. A f t e r 1 t o 2 m i n in t h i s s o l u t i o n t h e surface of t h e slice a c q u i r e d a l i g h t b r o w n color. E a c h slice was n e x t t r a n s f e r r e d to a pool of p h o s p h a t e , b u f f e r e d sucrose o n a clear p l a s t i c disk o n t h e t r a n s i l l u m i n a t i n g s t a g e of a diss e c t i n g microscope (Figure 1). T h e o s m i c a t e d m y e l i n a t e d
Fig. 1 and 2. Transverse slices through the diencephalon of the rat. The slice has been lightly osmieated. Note the prominent landmarks of myelinated fibre tracts and blood vessels. An arrow in Figure 2 indicates sample taken for eleetron microscopy. Dorsal and ventral lateral geniculate nuclei (DLGN, VLGN) ; superior thalamie radiation (str); crus cerebri (cc); blood vessels (b). • 12.
15.5. 1975
Speeialia
fibre b u n d l e s p r o v i d e d t h e n e c e s s a r y o r i e n t a t i o n so t h a t , in t h e s e studies, w e d g e - s h a p e d tissue s a m p l e s of t h e D L G N c o u l d b e t a k e n (Figure 2). T h e w e d g e - s h a p e d s a m p l e s were p l a c e d in i n d i v i d u a l glass vials a n d f u r t h e r processed b y p o s t - f i x a t i o n for 2 h in 1 % o s m i u m - t e t r o x i d e a n d 4 % sucrose in 0.1 M p h o s p h a t e buffer. T h e s a m p l e s were r a p i d l y d e h y d r a t e d t h r o u g h a g r a d e d series of e t h y l alcohols a n d p r o p y l e n e oxide t h e n i n f i l t r a t e d in E p o n , All wedges were c a r e f u l l y o r i e n t e d as t h e y were e m b e d d e d in f i a t m o l d s t h a t c o n t a i n e d E p o n so t h a t t r a n s v e r s e semit h i n a n d t h i n sections could b e cut. S e m i - t h i n sections of t h e e n t i r e b l o c k face, 1 t o 3 ~zm t h i c k , were c u t w i t h glass k n i v e s o n a Sorvall P o r t e r B l u m MT2 u l t r a - m i c r o t o m e . T h e s e sections were s t a i n e d w i t h M a l l o r y ' s a z u r e I f - m e t h y l e n e blue 1 a n d e x a m i n e d w i t h t h e l i g h t microscope. F r o m t h e s t u d y of t h e semit h i n s e c t i o n s a n a p p r o p r i a t e a r e a c o u d b e c h o s e n for subsequent thin sectioning and study. T h i s s a m p l i n g t e c h n i q u e p r o v i d e d for t h e precise localization w i t h i n t h e D L G N of t h e t h i n sections used in e l e c t r o n microscopy. T h e l o c a l i z a t i o n a n d o r i e n t a t i o n of a
623
D L G N tissue s a m p l e is i l l u s t r a t e d in F i g u r e s 1 a n d 2. T h e c u r v e d side of t h e w e d g e - s h a p e d s a m p l e (arrow in F i g u r e 2) p e r m i t t e d t h e i m m e d i a t e i d e n t i f i c a t i o n of t h e o p t i c t r a c t surface. This, a l o n g w i t h o r i e n t a t i o n d r a w i n g s c o m p l e t e d a t t h e t i m e of i n i t i a l o s m i c a t i o n , e n a b l e d one t o c o r r e c t l y e m b e d t h e w e d g e - s h a p e d s a m p l e in a f l a t mold. B y t h i s m e t h o d t h e t h i n sections could a l w a y s b e o b t a i n e d in t h e desired p l a n e of section. To d a t e t h e s a m p l e s were all e m b e d d e d so t h a t t r a n s v e r s e sections were m a d e t h r o u g h t h e n u c l e u s f r o m r o s t r a l to caudal. Since t h e t h i c k n e s s of e a c h s a m p l e was k n o w n , it was possible t o d e t e r m i n e t h e r o s t r a l t o c a u d a l l o c a t i o n of e a c h of t h e s e m i - t h i n a n d t h i n sections w i t h i n t h e D L G N . T h e n e u r o p i l of t h e D L G N is p a c k e d b e t w e e n t h e m y e l i n a t e d a x o n b u n d l e s . Careful e x a m i n a t i o n of s u c h s e m i - t h i n sections e n a b l e d us to i d e n t i f y p o c k e t s of n e u r o p i l ( d o t t e d line in F i g u r e 3) in t h e D L G N . I n t h e s e n e u r o p i l p o c k e t s t h e r e d u c e d n u m b e r of m y e l i n a t e d s t r u c t u r e s was n o t i c e a b l e . S u c h a r e a s were u s u a l l y s u r r o u n d e d b y several n e u r o n a l p e r i k a r y a . T h e s e areas of special i n t e r e s t were isolated i n t o p y r a m i d f o r m b y t h e ' m e s a - p y r a m i d ' t e c h n i q u e 2. T h i n sections of t h e s e n e u r o p i l p o c k e t s u s u a l l y d e m o n s t r a t e d m a n y a r e a s of c o m p l e x synaptic contacts.
Zusammen/assung. U m e i n g e h e n d e l e k t r o n e n m i k r o skopisctl d e n Verlauf, die E n d i g u n g e n u n d die V e r t e i l u n g d e r A x o n e y o n d e r R e t i n a des A u g e s i m N u c l e u s genic n l a t n s dorsalis l a t e r a l i s u n t e r s u c h e n zu k S n n e n , w u r d e die h i e r b e s c h r i e b e n e sichere M e t h o d e ffir Lrbersicht u n d L o k a l i s i e r u n g a u s g e a r b e i t e t . M i t dieser M e t h o d e k 6 n n e n n u n g e n a u lokalisierte Stellen i m Nucleus g e n i c u l a t u s dorsalis l a t e r a l i s a u f g e s u c h t u n d a n s c h l i e s s e n d m i t d e m E l e k t r o n e n m i k r o s k o p u n t e r s u c h t werden. R. M. t'{RI~Et~.EL3
Department o/ Anatomy, P. O. Box 906, Medical College o/Virginia, Richmond (Virginia 23298, USA), 73 November 1974.
Fig. 3. Transverse 1 fxm section through DLGN. Dashed line indicates pocket of neuropil. Note optic tract (OT) on surface of sample. • 600.
1 K. C. RICHARDSON,L. JANET and E. H. I~'INKE, Stain Techn. 35, 313 (1960). 2 S. M. M'cGEE-RussEL and G. GOSZTONYI,Nature, Loud. 274, 1204 (1967). 3 Supported by U.S.P.H.S. Grant No. 5 T01-GMI410-05. The author is grateful to Dr. H. R. SEIBEL for reading this report.
S e p a r a t i o n of H o m o v a n i l l i c Acid and V a n i l l y l m a n d e l i c Acid w i t h I o n - E x c h a n g e R e s i n C o l u m n I n t h e s t u d y o n t h e roles of d 0 p a m i n e (DA) a n d norep i n e p h r i n e (NE) in b r a i n f u n c t i o n s , it is e s s e n t i a l to e x a m i n e t h e m e t a b o l i c c h a n g e s of t h e s e a m i n e s . A l t h o u g h v a r i o u s m e t h o d s for t h e s i m u l t a n e o u s e s t i m a t i o n of catecholamines and their metabolites in the brain have b e e n p r e s e n t e d , t h e y l e a v e m u c h to b e i m p r o v e d w i t h regard to the separation, the recovery rate and the practical s i m p l i c i t y of procedures. A b o v e all, t h e s e p a r a t i o n of h o m o v a n i l l i c acid (HVA) a n d v a n i l l y l m a n d e l i c acid (VMA) h a s b e e n c o n s i d e r e d m o s t difficult. T h e r e are t w o s t u d i e s o n t h e s e p a r a t i o n m e t h o d of H V A a n d V M A b y c o l u m n c h r o m a t o g r a p h y . TAYLOR a n d LAVERTY 1 h a d s e p a r a t e d t h e s e acids of t h e b r a i n b y e l u t i o n of a D o w e x 1 - X 2 a n i o n e x c h a n g e r e s i n c o l u m n w i t h i n c r e a s i n g conc e n t r a t i o n s of HC1. U n d e r o u r e x p e r i m e n t a l c o n d i t i o n s ,
h o w e v e r , some o v e r l a p of t h e acids i n t o adj a c e n t f r a c t i o n s r e m a i n e d w i t h t h e i r m e t h o d . MESSI~IA et al. 2 r e p o r t e d s e p a r a t i o n of u r i n a r y H V A a n d V M A w i t h basic a l u m i n a column. W e h a v e successfully s e p a r a t e d H V A f r o m V M A u s i n g t h e m e t h o d h e r e given. I o n - e x c h a n g e resin e m p l o y e d was Dowex-l-X2 a n i o n e x c h a n g e resin, 200-400 m e s h (chloride form) (Dow C h e m i c a l Co.). T h e b r a i n e x t r a c t of ~vVistar r a t s was o b t a i n e d b y h o m o g e n i z i n g t h e b r a i n , e x c e p t for t h e o l f a c t o r y b u l b , p i n e a l g l a n d a n d cerebellum, in 5 - 6 v o l u m e s of ice-cold 0.4 N p e r c h l o r i c acid. A f t e r 1 K. IV[. TAYLOR and R. LAVERTY,J. Neurochem. 16, 1361 (1969). 2 F. S. MESSrHA, E. BAKUTIS and V. FRASKOS, Clinica chim. Aeta d5, 159 (1973).
Speeialia
v a r i e d f r o m n e r v e - l i k e t h r e a d s t o c o m p l e x p l e x u s e s in w h i c h i n d i v i d u a l fibres could b e seen (Figures 3 a n d 4). I n t h e 3 areas f r o m e a c h of t h e e x p e r i m e n t a l sections a t l e a s t 4 labelled s t r u c t u r e s were f o u n d , t y p i c a l p h o t o m e t e r r e a d i n g s b e i n g as s h o w n in t h e Table. No such s t r u c t u r e s were f o u n d in l u n g tissue f r o m t h e c o n t r a l a t e r a l side or t h e u n t r e a t e d c o n t r o l hen. Conclusion. W e c o n c l u d e t h a t t h i s t e c h n i q u e c a n b e used t o d e m o n s t r a t e t h e p e r i p h e r a l d i s t r i b u t i o n of v a g a l a f f e r e n t n e r v e fibres w h i c h h a v e t h e i r cell b o d i e s i n t h e nodose g a n g l i o n a n d t h i s w o r k is n o w in progress.
EXPERIENTIA 31/5
Rdsumd. P a r m a r q u a g e d u g a n g l i o n n o d o s u m avec la *H-leucine n o u s a v o n s 6tudi6 Ia r e p a r t i t i o n des aff6rences vagales a n n i v e a u de la p a t t i e inf6rieure du s y s t 6 m e r e s p i r a t o i r e d u p o u l e t p a r des t e c h n i q u e s d ' a u t o r a d i o graphic. A. J. BOWER, V. MOLONY a n d C. M. BROWN Department ol Anatomy and Department o] Veterinary d natomy, University of Liverpool, Brownlow Hill, P.O. Box 147, Liverpool L69 3 B X (England), 12 November 1974.
Samplin~ of the Rodent Dorsal Lateral Geniculate Nucleus for Electron Microscopy I n o u r l a b o r a t o r y we h a v e b e e n c o n c e r n e d w i t h t h e e l e c t r o n m i c r o s c o p y of r e t i n a l a x o n t e r m i n a t i o n a n d d i s t r i b u t i o n of t h e n e u r o n s of t h e dorsal l a t e r a l g e n i c u l a t e n u c l e u s ( D L G N ) in t h e r a t . I n o r d e r t h a t c o r r e l a t i o n c o u l d be m a d e w i t h o t h e r a n a t o m i c a l a n d physiological d a t a i t was n e c e s s a r y to d e v e l o p a n a c c u r a t e s a m p l i n g t e c h n i q u e t o i n s u r e precise localization w i t h i n t h e D L G N of i n f o r m a t i o n o b t a i n e d in u l t r a s t r u c t u r a l studies. T h e b r a i n s of l a b o r a t o r y r a t s were p e r f u s e d t h r o u g h t h e a o r t a w i t h a m i x t u r e of 4 % p a r a f o r m a l d e h y d e a n d 0.5% g l u t a r a l d e h y d e w i t h 4 % sucrose in 0.1 M p h o s p h a t e b u f f e r ( p H 7.3). T h e p e r f u s a t e was used a t r o o m t e m p e r a t u r e . Sufficient p r e s s u r e for g r a v i t y p e r f n s i o n was achieved by maintaining the perfusate reservoir at a h e i g h t of 3.5 feet (78.5 m m Hg) a b o v e t h e a n i m a l s . T h e p e r f u s i o n s c o n t i n u e d for a t o t a l of 20 min. I m m e d i a t e l y a f t e r p e r f u s i o n e a c h a n i m a l w a s dec a p i t a t e d b y first s e v e r i n g t h e soft tissue s t r u c t u r e s a r o u n d t h e n e c k region. T h i s w a s followed b y a l a m i n e c t o m y to expose t h e u p p e r cervical cord. T h e cervical spinal cord was t r a n s e c t e d w i t h a s h a r p r a z o r blade. T h i s step is p e r f o r m e d t o a v o i d s t r e t c h i n g a r t i f a c t of t h e b r a i n s t e m . T h e c a t v a r i u m was r e m o v e d w i t h a s m a l l p a i r of ' r o n g e u r s ' .
After removing the brain from the cranial cavity, the b r a i n was sliced s a g i t t a l y . E a c h half of t h e b r a i n was t r i m m e d so t h a t t h e e n t i r e desired n u c l e a r region was c o n t a i n e d in o n e t i s s u e block. T h e tissue b l o c k was imm e r s e d for t w o h o u r s in cold 4 % g l u t a r a l d e h y d e in t h e s a m e b u f f e r as t h e p e r f u s a t e . T h e b l o c k was t r a n s f e r r e d t o a rinse s o l u t i o n of 0.1 M p h o s p h a t e b u f f e r a n d 4 % sucrose. T h e b l o c k of b r a i n tissue was f u r t h e r t r i m m e d u n d e r a dissecting m i c r o s c o p e to t h e s m a l l e s t d i m e n s i o n s t h a t still c o n t a i n e d t h e e n t i r e n u c l e a r g r o u p of choice. W i t h t h e aid of a S m i t h - F a r q u h a r tissue s e c t i o n e r t h i s tissue b l o c k was c u t i n t o serial slices 250 [~m t h i c k . I n t e g r i t y of t h e slice series was m a i n t a i n e d b y t h e a g a r m e d i a used to s u p p o r t t h e block. T h e slices were p l a c e d serially in p a r t i t i o n e d p l a s t i c cases t h a t c o n t a i n e d t h e rinse solution. Sections were r i n s e d for a n a d d i t i o n a l 10 rain. P r i o r to p o s t - f i x a t i o n e a c h slice w a s t r a n s f e r r e d t o a s m a l l p e t r i d i s h t h a t c o n t a i n e d a s o l u t i o n of 0.5% o s m i u m - t e t r o x i d e a n d 4 % sucrose in 0.1 M p h o s p h a t e buffer. A f t e r 1 t o 2 m i n in t h i s s o l u t i o n t h e surface of t h e slice a c q u i r e d a l i g h t b r o w n color. E a c h slice was n e x t t r a n s f e r r e d to a pool of p h o s p h a t e , b u f f e r e d sucrose o n a clear p l a s t i c disk o n t h e t r a n s i l l u m i n a t i n g s t a g e of a diss e c t i n g microscope (Figure 1). T h e o s m i c a t e d m y e l i n a t e d
Fig. 1 and 2. Transverse slices through the diencephalon of the rat. The slice has been lightly osmieated. Note the prominent landmarks of myelinated fibre tracts and blood vessels. An arrow in Figure 2 indicates sample taken for eleetron microscopy. Dorsal and ventral lateral geniculate nuclei (DLGN, VLGN) ; superior thalamie radiation (str); crus cerebri (cc); blood vessels (b). • 12.
15.5. 1975
Speeialia
fibre b u n d l e s p r o v i d e d t h e n e c e s s a r y o r i e n t a t i o n so t h a t , in t h e s e studies, w e d g e - s h a p e d tissue s a m p l e s of t h e D L G N c o u l d b e t a k e n (Figure 2). T h e w e d g e - s h a p e d s a m p l e s were p l a c e d in i n d i v i d u a l glass vials a n d f u r t h e r processed b y p o s t - f i x a t i o n for 2 h in 1 % o s m i u m - t e t r o x i d e a n d 4 % sucrose in 0.1 M p h o s p h a t e buffer. T h e s a m p l e s were r a p i d l y d e h y d r a t e d t h r o u g h a g r a d e d series of e t h y l alcohols a n d p r o p y l e n e oxide t h e n i n f i l t r a t e d in E p o n , All wedges were c a r e f u l l y o r i e n t e d as t h e y were e m b e d d e d in f i a t m o l d s t h a t c o n t a i n e d E p o n so t h a t t r a n s v e r s e semit h i n a n d t h i n sections could b e cut. S e m i - t h i n sections of t h e e n t i r e b l o c k face, 1 t o 3 ~zm t h i c k , were c u t w i t h glass k n i v e s o n a Sorvall P o r t e r B l u m MT2 u l t r a - m i c r o t o m e . T h e s e sections were s t a i n e d w i t h M a l l o r y ' s a z u r e I f - m e t h y l e n e blue 1 a n d e x a m i n e d w i t h t h e l i g h t microscope. F r o m t h e s t u d y of t h e semit h i n s e c t i o n s a n a p p r o p r i a t e a r e a c o u d b e c h o s e n for subsequent thin sectioning and study. T h i s s a m p l i n g t e c h n i q u e p r o v i d e d for t h e precise localization w i t h i n t h e D L G N of t h e t h i n sections used in e l e c t r o n microscopy. T h e l o c a l i z a t i o n a n d o r i e n t a t i o n of a
623
D L G N tissue s a m p l e is i l l u s t r a t e d in F i g u r e s 1 a n d 2. T h e c u r v e d side of t h e w e d g e - s h a p e d s a m p l e (arrow in F i g u r e 2) p e r m i t t e d t h e i m m e d i a t e i d e n t i f i c a t i o n of t h e o p t i c t r a c t surface. This, a l o n g w i t h o r i e n t a t i o n d r a w i n g s c o m p l e t e d a t t h e t i m e of i n i t i a l o s m i c a t i o n , e n a b l e d one t o c o r r e c t l y e m b e d t h e w e d g e - s h a p e d s a m p l e in a f l a t mold. B y t h i s m e t h o d t h e t h i n sections could a l w a y s b e o b t a i n e d in t h e desired p l a n e of section. To d a t e t h e s a m p l e s were all e m b e d d e d so t h a t t r a n s v e r s e sections were m a d e t h r o u g h t h e n u c l e u s f r o m r o s t r a l to caudal. Since t h e t h i c k n e s s of e a c h s a m p l e was k n o w n , it was possible t o d e t e r m i n e t h e r o s t r a l t o c a u d a l l o c a t i o n of e a c h of t h e s e m i - t h i n a n d t h i n sections w i t h i n t h e D L G N . T h e n e u r o p i l of t h e D L G N is p a c k e d b e t w e e n t h e m y e l i n a t e d a x o n b u n d l e s . Careful e x a m i n a t i o n of s u c h s e m i - t h i n sections e n a b l e d us to i d e n t i f y p o c k e t s of n e u r o p i l ( d o t t e d line in F i g u r e 3) in t h e D L G N . I n t h e s e n e u r o p i l p o c k e t s t h e r e d u c e d n u m b e r of m y e l i n a t e d s t r u c t u r e s was n o t i c e a b l e . S u c h a r e a s were u s u a l l y s u r r o u n d e d b y several n e u r o n a l p e r i k a r y a . T h e s e areas of special i n t e r e s t were isolated i n t o p y r a m i d f o r m b y t h e ' m e s a - p y r a m i d ' t e c h n i q u e 2. T h i n sections of t h e s e n e u r o p i l p o c k e t s u s u a l l y d e m o n s t r a t e d m a n y a r e a s of c o m p l e x synaptic contacts.
Zusammen/assung. U m e i n g e h e n d e l e k t r o n e n m i k r o skopisctl d e n Verlauf, die E n d i g u n g e n u n d die V e r t e i l u n g d e r A x o n e y o n d e r R e t i n a des A u g e s i m N u c l e u s genic n l a t n s dorsalis l a t e r a l i s u n t e r s u c h e n zu k S n n e n , w u r d e die h i e r b e s c h r i e b e n e sichere M e t h o d e ffir Lrbersicht u n d L o k a l i s i e r u n g a u s g e a r b e i t e t . M i t dieser M e t h o d e k 6 n n e n n u n g e n a u lokalisierte Stellen i m Nucleus g e n i c u l a t u s dorsalis l a t e r a l i s a u f g e s u c h t u n d a n s c h l i e s s e n d m i t d e m E l e k t r o n e n m i k r o s k o p u n t e r s u c h t werden. R. M. t'{RI~Et~.EL3
Department o/ Anatomy, P. O. Box 906, Medical College o/Virginia, Richmond (Virginia 23298, USA), 73 November 1974.
Fig. 3. Transverse 1 fxm section through DLGN. Dashed line indicates pocket of neuropil. Note optic tract (OT) on surface of sample. • 600.
1 K. C. RICHARDSON,L. JANET and E. H. I~'INKE, Stain Techn. 35, 313 (1960). 2 S. M. M'cGEE-RussEL and G. GOSZTONYI,Nature, Loud. 274, 1204 (1967). 3 Supported by U.S.P.H.S. Grant No. 5 T01-GMI410-05. The author is grateful to Dr. H. R. SEIBEL for reading this report.
S e p a r a t i o n of H o m o v a n i l l i c Acid and V a n i l l y l m a n d e l i c Acid w i t h I o n - E x c h a n g e R e s i n C o l u m n I n t h e s t u d y o n t h e roles of d 0 p a m i n e (DA) a n d norep i n e p h r i n e (NE) in b r a i n f u n c t i o n s , it is e s s e n t i a l to e x a m i n e t h e m e t a b o l i c c h a n g e s of t h e s e a m i n e s . A l t h o u g h v a r i o u s m e t h o d s for t h e s i m u l t a n e o u s e s t i m a t i o n of catecholamines and their metabolites in the brain have b e e n p r e s e n t e d , t h e y l e a v e m u c h to b e i m p r o v e d w i t h regard to the separation, the recovery rate and the practical s i m p l i c i t y of procedures. A b o v e all, t h e s e p a r a t i o n of h o m o v a n i l l i c acid (HVA) a n d v a n i l l y l m a n d e l i c acid (VMA) h a s b e e n c o n s i d e r e d m o s t difficult. T h e r e are t w o s t u d i e s o n t h e s e p a r a t i o n m e t h o d of H V A a n d V M A b y c o l u m n c h r o m a t o g r a p h y . TAYLOR a n d LAVERTY 1 h a d s e p a r a t e d t h e s e acids of t h e b r a i n b y e l u t i o n of a D o w e x 1 - X 2 a n i o n e x c h a n g e r e s i n c o l u m n w i t h i n c r e a s i n g conc e n t r a t i o n s of HC1. U n d e r o u r e x p e r i m e n t a l c o n d i t i o n s ,
h o w e v e r , some o v e r l a p of t h e acids i n t o adj a c e n t f r a c t i o n s r e m a i n e d w i t h t h e i r m e t h o d . MESSI~IA et al. 2 r e p o r t e d s e p a r a t i o n of u r i n a r y H V A a n d V M A w i t h basic a l u m i n a column. W e h a v e successfully s e p a r a t e d H V A f r o m V M A u s i n g t h e m e t h o d h e r e given. I o n - e x c h a n g e resin e m p l o y e d was Dowex-l-X2 a n i o n e x c h a n g e resin, 200-400 m e s h (chloride form) (Dow C h e m i c a l Co.). T h e b r a i n e x t r a c t of ~vVistar r a t s was o b t a i n e d b y h o m o g e n i z i n g t h e b r a i n , e x c e p t for t h e o l f a c t o r y b u l b , p i n e a l g l a n d a n d cerebellum, in 5 - 6 v o l u m e s of ice-cold 0.4 N p e r c h l o r i c acid. A f t e r 1 K. IV[. TAYLOR and R. LAVERTY,J. Neurochem. 16, 1361 (1969). 2 F. S. MESSrHA, E. BAKUTIS and V. FRASKOS, Clinica chim. Aeta d5, 159 (1973).
