HISTOCHEMICAL OBSERVATIONS OF FLUORESCENT BIOGENIC
AMINES IN CRYOSTAT SECTIONS OF PERIPHERAL
AND CENTRAL NERVOUS TISSUE
O. DUARTE-ESCALANTE J. P . PICKETT
R . E . PENDERGRASS
In our present report w e describe a simple histochemical modification of the cryostat technique for demonstrating the catecholamine and serotonin fluorescence in thin sections of peripheral and central nervous tissue.
The histochemical fluorescence method of Falck and H i l l a r p 7
has proven to be a valuable histological tool to study the monoamine localization in central and peripheral nervous tissue. The method shews an extreme sensi-tivity when the tissue is embedded in Araldite and sectioned down to 1 ¡x of thickness ( H o k f e l t0
) . A high degree of specificity is demonstrated by the microspectrophotometric measurements of the several fluorescent compounds of catecholamines and serotonin (Corrodi and J o n s s o n1
) . However, the original procedure cannot b e practically adapted as a regular histologic technique. Besides others, the main disadvantages a r e : 1) the freeze-drying is time-consuming; 2) the enzymatic system (cholinergic and M A O - e n z y m e ) is practically destroyed in a procedure that heats the tissue twice around
M A T E R I A L A N D M E T H O D S
S m a l l pieces of fresh nervous tissue ( u p to 0.5 cm b y side) w e r e obtained f r o m decapitated small animals, f r o m l a r g e a n i m a l s u n d e r a l i g h t b a r b i t u r a t e anesthesia ( c a t , rabbit, d o g ) , f r o m m a t e r i a l obtained a t the s l a u g h t e r house ( p i g , c o w ) , a n d h u m a n specimens f r o m the hospital surgical room. T h e fresh tissue was a d e q u a t e l y oriented in a small flask, tightly closed a n d then quickly frozen by immersion in a m i x t u r e of acetone in dry ice. T h e frozen piece of tissue w a s
T h e r e s e a r c h r e p o r t e d in this p a p e r w a s d o n e w h i l e t h e s e n i o r a u t h o r w a s s u p p o r t e d in p a r t b y a P o s t - D o c t o r a l R e s e a r c h T r a i n i n g F e l l o w s h i p f r o m t h e N a t i o n a l I n s t i t u t e o f M e n t a l H e a l t h , G r a n t N o . 5 t l - M H 8394-02 a n d 2 F 1 1 N B 2006-02 N S R A a t t h e D e p a r t m e n t o f P s y c h i a t r y a n d D e p a r t m e n t o f P a t h o l o g y , D u k e U n i -v e r s i t y M e d i c a l C e n t e r , D u r h a m , N o r t h C a r o l i n a .
transferred to a cryostat ( A m e s L a b T e k ) a t — 30°C. a n d the sections w e r e m a d e of 5 to 20 microns thickness. T h e sections w e r e m o u n t e d o n pre-cooled slides, a n d w e r e dried o v e r phosphorous pentoxide in a s m a l l e v a c u a t e d desiccator o v e r -night a t r o o m t e m p e r a t u r e . N o difference in the fluorescent results w a s observed by storing the desiccator ( w i t h the sections a n d phosphorus pentoxide inside) in the r e f r i g e r a t o r a t — 4°C. o r in the freezer a t — 4 0 ° C , b u t in these conditions the sections w e r e left u n d e r the d r y i n g process f o r m o r e than three d a y s .
A f t e r d r y i n g the sections, the phosphorus pentoxide w a s replaced by 10-15 gms. of p a r a f o r m a l d e h y d e e q u i l i b r a t e d a t 60-70% of r e l a t i v e humidity under the conditions suggested by H a m b e r g e r et a l .8
T h e e x c h a n g e of p a r a f o r m a l d e h y d e f o r phosphorus pentoxide w a s accomplished in a f e w seconds under a h o o d . T h e desiccator w a s a g a i n e v a c u a t e d a n d heated in an oven a t 65°C. for 45 minutes, a n d then cooled u n d e r the hood a t 25°C. for one hour. A f t e r that, the sections w e r e r e m o v e d a n d directly mounted w i t h H a r l e c o nonfluorescent m o u n t a n t m e d i u m in x y l e n e . G l y cerin is useful as a m o u n t i n g m e d i u m , too. M i n e r a l oil, h o w e v e r , is not r e c o m m e n d -ed, as it often g i v e s too m u c h fluorescent artifact. T h e fluorescent p r e p a r a t i o n s w e r e studied w i t h a Zeiss fluorescent microscope fitted w i t h a p r i m a r y filter B G 12 and a secondary filter 470 m/x.
B l a c k a n d w h i t e pictures w e r e taken w i t h a p o l a r o i d film 3000 speed, being the e x p o s u r e time of 10 to 45 seconds. C o l o r pictures w e r e taken w i t h the h i g h speed K o d a k f i l m E k t a c h r o m e E H B , w h i c h w a s developed f o r 3000 speed.
R E S U L T S
a ) I n F i g u r e l a , a b u n d l e of varicose a d r e n e r g i c fibers can b e observed coursing a l o n g the l o n g i t u d i n a l smooth m u s c l e l a y e r of a h u m a n ureter (8 n t h i c k n e s s ) . I n other sections, both thick a n d delicate beaded a d r e n e r g i c fibers in the adventitia a n d epithelial layers of h u m a n ureter w e r e observed a l l a l o n g the w h o l e ureter, as w e l l as a g r o u p of g a n g l i o n cells fluorescing in v a r y i n g degrees of intensity a t the l o w e r u r e t e r a l s e g m e n t .
b) I n F i g u r e l b , the fluorescent varicosities in the g l o m e r u l i l a y e r of olfactory
b u l b of a n o r m a l cat can be observed, as w e l l as a f e w s m a l l e r cells s h o w i n g the same type of g r e e n i s h - y e l l o w fluorescence. T h e varicosities a r e observed to be of s e v e r a l sizes: 1) l a r g e r a n d m e d i u m size (12 n) w i t h a more greenish f l u o -rescence localized a r o u n d the g l o m e r u l i ( G ) and a r o u n d the m i t r a l ( M ) a n d e x t e r n a l g r a n u l a r cells; 2) small size (less than 1 fi) of y e l l o w i s h g r e e n f l u o -rescence localized t h r o u g h o u t the three c e l l u l a r layers, b u t predominantly a r o u n d the e x t e r n a l a n d internal g r a n u l a r cells.
c ) I n F i g u r e l c , a g r o u p of fluorescent neurons f r o m m e d u l l a o b l o n g a t a of a n o r m a l cat is s h o w n . Varicosities of s e v e r a l sizes a n d v a r y i n g degrees of f l u o -rescence intensity a r e observed a r o u n d the cells. Fluorescent cells a n d fluorescent varicosities w e r e observed in the other structures of pons, mesencephalon a n d d i e n c e p h a l o n .
D I S C U S S I O N A N D C O N C L U S I O N
T h e results of the reported procedure are comparable to the previous
procedure applied in peripheral nervous tissue ( E r ã n k o
6; Sprigs et a l .
1 2;
Csillik and K a l m a n
2) , and the attempts of Nelson and W a k e f i e l d
1 0and
Placidi and M a s u o k a
1 1in central nervous tissue. T h e fluorescence developed
in our material is very sensitive to the ultraviolet light irradiation. T h e
fluorescence is quickly abolished by the action of sodium borohydride and
gas. A s in the preliminary observations of Dahlstrõm and F u x e3
the fluo-rescence from our material treated with reserpine disappears from the cen-tral and peripheral nerve fibers, as well as from the cencen-tral and peripheral nerve cells. A n original observation is that in the material treated with one or two doses of amphetamine ( E l l i n w o o d and D u a r t e - E s c a l a n t e5
) , the fluorescent varicosities are the first to disappear from central and peripheral nerve tissue, while the fluorescence from the cell bodies disappears by the 6th to 8th weeks in the material daily treated with increasing doses of amphetamine, 10-40 m g / k g .
T h e r e are some difficulties in the development of the fluorescent technique even with the original freeze-drying procedure. T h e more critical problems are related to the handling of the tissue during the drying process and to the preparation of paraformaldehyde with the proper percentage of relative humidity. W e have found that in our environment the 60-70% of relative humidity is the useful value to develop the biogenic amine fluorescence in a well dried nervous tissue. Furthermore, the heating time at 65°C allows us the preservation of the enzymatic systems to develop subsequently in alternate sections the histochemical reactions for cholinesterases ( D u a r t e --Escalante et al. 4
) and M A O - e n z y m e (to be published).
R E S U M O
Verificação histoquímica de aminas biogênicas fluorescentes em secções
criostáticas de tecido nervoso central e periférico
R e l a t o de m o d i f i c a ç ã o da t é c n i c a criostáticohistoquímica para a v e r i f i
-c a ç ã o da fluores-cên-cia das -c a t e -c o l a m i n a s e da serotonina e m se-cções de
tecido n e r v o s o c e n t r a l e p e r i f é r i c o . S ã o discutidas as v a n t a g e n s desta m o d i
-f i c a ç ã o técnica e m r e l a ç ã o a o u t r a s propostas para a m e s m a -finalidade.
R E F E R E N C E S
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