Exp. Brain Res. 24, 97--101 (1975)
Experimental Brain Research 9 by Springer-Verlag 1975
t/61e of Axonal Transport in Maintaining
Central Synaptic Connections R. E, Cull Department of Physiology, University Medical School, Teviot Place, Edinburgh (Great Britain) S u m m a r y . S y n a p t i c b o u t o n s on t h e s o m a t a of r a t hypogtossal neurones have been s t a i n e d with Zinc I o d i d e - O s m i u m , a n d c o u n t e d using light microscopy. Two weeks after Silastie cuffs c o n t a i n i n g colehicine or v i n b l a s t i n e were a p p l i e d to t h e hypoglossal nerve, t h e n u m b e r of b o u t o n s on p a r e n t n e r v e cell bodies was significantly lower t h a n controls. These drugs i n h i b i t e d r e t r o g r a d e a x o n a l t r a n s p o r t of h o r s e r a d i s h peroxidase, b u t d i d n o t affect n e u r o m u s c u l a r c o n d u c t i o n or t h e n u m b e r o f a x o n s in t h e nerve t r u n k . Cuffs c o n t a i n i n g lignoeaine d i d n o t affect b o u t o n s or a x o n a l t r a n s p o r t significantly. K e y words: S y n a p t i c b o u t o n s - - AxonM t r a n s p o r t
Introduction A f t e r one hypoglossal nerve has been divided, t h e n u m b e r of s y n a p t i c b o u t o n s on t h e p e r i k a r y a of t h e p a r e n t neurones falls, a n d o n l y r e t u r n s to n o r m a l a f t e r t h e axons reform functioning n e u r o m u s c u l a r j u n c t i o n s (Cull, 1974, 1975 ; S u m n e r a n d S u t h e r l a n d , 1973; Sumner, 1975). W h e n silastic cuffs containing eolchicine or v i n b l a s t i n e - - drugs which i n h i b i t a x o n a l t r a n s p o r t (Karlsson et al., 1971 ; B u n t a n d L u n d , 1974) - - are a p p l i e d to a m o t o r nerve t r u n k , effective nerve-muscle c o n t a c t is m a i n t a i n e d , b u t t h e muscle develops signs of d e n e r v a t i o n ( A l b u q u e r q u e et al., 1972). The p r e s e n t s t u d y was u n d e r t a k e n to see if d r u g - c o n t a i n i n g cuffs w o u l d affect t h e synapses on m o t o r neurones. The results i n d i c a t e t h a t a x o n a l t r a n s p o r t has a n i m p o r t a n t role in m a i n t a i n i n g s y n a p t i c contacts.
Methods
Animals Adult male albino rats, aged three months at the time of operation, were studied. They had free access to standard food pellets and water.
Cu#s Silastic rings (approximate dimensions: outer diameter 2 mm; inner diameter 1 mm; thickness 0.5 mm) were made by squeezing Silastie RTV-A (Dow Coming; Lepetit Pharmaceuticals) through a 25 gauge hypodermic needle on to a glass dish coated with PTFE. The uncured silastic was used either directly or mixed with one of the following: colchieine (0.1% w/v); vinblastine sulphate (0.1% w/v); lignocaine hydrochloride (20% w/v). Cuffs were yuleanised at room temperature for 24--48 hours. 7
E x p . B r a i n Res. Vol. 24
98
R . E . Cull
Operations Under light ether anaesthesia, both hypoglossal nerves were carefully exposed as they passed deep to the posterior belly of the digastric muscle. A plain silastic cuff (drug free), slit at one point on its circumference, was applied around the right hypoglossal nerve. The left hypoglossal nerve received a cuff containing either 0.]~ colchicine (6 rats) or 0.1% vinblastine (5 rats) or 20o/0 lignocaine (6 rats).
Electrophysiology Fourteen days after receiving silastic cuffs, rats were anaesthetised with urethane and the electromyographic (EMG) responses to maximal stimulation of the hypoglossal nerves were recorded from the intrinsic tongue muscles, using bipolar needle electrodes. The voltage of the EMG spikes was measured, and pooled data from control and drug-treated nerves compared by Student's 't' test.
Boutons After EMG measurements, rats were killed and the hypoglossal area of their medullae removed and impregnated in Zinc Iodide-Osmium (ZIO), exactly as previously described (Cull, 1974, 1975). Miscroscopy and counting of boutons was carried out as before (Cull, 1975). Fifty neuronal profiles were assessed from each hypoglossal nucleus; the mean number of boutons/ neurone perimeter was calculated. Data from control and drug-treated sides was compared by Student's 't' test.
Axons Immediately after death, both hypoglossal nerves were removed, fixed in 10% FormolSaline, and stained for axons by Holmes' silver method (Drury and Wallington, 1967). Twenty l0 z m longitudinal sections from each nerve were examined under transmitted light at • 400 magnification. An eyepiece with a circular graticule was used to count the number of axons crossing a fixed area in the cuffed regions. Results from control and drug-treated nerveswere compared by Student's 't' test.
Axonal Transport Retrograde axonal transport was studied in a separate group of 12 rats by tracing horseradish peroxidase (HRP), injected into the tongue, to the hypoglossal neurones. Fourteen days before H R P injection, plain silastic cuffs were applied to the right hypoglossal nerves. At the same time, the left hypoglossal nerve received a cuff containing either 0.1% colchicine (4 rats) or 0.1% vinblastine (4 rats) or 20% lignocaine (4 rats). On the day before death, 10 mg H R P (British Drug Houses Limited) in 0.2 ml of 0.2 M phosphate buffer (pH 7.4) was injected into the intrinsic tongue muscle (5 mg into either side), under light ether anaesthesia. Twenty four hours later, rats were re-anaesthetised and the vascular system perfused with a pre-wash of 20 ml 0.2 )/[ phosphate buffer (pH 7.4) containing 1% Gum Acacia, followed by 40 ml of Karnovsky fixative (Karnovsky, 1965) which also contained 1% Gum Acacia. The medulla was then removed, left in the same fixative for 4 hours, and cut into 3 0 / l m transverse sections on a freezing microtome. After washing in water, sections were transferred to 75 ml of 0.08% Diaminobenzidine in 1/30 M phosphate buffer at pH 7.0. After 10 min, 0.25 ml of 20 vols. Hydrogen Peroxide was added, and the sections left for a further I0 rain. They were then dehydrated in graded alcohols, cleared in xylene and mounted in DPX. Sections were examined under phase contrast at • 400 magnification. Neurones containing H R P were identified by their content of small dark granules. The number of HRP-containing cells/section was counted for both right and left sides of the medulla. Data pooled from the various groups was compared by Student's 't' test.
Results Boutons Cuffs c o n t a i n i n g e i t h e r colchicine or v i n b l a s t i n e c a u s e d a s i g n i f i c a n t r e d u c t i o n in t h e n u m b e r o f b o u t o n s o n h y p o g l o s s a l n e u r o n e s . As a whole, t h e l i g n o c a i n e
Synaptie Boutons
99 P =
Experimental Brain Research 9 by Springer-Verlag 1975
t/61e of Axonal Transport in Maintaining
Central Synaptic Connections R. E, Cull Department of Physiology, University Medical School, Teviot Place, Edinburgh (Great Britain) S u m m a r y . S y n a p t i c b o u t o n s on t h e s o m a t a of r a t hypogtossal neurones have been s t a i n e d with Zinc I o d i d e - O s m i u m , a n d c o u n t e d using light microscopy. Two weeks after Silastie cuffs c o n t a i n i n g colehicine or v i n b l a s t i n e were a p p l i e d to t h e hypoglossal nerve, t h e n u m b e r of b o u t o n s on p a r e n t n e r v e cell bodies was significantly lower t h a n controls. These drugs i n h i b i t e d r e t r o g r a d e a x o n a l t r a n s p o r t of h o r s e r a d i s h peroxidase, b u t d i d n o t affect n e u r o m u s c u l a r c o n d u c t i o n or t h e n u m b e r o f a x o n s in t h e nerve t r u n k . Cuffs c o n t a i n i n g lignoeaine d i d n o t affect b o u t o n s or a x o n a l t r a n s p o r t significantly. K e y words: S y n a p t i c b o u t o n s - - AxonM t r a n s p o r t
Introduction A f t e r one hypoglossal nerve has been divided, t h e n u m b e r of s y n a p t i c b o u t o n s on t h e p e r i k a r y a of t h e p a r e n t neurones falls, a n d o n l y r e t u r n s to n o r m a l a f t e r t h e axons reform functioning n e u r o m u s c u l a r j u n c t i o n s (Cull, 1974, 1975 ; S u m n e r a n d S u t h e r l a n d , 1973; Sumner, 1975). W h e n silastic cuffs containing eolchicine or v i n b l a s t i n e - - drugs which i n h i b i t a x o n a l t r a n s p o r t (Karlsson et al., 1971 ; B u n t a n d L u n d , 1974) - - are a p p l i e d to a m o t o r nerve t r u n k , effective nerve-muscle c o n t a c t is m a i n t a i n e d , b u t t h e muscle develops signs of d e n e r v a t i o n ( A l b u q u e r q u e et al., 1972). The p r e s e n t s t u d y was u n d e r t a k e n to see if d r u g - c o n t a i n i n g cuffs w o u l d affect t h e synapses on m o t o r neurones. The results i n d i c a t e t h a t a x o n a l t r a n s p o r t has a n i m p o r t a n t role in m a i n t a i n i n g s y n a p t i c contacts.
Methods
Animals Adult male albino rats, aged three months at the time of operation, were studied. They had free access to standard food pellets and water.
Cu#s Silastic rings (approximate dimensions: outer diameter 2 mm; inner diameter 1 mm; thickness 0.5 mm) were made by squeezing Silastie RTV-A (Dow Coming; Lepetit Pharmaceuticals) through a 25 gauge hypodermic needle on to a glass dish coated with PTFE. The uncured silastic was used either directly or mixed with one of the following: colchieine (0.1% w/v); vinblastine sulphate (0.1% w/v); lignocaine hydrochloride (20% w/v). Cuffs were yuleanised at room temperature for 24--48 hours. 7
E x p . B r a i n Res. Vol. 24
98
R . E . Cull
Operations Under light ether anaesthesia, both hypoglossal nerves were carefully exposed as they passed deep to the posterior belly of the digastric muscle. A plain silastic cuff (drug free), slit at one point on its circumference, was applied around the right hypoglossal nerve. The left hypoglossal nerve received a cuff containing either 0.]~ colchicine (6 rats) or 0.1% vinblastine (5 rats) or 20o/0 lignocaine (6 rats).
Electrophysiology Fourteen days after receiving silastic cuffs, rats were anaesthetised with urethane and the electromyographic (EMG) responses to maximal stimulation of the hypoglossal nerves were recorded from the intrinsic tongue muscles, using bipolar needle electrodes. The voltage of the EMG spikes was measured, and pooled data from control and drug-treated nerves compared by Student's 't' test.
Boutons After EMG measurements, rats were killed and the hypoglossal area of their medullae removed and impregnated in Zinc Iodide-Osmium (ZIO), exactly as previously described (Cull, 1974, 1975). Miscroscopy and counting of boutons was carried out as before (Cull, 1975). Fifty neuronal profiles were assessed from each hypoglossal nucleus; the mean number of boutons/ neurone perimeter was calculated. Data from control and drug-treated sides was compared by Student's 't' test.
Axons Immediately after death, both hypoglossal nerves were removed, fixed in 10% FormolSaline, and stained for axons by Holmes' silver method (Drury and Wallington, 1967). Twenty l0 z m longitudinal sections from each nerve were examined under transmitted light at • 400 magnification. An eyepiece with a circular graticule was used to count the number of axons crossing a fixed area in the cuffed regions. Results from control and drug-treated nerveswere compared by Student's 't' test.
Axonal Transport Retrograde axonal transport was studied in a separate group of 12 rats by tracing horseradish peroxidase (HRP), injected into the tongue, to the hypoglossal neurones. Fourteen days before H R P injection, plain silastic cuffs were applied to the right hypoglossal nerves. At the same time, the left hypoglossal nerve received a cuff containing either 0.1% colchicine (4 rats) or 0.1% vinblastine (4 rats) or 20% lignocaine (4 rats). On the day before death, 10 mg H R P (British Drug Houses Limited) in 0.2 ml of 0.2 M phosphate buffer (pH 7.4) was injected into the intrinsic tongue muscle (5 mg into either side), under light ether anaesthesia. Twenty four hours later, rats were re-anaesthetised and the vascular system perfused with a pre-wash of 20 ml 0.2 )/[ phosphate buffer (pH 7.4) containing 1% Gum Acacia, followed by 40 ml of Karnovsky fixative (Karnovsky, 1965) which also contained 1% Gum Acacia. The medulla was then removed, left in the same fixative for 4 hours, and cut into 3 0 / l m transverse sections on a freezing microtome. After washing in water, sections were transferred to 75 ml of 0.08% Diaminobenzidine in 1/30 M phosphate buffer at pH 7.0. After 10 min, 0.25 ml of 20 vols. Hydrogen Peroxide was added, and the sections left for a further I0 rain. They were then dehydrated in graded alcohols, cleared in xylene and mounted in DPX. Sections were examined under phase contrast at • 400 magnification. Neurones containing H R P were identified by their content of small dark granules. The number of HRP-containing cells/section was counted for both right and left sides of the medulla. Data pooled from the various groups was compared by Student's 't' test.
Results Boutons Cuffs c o n t a i n i n g e i t h e r colchicine or v i n b l a s t i n e c a u s e d a s i g n i f i c a n t r e d u c t i o n in t h e n u m b e r o f b o u t o n s o n h y p o g l o s s a l n e u r o n e s . As a whole, t h e l i g n o c a i n e
Synaptie Boutons
99 P =
