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A M I N O A C IDS AS C E N T R A L T R A N S M I T T E R S

by

A.W. D U G G A N

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M o s t of these e x p e r i m e n t s w e r e p e r f o r m e d in c o l l a b o r a ­ tion w i t h P r o f e s s o r D.R. Curtis. T h o s e d e a l i n g wi t h the s e n s i t i v i t y of spinal n e u r o n e s to L g l u t a m a t e and L a s p a r tate and p a r t s of the e x p e r i m e n t s on m o r p h i n e and b i c u c u l -line were my own work. The n e u r o c h e m i c a l st u d i e s on the d i s t r i b u t i o n of free amino acids w e r e done w i t h the a s s i s ­ tance of Dr. G.A.R. J o h n s t o n w h i l s t the e x p e r i m e n t s d e a l ­ ing w i t h the acti o n of b i c u c u l l i n e on t h a l a m i c n e u r o n e s were p e r f o r m e d w i t h P r o f e s s o r J. M c L e n n a n of the U n i v e r s i t y of B r i t i s h Colum b i a .

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D u r i n g the tenu r e of my A u s t r a l i a n N a t i o n a l U n i v e r s i t y and C o m m o n w e a l t h P o s t g r a d u a t e S c h o l a r s h i p s , the f o l l o w i n g p a p e r s have been p u b l i s h e d or s u b m i t t e d for p u b l i c a t i o n .

J o h n s t o n , G.A.R., Curtis, D.R., de Groat, W.C. and Duggan, A.W. (1968) C e n t r a l a c t i o n of i b o t e n i c acid and

m u s c i m o l . Biochem. P h a r m a c o l . 17, 2488.

Curtis, D.R. and Duggan, A.W. (1969) The d e p r e s s i o n of spinal i n h i b i t i o n by m o r p h i n e . A g e n t s and A c t i o n s , 1, 414-419.

Curtis, D.R., Duggan, A.W. and J o h n s t o n , G.A.R. (1969)

Glycine, s t r y c h n i n e , p i c r o t o x i n and s p i n a l i n h i b i t i o n . B r a i n R e s e a r c h , 14, 759-762.

Curtis, D.R. and Duggan, A.W. (1969). On the e x i s t e n c e of R e n s h a w cells. B r a i n R e s e a r c h , 15, 597-599.

Duggan, A.W. and J o h n s t o n , G.A.R. ( 1970) . G l u t a m a t e and r e l a t e d amino acids in cat, dog and rat spin a l roots. Comp, and G e n e r a l P h a r m a c o l . 1, 127-128.

Duggan, A.W. and J o h n s t o n , G.A.R. (1970) G l u t a m a t e and r e l a t e d amino acids in cat spi n a l roots, d o r s a l root g a n g l i a and p e r i p h e r a l n e rves. J .N e u r o c h e m . 17, 1205-1208.

Curtis, D.R. , Duggan, A.W. and J o h n s t o n , G.A.R. (1970) . The i n a c t i v a t i o n of e x t r a c e l l u l a r l y a d m i n i s t e r e d amino acids. E x p . B r a i n R e s . 10, 447-462.

Curtis, D.R., Duggan, A.W., Felix, D. and J o h n s t o n , G.A.R. (1970) GABA, b i c u c u l l i n e and c e n t r a l i n h i b i t i o n .

N a t u r e , 226, 1222-1224.

Curtis, D.R., Duggan, A.W. and Felix, D. (1970). GABA and i n h i b i t i o n of D e i t e r s n e u r o n e s . B r a i n R e s . 23, 117-120.

Duggan, A.W. and M c L e n n a n , H. (1970) B i c u c u l l i n e and

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(1970) Curtis, D.R., Duggan, A.W. and J o h n s t o n , G.A.R.

The s p e c i f i c i t y of s t r y c h n i n e as a g l y c i n e a n t a g o n i s t . S u b m i t t e d for p u b l i c a t i o n .

Curtis, D.R., D u ggan, A.W., Felix, D. and J o h n s t o n , G.A.R. (1970) B i c u c u l l i n e and c e n t r a l G A B A rece p t o r s .

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T A B L E OF C O N T E N T S

Page N o . A C K N O W L E D G E M E N T S ...

I. G E N E R A L I N T R O D U C T I O N ... 1

II. M E T H O D S ... 3

(a) G e n e r a l M e t h o d s ... 3

(i) M i c r o e l e c t r o p h o r e s i s ... 3

(ii) The r e c o r d i n g of act i o n p o t e n t i a l s e x t r a c e l l u l a r l y ... 4

(iii) The feline s p i n a l cord p r e p a r a t i o n ... 4

(b) The s e n s i t i v i t y of s p i n a l i n t e r ­ n e u r o n e s to L - g l u t a m a t e and L - a s p a r t a t e ... 5

(c) M o r p h i n e , g l y c i n e and spi n a l r e f l e x e s .. 5

(i) M i c r o e l e c t r o p h o r e t i c e x p e r i m e n t s . .. 8

(ii) R e f l e x e x p e r i m e n t s ... 8

( i i i )M o r p h i n e and i n h i b i t i o n of R e n s h a w cells ... 9

(iv) M o r p h i n e and the a c t i v a t i o n of R e n s h a w cells by d o r s a l and v e n t r a l root s t i m u l i ... 9

(d) The p o t e n t i a t i o n of a m i n o a c i d e x c i t a t i o n and d e p r e s s i o n by o r g a n i c m e r c u r i a l s and t h i o s e m i c a r b a z i d e ... 10

(i) O r g a n i c m e r c u r i a l s ... 10

(ii) T h i o s e m i c a r b a z i d e ... 12

(e) The s p e c i f i c i t y of s t r y c h n i n e as a g l y c i n e a n t a g o n i s t in the s p i n a l cord. .. 12

(f) B i c u c u l l i n e , GA B A and c e n t r a l i n h i b i t i o n S o l u t i o n s and e l e c t r o d e s ... 13

Deiters' n u c l e u s ... 14

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ii Page No. (g) The d i s t r i b u t i o n of free a m ino acids in

spinal r o o t s , dor s a l ro o t g a n g l i a and

p e r i p h e r a l n e r v e s ... 15

III. THE S E N S I T I V I T Y OF S P I N A L I N T E R N E U R O N E S TO L - G L U T A M A T E AND L - A S P A R T A T E ... 18

I n t r o d u c t i o n ... 18

R e s u l t s ... 21

D i s c u s s i o n ... 23

IV. M O R P H I N E , G L Y C I N E AND S P I N A L I N H I B I T I O N ... 26

I n t r o d u c t i o n ... 26

R e s u l t s ... 30

(i) E l e c t r o p h o r e t i c a l l y a d m i n i s t e r e d m o r p h i n e and g l y c i n e ... 30

(ii) M o r p h i n e and s p i n a l i n h i b i t i o n . . .. 30

(iii) M o r p h i n e and i n h i b i t i o n of R e n s h a w c e l l s ... 32

(iv) E l e c t r o p h o r e t i c a l l y a d m i n i s t e r e d m o r p h i n e and the s y n a p t i c a c t i v a t i o n of R e n s h a w cells ... 32

(a) V e n t r a l root s t i m u l u s ... 32

(b) D o r s a l root s t i m u l u s ... 36

D i s c u s s i o n ... 3 8 V. THE P O T E N T I A T I O N OF A M I N O AC I D E X C I T A T I O N AND D E P R E S S I O N BY O R G A N I C M E R C U R I A L S AND T H I O S E M I C A R -BAZ I D E ... 42

I n t r o d u c t i o n ... 42

R e s u l t s ... 44

(i) p - c h l o r o m e r c u r i p h e n y l s u l p h o n a t e and e x c i t a t i o n and d e p r e s s i o n of s p i n a l n e u r o n e s by a m i n o a c i d s ... 44

(ii) p - c h l o r o m e r c u r i p h e n y l s u l p h o n a t e and the i n h i b i t i o n of R e n s h a w c e l l s ... 47

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iii Page N o . (iv) H y d r az i n o p r o p i o n i c a c i d ... 48 Dis c u s s i o n • . ... 49 VI. THE S P E C I F I C I T Y OF S T R Y C H N I N E AS A G L Y C I N E

A N T A G O N I S T IN THE S P I N A L C O R D ... 55 Res u l t s . . . . ... 59

(i) The e f f e c t of e l e c t r o p h o r e t i c a l l y

a d m i n i s t e r e d s t r y c h n i n e on the g l y c i n e

d o s e - r e s p o n s e curve ... 59 (ii) The e f f e c t of i n t r a v e n o u s l y a d m i n i s t e r e d

s t r y c h n i n e on the g l y c i n e d o s e - r e s p o n s e

curve . . ... 61 (iii) The e f f e c t of s t r y c h n i n e on the d e p r e s s i o n

of s p i n a l n e u r o n e s by G A B A ... 62 Dis c u s s i o n . . ... 6 5 VII. B I C U C U L L I N E , G A B A AND C E N T R A L I N H I B I T I O N .. .. 77

I n t r o d u c t i o n . . ... 77 R e s u l t s . . . . ... 84 (i) S p i n a l c o r d ... 84

(a) S p i n a l r e f l e x e s 84

(b) B i c u c u l l i n e and e l e c t r o p h o r e t i c a l l y a d ­ m i n i s t e r e d d e p r e s s a n t s 84

(1) G l y c i n e and G A B A 84

(2) O t h e r s u b s t a n c e s 87

(ii) Deiters' n u c l e u s . ... 89 (a) The e f f e c t of b i c u c u l l i n e on the

a c t i o n of d e p r e s s a n t a m i n o acids. .. 89 (b) B i c u c u l l i n e and the i n h i b i t i o n of

<

Deiters' n e u r o n e s by i m p u l s e s from

the a n t e r i o r c e r e b e l l u m ... 90

( i i i ) T h a l a m u s . 92

(a) B i c u c u l l i n e and the a c t i o n of

d e p r e s s a n t amino acids ... 92 (b) B i c u c u l l i n e and the p a t t e r n of

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iv P a g e No. Dis c u s s i o n ... 9 7 VIII. THE D I S T R I B U T I O N OF FREE A M I N O A C IDS IN S P I N A L

CORD, S P I N A L ROOTS, D O R S A L ROOT G A N G L I A AND

P E R I P H E R A L NERV E S ... 103 I n t r o d u c t i o n ... 103 R e s u l t s ... 104

(i) Free amino acids in spinal roots, d o r s a l root g a n g l i a and p e r i p h e r a l ner v e s of

the c a t ... 104 (ii) Free amino acids in the d o r s a l and

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A C K N O W L E D G E M E N T S

My thanks go to P r o f e s s o r D.R. Curt i s for s u p e r v i s i n g this w o r k and for an e x c e l l e n t i n t r o d u c t i o n to n e u r o p h a r m a ­ cology. C h e m i s t s are i n d i s p e n s a b l e to p h a r m a c o l o g i s t s

and I am i n d e b t e d to Dr. G.A.R. J o h n s t o n for his a s s i s t a n c e in this aspect. I thank P r o f e s s o r H. M c L e n n a n and Dr. M.J. C r a w f o r d for t h e i r c o l l a b o r a t i o n .

For t e c h n i c a l a s s i s t a n c e my t h a n k s go to Mrs. A. D a d a y , Mr. L. Davies, Mr. A. Ch a p m a n , Mr. B. Maher, Mrs. A. G r e i n e r

and Mrs. P. Searle.

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1

I. G E N E R A L I N T R O D U C T I O N

The various s e c t i o n s of this T h e s i s all relate to the i d e n t i f i c a t i o n of s y n a p t i c t r a n s m i t t e r s w i t h i n the m a m m a l ­ ian c e n t r a l n e r v o u s system. S e v e r a l a u thors have p r o p o s e d c r i t e r i a that a s u b s t a n c e needs to s a t i s f y for it to be r e g a r d e d as a t r a n s m i t t e r w i t h i n the c e n t r a l ner v o u s s y s t e m

( Paton, 1958; Curtis, 1961; C o s t a and Bloom, 1965;

W e rman, 1966; Br a d l e y , 1968 ; C u r t i s and J o h n s t o n , 1970) . T hose lis t e d b e l o w are those of C u r t i s and J o h n s t o n (1970) .

(a) The s u b s t a n c e and the e n z y m e s for its s y n t h e s i s s h o u l d be p r e s e n t in the a p p r o p r i a t e n e rve endings.

(b) The s u b s t a n c e s h o u l d be r e l e a s e d f o l l o w i n g i m p u l s e s in the a p p r o p r i a t e nerve fibres.

(c) The p o s t s y n a p t i c a c t i o n of the s u b s t a n c e s h o u l d be i d e n t i c a l w i t h that of the t r a n s m i t t e r .

(d) C o m p o u n d s w h i c h b l o c k s y n a p t i c t r a n s m i s s i o n by a s p e c i ­ fic p o s t s y n a p t i c e f f e c t m u s t also a n t a g o n i z e the act i o n of the s u s p e c t e d t r a n s m i t t e r .

(e) The p r o c e s s e s w h i c h i n a c t i v a t e and rem o v e r e l e a s e d t r a n m i t t e r s h o u l d also t e r m i n a t e the a c t i o n of the a r t i ­ f i c i a l l y a d m i n i s t e r e d s u b s tance.

T r a n s m i t t e r i d e n t i f i c a t i o n thus d e p e n d s on a body of n e u r o c h e m i c a l , n e u r o p h y s i o l o g i c a l and a n a t o m i c a l e v i d e n c e w h i c h c o l l e c t i v e l y , bu t not s e p a r a t e l y , p o i n t s to a s u b ­ stance b e i n g a t r a n s m i t t e r . The v a r i o u s s e c t i o n s of this Thesis c o n t a i n data r e l e v a n t to a p o s s i b l e e x c i t a t o r y

t r a n s m i t t e r role for L - g l u t a m a t e and L - a s p a r t a t e and an i n h i b i t o r y t r a n s m i t t e r role for g l y c i n e and g a m m a - a m i n o ­ b u t y r i c acid.

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2

a c t i o n of t r a n s m i t t e r suspects.

The work on o r g a n i c m e r c u r i a l s and the a c t i o n of a m i n o acids (V) is r e l e v a n t to c r i t e r i o n (e) w h ich deals w i t h the i n a c t i v a t i o n of t r a n s m i t t e r s , a l t h o u g h a t t e m p t s to i n f l u e n c e s y n a p t i c events w i t h the m e r c u r i a l s we r e u n s u c c e s s f u l .

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3

II. M E T H O D S

(a) G e n e r a l M e t h o d s

The p r e p a r a t i o n of the s p i n a l cord for e x t r a c e l l u l a r r e c o r d i n g of action p o t e n t i a l s , and the e l e c t r o p h o r e t i c a p p l i c a t i o n of s u b s t a n c e s to n e u r o n e s we r e c o m m o n to mo s t s e c t i o n s of this Thesis.

( i ) M i c r o e l e c t r o p h o r e s i s

This t e c h n i q u e c o n sists of the a d m i n i s t r a t i o n of c o m p o u n d s to the e x t r a c e l l u l a r e n v i r o n m e n t of single n e rve cells by e j e c t i n g ions from glass m i c r o p i p e t t e s . The m e t h o d s u s e d were those d e s c r i b e d by Curtis (1964) . Five and seven b a r r e l m i c r o p i p e t t e s were d r a w n in a v e r t i c a l m i c r o e l e c t r o d e p u l l e r and b r o k e n back to the d e s i r e d size (commonly 5-7yM) by s t r i k i n g their tips w i t h a fine glass rod, the m o v e m e n t s of w h i c h were c o n t r o l l e d by a " j o y s t i c k " m i c r o m a n i p u l a t o r . The s o l u t i o n s to be u s e d were c e n t r i f u g e d , then t r a n s ­ ferred to the b a r r e l s of the m i c r o p i p e t t e s , and d r i v e n to the tips by f u r t h e r c e n t r i f u g a t i o n . The b a r r e l s we r e then i n s p e c t e d m i c r o s c o p i c a l l y and the e l e c t r i c a l r e s i s t a n c e of each m e a s u r e d .

The centre b a r r e l of each m i c r o p i p e t t e was fill e d w i t h 4M NaCl, and e l e c t r i c a l c o n t a c t made w i t h this s o l u t i o n w i t h a A g - A g C l wire. C o n t a c t w i t h drug

s o l u t i o n s was made u s i n g s i l v e r wires. The r e s i s t a n c e of each b a r r e l w i t h the e l e c t r o d e in the tis s u e u n der s tudy was m e a s u r e d by d e t e r m i n i n g the c u r r e n t r e q u i r e d to e s t a b l i s h 0.5 V p o t e n t i a l d i f f e r e n c e across the s o lution. The p o l a r i t y of this r e t a i n i n g v o l t a g e was such as to r e t a i n the a c t i v e ion w i t h i n the m i c r o ­ p i p e t t e , and the r e t a i n i n g c u r r e n t was m a i n t a i n e d

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4

of the active substance. The magnitudes of currents -9

used are expressed in nano amps {nA, 10 A) , a current which ejected cations from a micropipette being called cationic (+), an anionic (-) current that ejecting anions. A current of O nA indicates removal of the retaining current from a barrel, thus allowing diffus- ional efflux of a compound; with potent compounds,

this was often sufficient to produce measurable effects. (i l ) The recording of action potentials extracellularly

A cathode follower first stage with provision for capacitance neutralisation was followed by two p r e ­ amplifier units (time constant .003 sec) and an

oscilloscope. The output of the cathode follower was also connect.' . to another amplifying system, the o u t ­ put of which was used to trigger a pulse generator. A voltage discriminator was used to select the size of action potentials which triggered this generator, the output pulses of which were fed to a ratemeter and to an oscilloscope for monitoring. The output of the ratemeter was connected to a rectilinear pen recorder thus providing a continuous display of the firing rate of the cell being studied.

(iii) The feline spinal cord preparation

Following the induction of anaesthesia, the left e x ­ ternal radial vein, the R common carotid artery and the trachea were cannulated. The animal's temperature was maintained between 36-38°C by heating pads placed under the abdomen and chest. The current through these pads was controlled by a solid state regulating unit, the temperature sensitive probe of which was placed between the rib cage and R scapula.

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5

after i n f i l t r a t i o n w i t h 1% p r o c a i n e s o lution. The dural sac was o p e n e d dor sally over the lumb a r and sacr a l cord and, if R e n s h a w cells were b e i n g sought, the sev e n t h lumbar and f i r s t sac r a l v e n t r a l roots were d i v i d e d at the inter v e r t e b r a l f o r a m e n and m o u n ­ ted on p l a t i n u m e l e c t r o d e s . V o l l e y s e n t e r i n g the spin a l cord were m o n i t o r e d by a p l a t i n u m ball e l e c ­ trode p l a c e d on the d o r s a l columns. The i n d i f f e r e n t e l e c t r o d e , a coil of c h l o r i d e d s i l v e r wire e n c l o s e d in gauze, was s u t u r e d to the v e r t e b r a l muscl e s . The spin a l cord was i m m e r s e d in l i q u i d p a r a f f i n (BP), a p o o l b e i n g formed by the r a i s i n g and a n c h o r i n g of skin

flaps. Small areas of p i a w e r e r e m o v e d w i t h fine f o r ­ ceps at the sites of p r o p o s e d e l e c t r o d e p e n e t r a t i o n . (b ) The s e n s i t i v i t y of s p i n a l i n t e r n e u r o n e s to L - g l u t a m a t e and L - a s p a r t a t e .

The s o l u t i o n s used were:

L - A s p a r t a t e 1.0 M pH 7 NaOH L - G l u t a m a t e 1.0 M pH 7 NaOH

D L - H o m o c y s t e i c acid 0.2 M pH 8 NaOH A c e t y l c h o l i n e c h l o r i d e i.OM

Cats w e r e a n a e s t h e t i s e d w i t h p e n t o b a r b i t o n e s o d i u m (35 m g / k g i n i t i a l l y , and s u p p l e m e n t e d w h e n n e c e ssary) and the spinal cord was p r e p a r e d for r e c o r d i n g . The left sural, com m o n p e r o n e a l and c o m m o n tibi a l nerves we r e d i s s e c t e d , m o u n t e d on p l a t i n u m e l e c t r o d e s , and w e r e s t i m u l a t e d at a s t r e n g t h three to five times

thr e s h o l d . R e n s h a w cells w e r e i d e n t i f i e d by r e s p o n s e s w h i c h f o l l o w e d a n t i d r o m i c s t i m u l a t i o n of v e n t r a l roots.

I n t e r n e u r o n e s and R e n s h a w cells of the left s e v e n t h l u m b a r s e g m e n t (L7) w e r e studied. The m i c r o p i p e t t e was m a n o e u v e r e d w i t h a m i c r o m a n i p u l a t o r w h i c h p e r m i t t e d

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6

and Winsbury, 1954). The sensitivity of these cells to excitant amino acids was assessed in the following manner .

The cell was initially excited with a range of amino acid concentrations (ejecting currents) , and a rough potency assessed for each substance on the basis of the ratio of ejecting currents. A firing rate which was easily maintained by each substance was then

selected, and the currents of each required to attain this firing rate were determined. This, idealised method was disturbed by the following factors.

(1) The cell could not be fired by one substance with the currents that could be passed through the p a r t i c u ­ lar micropipette.

(2) The cell did not maintain a constant firing rate, but the rate "faded" with continued ejection of an e x ­ citant. This was most common with L-glutamate.

(3) The cell spike became positive before any constant firing rate was attained. This "depolarisation block" did not prevent gross comparisons of potency being

made, if the other compound was scarcely active with the same current.

After the necessary data were obtained from each cell, the following measurements were taken, using the micromanipulator where appropriate, in order to map the position of neurones within the spinal cord.

(1) The distance between the midline and the most medial point of entry of the dorsal roots.

(2) The angle between the microelectrode and the m i c romanipulator vertical.

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the "mapp i n g v e r t i c a l " and u s u a l l y d i f f e r e d from true v e r t i c a l , as i n d i c a t e d by the m i c r o m a n i p u l a t o r , as a r e s u l t of s l i g h t t w i s t i n g of the v e r t e b r a e by the f i x a ­ tion clamps. All m i c r o e l e c t r o d e tra c k s were then c o r r e c t e d for this error.

(4) The d i s t a n c e from the p o i n t of e n t r y of the m i c r o ­ e l e c t r o d e into the spi n a l cord and the midline.

(5) The depth of each cell from the surface, m e a s u r e d a l ong its p a r t i c u l a r track.

(6) The m a x i m a l a n t e r o - p o s t e r i o r t h i c k n e s s of the c o r d .

For each a n i m a l the cells s t u d i e d w e r e p l o t t e d on a s t a n d a r d map d rawn from a s e c t i o n t a k e n from u p per L7. The ratio

d i s t a n c e from m i c r o e l e c t r o d e e n try p o i n t to m i d l i n e , d i s t a n c e b e t w e e n line of d o r s a l root e n t r y and m i d l i n e d e t e r m i n e d the p o i n t from w h i c h each e l e c t r o d e track was drawn at a k n o w n angle to the " m a p p i n g v e r t i c a l "

The s t a n d a r d map was d i v i d e d into 1320 squ a r e s by g r i d lines, there b e i n g 45 lines d r awn in an a n t e r o ­ p o s t e r i o r d i r e c t i o n and 31 in a m e d i o l a t e r a l d i r e c t i o n .

Cells w e r e thus g i v e n c o o r d i n a t e s . T h ese c o o r d i n a t e s t o g e t h e r w i t h o t h e r v a r i a b l e s m e a s u r e d were e n c o d e d on p u n c h cards. A c o m p u t e r p r o g r a m was w r i t t e n w h i c h p r i n t e d out a map of the spi n a l cord t o g e t h e r w i t h the n u m b e r of cells in d i f f e r e n t p o s i t i o n s w h i c h met c e r t a i n r e q u i r e m e n t s . Up to six r e q u i r e m e n t s could be n o m i n a t e d to be met by a cell for it to be i n c l u d e d in a map.

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w h i t e matter, w h e n p l o t t e d , a p p e a r e d in the e x p e c t e d l o c a t i o n .

(c ) M o r p h i n e , g l y c i n e and s p i n a l r e f l e x e s (i ) M i c r o e l e c t r o p h o r e t i c e x p e r i m e n t s

The s o l u t i o n s u s e d were:

M o r p h i n e s u l p h a t e - 70 mM in H^O S t r y c h n i n e HC1 - 2 mM in 165 mM N a C l G l y c i n e 0.5 M pH 3 HC1

Y - A m i n o b u t y r i c acid (GABA) 0 . 5M pH 3 HC1 D L - H o m o c y s t e a t e 0.2 M pH 7, NaOH

A c e t y l c h o l i n e c h l o r i d e 1.0 M (i i ) R e f l e x e x p e r i m e n t s

M o n o s y n a p t i c r e f l e x e s w e r e r e c o r d e d p e r i p h e r a l l y from m u s c l e n e r v e s in r e s p o n s e to s u p r a m a x i m a l e l e c ­ t r i c a l s t i m u l a t i o n (1/sec) of the t r a n s e c t e d left s e v e n t h lumb a r and first s a c r a l d o r s a l roots. The r e f l e x e s were u s u a l l y s t a b i l i z e d by a p r e c e d i n g sub- m a x i m a l s t i m u l u s w h i c h f a c i l i t a t e d the r e s p o n s e to a

s e c o n d v o 1 l e y .

I n h i b i t o r y cur v e s we r e ..plotted d i r e c t l y on an X-Y r e c o r d e r (Varian F 80 - c o n v e r t e d to a p o i n t p l o t t e r ) . The X v o l t a g e was p r o p o r t i o n a l to the i n t e r v a l b e t w e e n the t e s t i n g and the i n h i b i t i n g volley, the Y v o l t a g e b e i n g p r o p o r t i o n a l to the area of the m o n o -p h a s i c r e f l e x res-po n s e . This ar e a was m e a s u r e d u s i n g a v o l t a g e to f r e q u e n c y c o n v e r t e r (Vidar 260) and a g a t e d c o u n t e r w i t h a n a l o g u e o u t p u t (Hewlett P a c k a r d c o u n t e r 5214L, D i g i t a l R e c o r d e r H 2 3 - 5 6 2 A ) .

D i r e c t I n h i b i t i o n . C u r v e s w e r e p l o t t e d of the i n ­ h i b i t i o n of the m o n o s y n a p t i c r e f l e x r e c o r d e d from the p o s t e r i o r b i c e p s s e m i t e n d i n o s u s m u s c l e n e r v e (BST) by v o l l e y s in the i p s i l a t e r a l q u a d r i c e p s n e rve ( Q ) , s t imu

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R e c u r r e n t I n h i b i t i o n . C u r v e s were p l o t t e d of the i n h i b i t i o n of the m o n o s y n a p t i c r e f l e x r e c o r d e d from e i t h e r the l a teral or m e d i a l g a s t r o c n e m i u s n e r v e (LG, MG) by s u p r a m a x i m a l s t i m u l a t i o n of the other, d o r s a l roots h a v i n g b e e n t r a n s e c t e d . This i n h i b i t i o n was o ften i n c r e a s e d by a d d i n g o t h e r h i n d limb n e r v e s to the b r a n c h p r o v i d i n g the i n h i b i t o r y volley.

Both types of i n h i b i t i o n w e r e s t u d i e d in each animal. This n e c e s s i t a t e d l e a v i n g L6 d o r s a l r o o t i n t a c t b e c a u s e of its c o n t e n t of q u a d r i c e p s fibres. As the m u s c l e ner v e s used to study r e c u r r e n t i n h i b i t i o n may have c o n t a i n e d a f f e r e n t fibres w h i c h e n t e r e d the spinal cord via the L6 d o r s a l root, one a n i m a l was

p r e p a r e d in the usual way, but r e c u r r e n t i n h i b i t i o n was also s t u d i e d on the o p p o s i t e (right) side w i t h all d o r ­ sal roots (L6 to S3 inclusive) t r a n s e c t e d .

(iii) M o r p h i n e and the i n h i b i t i o n of R e n s h a w cells

R e n s h a w cells w e r e i n h b i i t e d by s q u e e z i n g the h i n d p a w w i t h a m o d i f i e d b i c y c l e w h e e l rim brake. An a d j u s t a b l e stop was i n c o r p o r a t e d in the h a n d l e of this b r a k e to fix the s e p a r a t i o n of the jaws, w h e n fully applied, at any d e s i r e d d i s t a n c e . The i n h i b i t i o n of firi n g p r o d u c e d in this way was c o m m o n l y r e p r o d u c e a b l e to w i t h i n +20%.

(i v ) M o r p h i n e and the a c t i v a t i o n of R e n s h a w cells by d o r s a l and v e n t r a l root sti m u l i

In a n i m a l s a n a e s t h e t i s e d w i t h p e n t o b a r b i t o n e , R e n ­ shaw cells were r a r e l y a c t i v a t e d by d o r s a l root stimuli, and hence m o s t s t u d i e s we r e p e r f o r m e d w i t h a n i m a l s d e ­ c e r e b r a t e d by c o a g u l a t i o n of the b r a i n stem u n d e r h a l o - thane a n a e s t h e s i a ( C rawford and C u r t i s , 1964) , the

a n a e s t h e t i c then b e i n g w i t h d r a w n .

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root stim u l u s , were r e c o r d e d on film, ten sweeps b e i n g s u p e r i m p o s e d . W h e n u s ing s u b m a x i m a l v e n t r a l ro o t s t i m u l i and d o r s a l root stimuli, i n d i v i d u a l r e s p o n s e s we r e r e c o r d e d on m o v i n g film and a v e r a g e d a r i t h m e t i c a l l y . L a t e n c i e s were m e a s u r e d from the a r r i v a l of the v e n t r a l root v o l l e y at the spin a l cord. To o b t a i n any a c c u r a c y in these m e a s u r e m e n t s , it was n e c e s s a r y to e x p a n d the sweep, with the r e s u l t that only the first six to e i g h t s p i k e s of a v e n t r a l root r e s p o n s e were i n c l u d e d in the records. The first a c t i o n p o t e n t i a l was o f t e n o b s c u r e d by the field p o t e n t i a l r e s u l t i n g from the a n t i d r o m i c

f i r i n g of m o t o n e u r o n e s and h e n c e its l a t e n c y c o u l d r a r e l y be m e a s u r e d a c c u r a t e l y .

In one e x p e r i m e n t , a c o m p u t e r ( E n h a n c e t r o n , N u c l e a r D a t a ) , was us e d to p r e p a r e a p o s t s t i m u l u s h i s t o g r a m of cell firing in r e s p o n s e to s u b m a x i m a l v e n t r a l r o o t

stimuli. Only the first three a c t i o n p o t e n t i a l s w e r e r e c o r d e d in this way.

(d ) The p o t e n t i a t i o n of a m i n o acid e x c i t a t i o n and d e p r e s s i o n by o r g a n i c m e r c u r i a l s and t h i o s e m i c a r b a z i d e

(i ) O r g a n i c m e r c u r i a l s

The f o l l o w i n g s o l u t i o n s w e r e used:

p - C h l o r o m e r c u r i p h e n y l s u l p h o n a t e Na salt ( p C S ) , l O m M in 16 5mM N a C 1, pH 8-8.5

G l y c i n e 0 . 5M pH 3 (HCl) G A B A 0 . 5M pH 3 (HCl)

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C e lls were s t u d i e d in the s e v e n t h lumbar and first s a c r a l s e g m e n t s of the s p i n a l cords of cats a n a e s t h e ­ t i s e d w i t h p e n t o b a r b i t o n e . In s t u d y i n g the e f f e c t of i m p u l s e s in h i n d p a w a f f e r e n t fibres on R e n s h a w cell firing, the d i s s e c t i o n was m o d i f i e d by r e p l a c i n g the c l a m p that n o r m a l l y h e l d the left hi n d limb by a sling, to p r e v e n t d a m a g e to the n e r v e s of the foot.

In a s s e s s i n g p o s s i b l e p o t e n t i a t i o n of one c o m p o u n d by a n o t h e r the f o l l o w i n g d i f f i c u l t i e s w e r e a v o i d e d as far as p o s s i b l e .

(1) If the d i s t a n c e b e t w e e n the d r u g - c o n t a i n i n g p i p e t t e and the cell d e c r e a s e s a f t e r c o n t r o l o b s e r v a t i o n s have b e e n made, dr u g r e s p o n s e s wi l l a p p e a r to be e n h a n c e d . Such an e f f e c t can be m i n i m i z e d by m o v i n g the e l e c t r o d e in o r d e r to keep the spike a m p l i t u d e c o n s t a n t , and to m a i n t a i n a c o n s t a n t r e s p o n s e to a n o t h e r s u b s t a n c e w h ose a c t i o n is u n a f f e c t e d by the p o t e n t i a t i n g agent. This l a t t e r m a n o e u v r e may not be always p o s s i b l e and de p e n d s on the s p e c i f i c i t y of the p o t e n t i a t i n g s u b s t a n c e .

(2) W h e n a c o m p o u n d has not b e e n e j e c t e d for some time, its c o n c e n t r a t i o n w i t h i n the tip of the m i c r o ­ p i p e t t e will be l o wer than e l s e w h e r e in the b a r r e l b e ­ cause of the r e t a i n i n g current# the drug b e i n g d i l u t e d by the ions from the e x t e r n a l m e dium. S u b s e q u e n t

e j e c t i n g c u r r e n t s w i l l e j e c t i n c r e a s i n g a m o u n t s of the drug as the c o n c e n t r a t i o n w i t h i n the tip a p p r o a c h e s that of the bu l k of the s o l u t i o n and unl e s s time is

a l l o w e d for the tip c o n c e n t r a t i o n to r each this c o n s t a n t level, an a p p a r e n t w e a k e n i n g of the a c t i o n of the c o m ­ p o u n d w i l l be o b s e r v e d . Once e q u i l i b r i u m c o n d i t i o n s are reach e d , this e r r o r can be m i n i m i z e d by e j e c t i n g the

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c o n t r o l o b s e r v a t i o n s have be e n made may i n t r o d u c e e r r o r s in e s t i m a t i n g the e f f e c t i v e n e s s of a compound. This was a m i n o r p r o b l e m w i t h the o r g a n i c m e r c u r i a l s , but the i n c r e a s e is b a s a l firing rate f o l l o w i n g s y s ­ temic i n j e c t i o n of t h i o s e m i c a r b a z i d e was s i g n i f i c a n t . By s t u d y i n g a cell w h i c h r e q u i r e d the c o n t i n u o u s e j e c ­ tion of an e x c i t a n t , a c o n s t a n t f i r i n g rate c o u l d be m a i n t a i n e d by v a r y i n g the e j e c t i n g c u r r e n t of this e x c i t a n t .

T h i o s e m i c a r b a z i d e

This c o m p o u n d was i n j e c t e d i n t r a v e n o u s l y u s i n g a 2 0 m g / m l a q u e o u s solut i o n . E x t r a c e l l u l a r l y r e c o r d e d a c t i o n p o t e n t i a l s we r e p h o t o g r a p h e d to p e r m i t d i r e c t and a c c u r a t e m e a s u r e m e n t s of the time b e t w e e n the t e r ­ m i n a t i o n of the c u r r e n t e j e c t i n g a drug and the r e s u m p ­

tion of the c o n t r o l level of cell firing. Q u a n t i t a t i v e s t u d i e s on c h a n g e s in the e f f e c t i v e n e s s of e x c i t a n t and d e p r e s s a n t a m i n o acids r e q u i r e that the b a s a l level of fir i n g be c o n s t a n t , as c h a n g e s in this alone w i l l p r o ­ duce a p p a r e n t c h a n g e s in the a c t i o n of these s u b s t a n c e s . An i n c r e a s e in the b a s a l f r e q u e n c y tends to p r o l o n g the a c t i o n of e x c i t a n t s and m a k e s the m e a s u r e m e n t of r e ­ cov e r y times less p r e c i s e .

B e c a u s e of the t e n d e n c y of t h i o s e m i c a r b a z i d e to i n c r e a s e the s p o n t a n e o u s fir i n g rate of n e u r o n e s , cells were s t u d i e d w h i c h r e q u i r e d the c o n t i n u o u s e j e c t i o n of an e x c i t a n t to p r o d u c e a s a t i s f a c t o r y b a s a l level of firing in the c o n t r o l p e riod. A d j u s t m e n t of the a m o u n t of the e x c i t a n t e j e c t e d a f ter t h i o s e m i c a r b a z i d e , e n a b l e d a c o n s t a n t b a s a l fir i n g rate to be m a i n t a i n e d .

(e ) The s p e c i f i c i t y of s t r y c h n i n e as a g l y c i n e a n t a g o n i s t in the spi n a l cord.

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G l y c i n e 0.5M, pH 3, HCl G A B A 0.5M, pH 3, HCl DLH 0. 2 M pH 8, NaOH

A c e t y l c h o l i n e c h l o r i d e 1.0M.

S t r y c h n i n e HCl 2mM or 10 mM in 165 mM NaCl

E x p e r i m e n t s w e r e p e r f o r m e d on spinal i n t e r n e u r o n e s and R e n s h a w cells of the l ower lumbar s e g m e n t s of cats. The m a j o r i t y of ani m a l s w e r e a n a e s t h e t i s e d wi t h p e n t o ­ b a r b i t o n e s o d i u m (35 m g / k g i n t r a p e r i t o n e a l l y , i n i t i a l l y ) ; some an i m a l s were d e c e r e b r a t e d by c o a g u l a t i o n of the b r a i n stem u n d e r h a l o t h a n e a n a e s t h e t i c (Crawford and Cu r t i s , 1966) and w e r e u n a n a e s t h e t i s e d d u r i n g the e x ­ p e r i m e n t .

In s t u d y i n g a n t a g o n i s m of one c o m p o u n d by another, the same p r e c a u t i o n s n e e d e d in o b s e r v i n g p o t e n t i a t i o n and o u t l i n e d p r e v i o u s l y w h e n c o n s i d e r i n g o r g a n i c m e r ­ c u r ials, were taken. S u f f i c i e n t time was a l l o w e d for the e f f e c t s of each a d m i n i s t e r e d c o m p o u n d to be o b s e r v e d u n d e r e q u i l i b r i u m c o n d i t o n s . Care was t a ken to m a i n ­ tain a c o n s t a n t b a s a l firi n g rate b e f o r e and d u r i n g the a c t i o n of s t r y c h n i n e , as a g i v e n dose of a d e p r e s s a n t p r o d u c e d less p e r c e n t a g e d e p r e s s i o n as the b a s a l firing rate rose.

(f ) B i c u c u l l i n e G A B A and C e n t r a l I n h i b i t i o n

B i c u c u l l i n e h y d r o c h l o r i d e is s p a r i n g l y s o l u b l e in water. S o l u t i o n s we r e p r e p a r e d in 165 mM NaCl at pH 3, and are e s t i m a t e d to have a c o n c e n t r a t i o n of the a l k a ­ loid not g r e a t e r than 5 m M . Hence, a s s u m i n g equal

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e l e c t r i c a l r e s i s t a n c e . These p r e s u m a b l y r e s u l t e d from the r e d u c e d s o l u b i l i t y of b i c u c u l l i n e in t i s s u e fluids at a pH of 7.4, the s o l u b i l i t y of this a l k a l o i d

-4 in w a t e r at this pH b e i n g less than 3 x 10 M.

The s o l u t i o n s u s e d i n t r a v e n o u s l y c o n t a i n e d 0.2 m g / m l of the base.

The m u l t i b a r r e l m i c r o p i p e t t e s used in the s p i n a l cord and t h a l a m u s w e r e of 5-7 y M tip d i a m e t e r . T h o s e u s e d in Deiters' n u c l e u s w e r e 7-9 P M as the cells of this n u c l e u s are large (C.120y) and c o m m o n l y r e q u i r e d r e l a t i v e l y high e l e c t r o p h o r e t i c c u r r e n t s to p r o d u c e m e a s u r a b l e effects. The a n i m a l s were a n a e s t h e t i s e d wi t h p e n t o b a r b i t o n e and a b i l a t e r a l p h e u m o t h o r a x p e r ­ f o r m e d for e x p e r i m e n t s on the t h a l a m u s and D e i t e r s n u c l e u s .

D e i t e r s 1 N u c l e u s

The a p p r o a c h to Deiters' n u c l e u s was that d e s c r i b e d by Ito, Hongo, Yo s h i d a , O k a d a and O b a t a (1964). The p h a r y n x and o e s o p h a g u s were d i v i d e d at C3 level and r e f l e c t e d c r a n i a l l y to e x p o s e the b a s i o c c i p u t , a flap

of w h i c h was r e m o v e d , e x p o s i n g the lower pons and m e d u l l a . The a n i m a l s h e a d was v e n t r o f l e x e d 20° so that the e l e c ­ trode, w h e n i n t r o d u c e d a p p r o x i m a t e l y 5 mm c a u d a l to the p o n t o m e d u l l a r y junct i o n , a v o i d e d the i n f e r i o r c e r e b e l l a r artery. The v e n t r a l spinal cord was e x p o s e d by p a r t i a l r e m o v a l of the i n t e r v e r t e b r a l disc b e t w e e n C3 and C4 v e r t e b r a l b o dies; this e x p o s u r e t h r o u g h the disk was c h o s e n b e c a u s e the a n t e r i o r d u ral s i n u s e s of the s p i n a l cord are m a x i m a l l y s e p a r a t e d at this site. A b i p o l a r s t i m u l a t i n g e l e c t r o d e was p l a c e d on the v e n t r a l s u r f a c e of the spi n a l cord to s t i m u l a t e the v e n t r a l s p i n o c e r e ­ b e l l a r t r a c t so a c t i v a t i n g D e i t e r s n e u r o n e s a n t i d r o m i

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-15

t a x i c a l l y in lobules II, III and IV of the i p s i l a t e r a l c e r e b e l l a r verm i s 2mm from the m i d l i n e , and c e m e n t e d w i t h d e n t a l a c r y l i c to a p e r s p e x p l a t e r i g i d l y a t t a c h e d to the skull and s t e r e o t a x i c h e a d frame.

T h a i a m u s

T h a l a m i c n e u r o n e s were a p p r o a c h e d s t e r e o t a x i c a l l y , the o v e r l y i n g c e r e b r a l tiss u e b e i n g r e m o v e d by s u c t i o n and the e x p o s e d t i s s u e s c o n t i n u a l l y i r r i g a t e d wi t h a c a r b o g e n a t e d m a m m a l i a n R i n g e r s o l u t i o n at 37 ° C . The c o n t r a l a t e r a l ulnar, s u p e r f i c i a l radial, m e d i a n , tibial and p e r o n e a l n e r v e s w e r e p r e p a r e d for e l e c t r i c a l s t i m u ­ lation, so e n a b l i n g n e u r o n e s of the v e n t r a l i s p o s t e r o -l a t e r a -l i s (VPL) and v e n t r a -l i s p o s t e r o m e d i a -l i s (VPM) t h a l a m i c n u c l e i to be a c t i v a t e d . T h a l a m o c o r t i c a l r e ­ lay n e u r o n e s w e r e i d e n t i f i e d as those e x c i t e d w i t h short l a t e n c y (4-5 m s e c for the fore limb, 8-10 m s e c for the h i n d limb) by i m p u l s e s in only one of the p e r i p h e r a l n e r v e s .

(g ) Free a m i n o a c i d s in spi n a l roots, d o r s a l root g a n g l i a and p e r i p h e r a l n e r v e s

(l ) Cat

The c h e m i c a l p r o c e d u r e s in these s t u d i e s we r e p e r f o r m e d by Dr. G.A.R. J o h n s t o n of this d e p a r t m e n t . D o r s a l root g a n g l i a and v e n t r a l roots w e r e o b t a i n e d from the u p p e r s a c r a l and l ower l u m b a r s p i n a l s e g m e n t s of m a t u r e cats (4 to 5 kg) a n a e s t h e t i s e d w i t h p e n t o b a r b i t o n e

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16

union. Care was taken to m i n i m i s e v e n t r a l root c o n ­ t a m i n a t i o n of this segment. S a m p l e s of g a s t r o c n e m i u s and sural n e r v e were o b t a i n e d from the left h i n d limb and s a m p l e s of rad i a l n e rve from the r i ght fore limb.

The s p e c i m e n s w e r e r i n s e d in R i n g e r s o l u t i o n , b l o t t e d , froz e n in l i q u i d n i t r o g e n and s t o r e d at -28°

for no t more than one w e e k b e f o r e e x t r a c t i o n of free amino acids. The s p e c i m e n s we r e w e i g h e d w h i l e still fro z e n and not less than 18 mg u s e d for each e x t r a c t i o n . E a c h s p e c i m e n was h o m o g e n i s e d in a P o t t e r - E 1v e h j e m

h o m o g e n i s e r (glass m o r t a r and T e f l o n pestle) in 2 ml of i c e - c o l d 1% a q u e o u s p i c r i c acid and a l l o w e d to s t a n d at o° for 30 min. D o w e x 2 x 10 (Cl form) resin,

2 0 0 - 4 0 0 mesh, was a d d e d p o r t i o n w i s e to the h o m o g e n a t e s u f f i c i e n t to d i s c h a r g e the y e l l o w c o l o u r in the s o l u ­ tion. The r e sin was f i l t e r e d off w i t h W h a t m a n No. 1 paper, w a s h e d w i t h 2 x 10 ml of 0.02 N - h y d r o c h l o r i c acid and the c o m b i n e d f i l t r a t e and w a s h i n g s e v a p o r a t e d to d r y n e s s b e l o w 40° in v a c u o on a r o t a t o r y e v a p o r a t o r . The r e s i d u e was d i s s o l v e d in a k n o w n v o l u m e of pH 2.2 c i t r a t e bu f f e r , f i l t e r e d , and an a l i q u o t of the f i l t ­ rate a n a l y s e d for a c i d i c and n e u t r a l amino acids on a T e c h n i c o n Auto A n a l y s e r (mod i f i e d by Mr. L.B. James, D e p a r t m e n t of B i o c h e m i s t r y ; o p t i m u m s e n s i t i v i t y range 0.01 to 0.10 y m o l e , a c c u r a c y +_3% w i t h i n this range) u s i n g the two b u f f e r s y s t e m of S p a c k m a n , M o o r e and S t ein (1958). The size of each a l i q u o t a n a l y s e d was such that the c o n c e n t r a t i o n s of a l a n i n e and g l y c i n e each e x c e e d e d 0.01 y m o l e . S e v e r a l s a m p l e s of e x t r a c t s from both d o r s a l and v e n t r a l roots w e r e a n a l y s e d c o m p l e t e l y by the sin g l e c o l u m n p r o c e d u r e of H a m i l t o n (1963) . C o n c e n t r a t i o n s w e r e e x p r e s s e d as y m o l e / g w e t tissue.

( i i ) Dog and rat

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17

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18

III. THE S E N S I T I V I T Y OF S P I N A L I N T E R N E U R O N E S TO L - G L U T A M A T E AND L - A S P A R T A T E

N e u r o c h e m i c a l st u d i e s by Graham, Shank, W e r m a n and A p r i s o n (1967) of the d i s t r i b u t i o n of L - g l u t a m a t e and L-as- p a r t a t e in the feline spi n a l cord have shown that L g l u t a -mate is p r e s e n t in g r e a t e s t amo u n t s in the d o r s a l grey m a t t e r and the h i g h e s t levels of L - a s p a r t a t e o c c u r in the v e n t r a l grey matter. The a u t h o r s b e l i e v e these levels to be c o n s i s t e n t w i t h L - g l u t a m a t e and L - a s p a r t a t e o p e r a t i n g as e x c i t a t o r y t r a n s m i t t e r s at the t e r m i n a l s of p r i m a r y a f f ­ e r e n t fibres and s p i n a l e x c i t a t o r y i n t e r n e u r o n e s r e s p e c t i v e l y .

In the o r i g i n a l s t u d i e s of the a c t i o n of acid i c a m i n o acids w h i c h e x c i t e s p i n a l n e u r o n e s (Curtis and W a t k i n s ,

1961, 1963) , L - g l u t a m a t e and L - a s p a r t a t e were r a ted as b e i n g of a p p r o x i m a t e l y e q ual p o t e n c y as e x c i t a n t s of b o t h R e n s h a w

cells and o t h e r s p i n a l i n t e r n e u r o n e s . In the c e r e b r a l c o r ­ tex, L g l u t a m a t e was r e p o r t e d to be m o r e p o t e n t than L a s p -artate (Krnjevic and P h i l l i s , 1961, 1963) , but o t h e r w o r k e r s found them to be r o u g h l y e q u i p o t e n t (Cra w f o r d and Cur t i s , 1964). These two a m i n o acids h a v e p r o v e d to be e x c i t a n t s of all cells t e s t e d in the b r a i n of the cat (revi e w e d by Curtis and C r a w f o r d , 1969) .

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19

Such c o n c l u s i o n s , ho w e v e r , are b a s e d on s e v e r a l a s s u m p t i o n s . F i r s t l y it is a s s u m e d that these e x c i t a n t s act only on the p o s t s y n a p t i c m e m b r a n e , and not on i n t e r v e n i n g areas of cell m e m b r a n e or p r e s y n a p t i c s t r u c t u r e s . W h i l s t there is e v i ­ dence that this is true for the act i o n of L - g l u t a m a t e and GA B A on i n s e c t and c r u s t a c e a n m u s c l e (Usherwood, M a c h i l i and Leaf, 1968; T a k e u c h i and T a k e u c h i , 1967) the s i t u a t i o n for m a m m a l i a n c e ntral n e u r o n e s r e m a i n s u n k n o w n , a l t h o u g h first o r d e r s e n s o r y cells of the l a m p r e y ( f u n c t i o n a l l y e q u i v a l e n t to dors a l root g a n g l i o n cells of o t h e r v e r t e b r a t e s ) , w h i c h have no s ynapses, are u n r e s p o n s i v e to GA B A and L - g l u t a m a t e a d m i n i s t e r e d e l e c t r o p h o r e t i c a l l y (Martin, W i c k e l g r e n and B e r anek, 1970). It is also a s s u m e d that the s u b s t a n c e s u s e d are not s t r u c t u r a l a n a l o g u e s of the t r a n s m i t t e r , c a p ­ able of c o m b i n i n g w i t h the l a t t e r ' s r e c e p t o r and p r o d u c i n g a re spon s e .

A b s o l u t e s e n s i t i v i t y is of no use in i n f e r r i n g a t r a n s m i t t e r role for a s u b s t a n c e . For e x a m p l e R e n s h a w cells w h i l s t b e i n g very s e n s i t i v e to a c e t y l c h o l i n e , the

likely t r a n s m i t t e r r e l e a s e d at m o t o n e u r o n e axon c o l l a t e r a l s , are more s e n s i t i v e to a n u m b e r of c h o l i n o m i m e t i c s w h i c h do not o c c u r in the c e n t r a l n e r v o u s s y s t e m (Curtis and Ryall,

1 9 6 6 a ) .

Stu d i e s of r e l a t i v e s e n s i t i v i t y h o w e v e r , can p r o v i d e e v i d e n c e for a t r a n s m i t t e r role if d i f f e r e n c e s in s e n s i v i t y can be c o r r e l a t e d w i t h some o t h e r d i f f e r e n c e b e t w e e n the groups of cells b e i n g s t u died, for e x a m p l e in a f f e r e n t c o n n e c t i o n s . Thus R e n s h a w cells are e x c i t e d by A C h , yet d o r s a l horn i n t e r n e u r o n e s are not (Curtis and E c c l e s , 1958).

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(a) The n e u r o c h e m i c a l s t u d i e s of G r a h a m et a 1 . (1967) (b) The p a u c i t y of p r i m a r y a f f e r e n t fibre c o n n e c t i o n s to

R e n s h a w cells w h e n c o m p a r e d w i t h o t h e r s p i n a l i n t e r ­ n e u r o n e s , as j u d g e d by the long l a t e n c y of a c t i v a t i o n of R e n s h a w cells by s t i m u l a t i n g d o r s a l roots (Curtis, P h i l l i s and W a t k i n s , 1961) , g r o u p III fibres in ipsi- l a teral n e r v e s (Eccles, F a t t and K o k e t s u , 1954) or high t h r e s h o l d a f f e r e n t s in c o n t r a l a t e r a l n e r v e s (Wil­ son, T a l b o t and Kato, 1964; Wil s o n , 1966).

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21

R E S U L T S

Data were o b t a i n e d from 176 cells, thi r t y one of w h i c h we r e R e n s h a w cells and the r e m a i n d e r i n t e r n e u r o n e s . Ma n y c o m p u t e r c o m p i l e d maps of the spi n a l cord w e r e o b t a i n e d from these results, and the more s i g n i f i c a n t are i l l u s t r a t e d in the

Fig . Fig .

f o 1 lowing 1

2

o r d e r

:-The d i s t r i b u t i o n of all i n t e r n e u r o n e s The d i s t r i b u t i o n of all R e n s h a w cells Fig . 3 The d i s t r i b u t i o n of i n t e r n e u r o n e s more

sitive to a s p a r a t e than to g l u t a m a t e

sen-Fig . 4 The d i s t r i b u t i o n of i n t e r n e u r o n e s m o r e sitive to g l u t a m a t e than to a s p a r t a t e

sen-Fig . 5 The d i s t r i b u t i o n of i n t e r n e u r o n e s e q u a l l y s e n s i t i v e to a s p a r t a t e and to g l u t a m a t e Fig . 6 The d i s t r i b u t i o n of R e n s h a w cells more

sitive to a s p a r t a t e than to g l u t a m a t e

sen-Fig . 7

It can be

The d i s t r i b u t i o n of R e n s h a w cells m o r e s e n ­ sitive to g l u t a m a t e than to a s p a r t a t e , seen that the d i s t r i b u t i o n of i n t e r n e u r o n e s i n v e s t i g a t e d w i t h i n the spi n a l cord b e a r s no r e l a t i o n to s e n s i t i v i t y to the two a m ino acids (Figs. 3, 4, 5) . In all three c a t e g o r i e s of s e n s i t i v i t y the cells w e r e e v e n l y d i s ­ t r i b u t e d t h r o u g h o u t the areas of spin a l cord studied. H o w ­ ever w i t h R e n s h a w cells, a s i g n i f i c a n t l y h i g h e r p r o p o r t i o n were more s e n s i t i v e to a s p a r t a t e wh e n c o m p a r e d w i t h o t h e r i n t e r n e u r o n e s . These p r o p o r t i o n s are l i s t e d in T a b l e 1. In few cases h o w e v e r was any one c o m p o u n d m o r e than twice as p o t e n t as the o t h e r as shown in Table 2.

[image:30.534.54.516.86.701.2]
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* # * * * * ************* 1 ** * * * ** * *

* *1** 1

* 3111 ** l 222*

***11 112 1

*2 1 1 1 1*

232432 * 1123412*

wmv

*112211 ** ** ** ** ** * ** * ** * ** * M * ** * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *

* * 21 2131 ** *

♦*1 1 2211 *** * *

♦ 1 1 11 11 * * * * *

* 1 1211 * * * * *

* 1 2111* * * * *

*

* 2{f}.* ** ** * ** *

* 2 2 ♦ * * * *

* 11 l* * * * *

* * * ** ** *

* 1 ** * **** *

* **** * *

* ** **

* * * **

** *** ********

** *****

Fig. 1

The distribution of all interneurones

************* ** ** * * * * * * * ** * * * * * * * * ** * * * * * * ** ** * * * * * ** * * * * * * * ** * * * * * * * ** * * * * * * * ** * * *

* * * * 4* * * *

* * * * *

* * * * * * * * * *

* * *

* * * *

** * * * *

* 1 * * * *

* * * * *

* 1 * * * *

* 2 11 * * * *

11

> *1

****1

2 2 1 1 1*

2 411* 42 **

* * * * * * ** **** * * * * ** *****

Fig. 2

The distribution of all Renshaw cells

[image:31.534.89.519.62.694.2]
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* * * *

Fig. 3

••***1 »*

► 1 1 1* »*

21 * ** *

**1

*2 ^

221211 *

1 23 l ♦

* 1 4 * 12 1241 *1 11 1 * 1 212

111

1 11 11 *

1 2 1 * 121 «

11 * 2 * 1*

1 **

• **

********

The distribution of

interneurones more sensitive

to L-aspartate than to

L-glutamate.

**i*** ► 2 *1

► 1*

1*

• 11 * 11 1 11

*.*

****

Fig. U

The distribution of

interneurones more sensitive

to L-glutamate than to

L-aspartate.

* * * u i

* * *1 11 ♦ * * * *

* * n li ♦ ♦ ♦

t i l * * «

*

i ♦*

« i*

* * * *

Fig. 5

The distribution of

interneurones equally sensitive

to L-aspartate and L-glutamate.

[image:32.534.89.514.64.703.2]
(33)

* * ************* ** ** * ** * * ** * * ****** ** **** *** * ** * ** * * * * * * * * * * ** *♦ * * ♦ * * * ** * * ** * * ** * ♦ ** * * * **** * * * * * * * * * * * * * *** * * * * * * * ** * * ♦ * * * * * *

* * 1 * * * * *

* * * * * * *

* * 1 ♦ * * * *

* * l ♦ * * * *

* * 2 1 1 1 1 * * * * * * * 2 3 1 1 * * * * * * * 42 ♦* * * * * * * 1 * * ** * * * * * *1 * * * * * *

* * * * * * *

* ** **

* * * **

** * * * * * * * * * * * ** *****

Fig. 6

The distribution of

Renshaw cells more

sensitive to L-aspartate

than to L-glutamate.

************* ** ** ** * ** * * ** * * * * * * * * ** * * * * * * ** ** * * ** * ** * * * *** * ** * * * * ** ♦ ♦ * * * * * * * * ** * * * ** * * ** * * * * * * * * * * * * ♦ * * * * * * * * * * * * ** * ** **♦ * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *

* 1 * * * * *

* ** * * * * * * * ** ** * * ** * *** * * * * * * * * * * ** ** * * * ** * * * * * * * * * * * * * ** *****

Fig. 7

The one Renshaw cell

more sensitive to

L-glutamate than to

[image:33.534.92.494.79.671.2]
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22

T a b l e 1

N o . w i t h L - a s p .

No. w i t h No. w i t h N o t re- T o t a l L - G l u t . L - A s p . c o r d e d

m o r e p o t - m o r e p o t - e q u i p o t e n t e n t t h a n

L - G l u t .

e n t t h a n w i t h L - G l u t . L - A s p .

R e n s h a w

it 24

c e l l s 1 3 3 31

O t h e r

I n t e r - 81

n e u r o n e s

30 28 6 145

T o t a l 105 31 31 9 176

X 2 = 2 2 . 2 2 6 4 P< .001

T a b l e 2 : I n t e r n e u r o n e s

P o t e n c y r a t i o 1 to 2 P o t e n c y r a t i o >2

A s p a r t a t e > G l u t a m a t e 73 8

G l u t a m a t e > A s p a r t a t e 25 5

R e n s h a w C e l l s

A s p a r t a t e > G 1 u t a m a t e 21 3

[image:34.534.57.525.105.709.2]
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2 3

D I S C U S S I O N

If a c e l l is m o r e s e n s i t i v e , as d e f i n e d in M e t h o d s , to

a s u b s t a n c e A t h a n t o a s u b s t a n c e B, b o t h b e i n g a d m i n i s t e r e d

e l e c t r o p h o r e t i c a l l y , a n y o n e o r c o m b i n a t i o n o f t h e f o l l o w i n g

f a c t o r s c o u l d b e t h e c a u s e , a s s u m i n g o f c o u r s e t h a t e q u a l

c u r r e n t s e j e c t A a n d B a t a p p r o x i m a t e l y e q u a l r a t e s .

(1) A d i f f u s e s m o r e f r e e l y t h a n B, a n d c a n a f f e c t a g r e a t e r

a r e a of t h e c e l l .

(2) A is n o t r e m o v e d as r e a d i l y as B f r o m t h e e x t r a c e l l u l a r

e n v i r o n m e n t , a n d c a n d i f f u s e to a g r e a t e r a r e a of t h e

c e l l or a t t a i n a h i g h e r c o n c e n t r a t i o n o v e r t h e s a m e

a r e a .

(3) T h e r e a r e m o r e r e c e p t o r s o f t h e A t y p e t h a n of t h e B

t y p e w i t h i n a g i v e n a r e a .

(4) A is of a h i g h e r i n t r i n s i c a c t i v i t y ( A r i e n s a n d S i m o n i s ,

1 9 6 4 ) at i t s r e c e p t o r t h a n is B a t i t s r e c e p t o r .

(5) A c a n r e a c t w i t h b o t h t y p e A a n d t y p e B r e c e p t o r s w h e r e a s

B c a n n o t .

(6) T y p e A r e c e p t o r s a r e in g e n e r a l c l o s e r to t h e a d m i n i s ­

t e r i n g m i c r o p i p e t t e t h a n a r e t h o s e o f t y p e B.

If B is t h e n f o u n d t o b e m o r e p o t e n t t h a n A o n a n o t h e r

c e l l , t h e d i f f e r e n c e s in c e l l s e n s i t i v i t y c o u l d b e d u e t o :

-(1) T h e s e c o n d c e l l h a s a h i g h e r p r o p o r t i o n o f t y p e B r e ­

c e p t o r s t h a n t h e f i r s t w i t h i n t h e v o l u m e o f t i s s u e

a f f e c t e d b y e j e c t e d s u b s t a n c e s . T h i s c o u l d b e t h e r e ­

s u l t o f a h i g h e r n u m b e r o f B r e c e p t o r s p r e s e n t u n i f o r m l y

o v e r t h e c e l l , o r t h e t y p e B r e c e p t o r s b e i n g c l o s e r to

t h e m i c r o p i p e t t e .

(2) E i t h e r s u b s t a n c e h a s a d i f f e r e n t i n t r i n s i c a c t i v i t y on

d i f f e r e n t c e l l t y p e s . T h i s i m p l i e s a c h a n g e in t h e

r e c e p t o r .

(36)

e n v i r o n m e n t of d i f f e r e n t cells.

If it is a s s u m e d that these c o m p o u n d s act only on s y n a ­ pses and that the cells being s t u d i e d are of r o u g h l y u n i ­

form size and shape, then d i f f e r e n c e s in s e n s i t i v i t y to d i f ­ f e r e n t c o m p o u n d s c o uld r e a s o n a b l y be i n t e r p r e t e d in terms of r e l a t i v e p r o p o r t i o n s of the d i f f e r e n t synapses.

G i v e n all these a s s u m p t i o n s , the results o b t a i n e d for R e n s h a w cells and i n t e r n e u r o n e s can be i n t e r p r e t e d as s u g g e s ­ ting that R e n s h a w cells have a h i g h e r p r o p o r t i o n of s y n a p s e s at w h i c h a s p a r t a t e is a t r a n m i t t e r than i n t e r n e u r o n e s . In c o n t r a s t i n t e r n e u r o n e s a p p e a r to have more s y n a p s e s at w h i c h g l u t a m a t e o p e r a t e s as an e x c i t a t o r y t r a n s m i t t e r . Hence these findi n g s are c o n s i s t e n t w i t h the h y p o t h e s i s of G r a h a m et al . ( 1967) , that g l u t a m a t e is the t r a n s m i t t e r r e l e a s e d from p r i m a r y a f f e r e n t t e r m i n a l s and a s p a r t a t e is that r e ­ lea s e d by spinal i n t e r n e u r o n e s .

H o w e v e r , in only two of 24 R e n s h a w cells in w h i c h a s p a r ­ tate was more p o t e n t than g l u t a m a t e was the p o t e n c y ratio g r e a t e r than two. I n t e r p r e t i n g this in terms of n u m b e r of sy n a p s e s i m plies that, w h i l s t there may be few p r i m a r y a f f e r ­ ent c o n n e c t i o n s w i t h R e n s h a w cells there is a s i g n i f i c a n t p r o p o r t i o n from o t h e r sou r c e s at w h i c h g l u t a m a t e is released. This b r i n g s out a n o t h e r w e a k n e s s in s tudies of this n a t u r e -the r e l a t i v e p r o p o r t i o n s of s y n a p s e s from v a r i o u s sources onto any one cell type of the m a m m a l i a n c e n t r a l n e r v o u s s y s ­ tem are not k n own w i t h any a c c u r a c y and h e nce c o m p a r i s o n s b e t w e e n these p r o p o r t i o n s and s e n s i t i v i t y to t r a n s m i t t e r su s p e c t s can only be made in a very gross m a nner, even a l l o w ­ ing the a s s u m p t i o n s above.

Figure

Fig . 1
♦ * ** * * *** ******11** *** *M  * *2    232432 *1123412*  wmv1121111  *1 **Fig. 1
••***1»** *Fig. 3►  111 * »* * * * ** *
****** *** *♦ *** ♦* ** ** * **♦** **** * *Fig. 6
+7

References

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