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UBC Theses and Dissertations

Electrical activity of the olfactory bulb Graystone, Peter 1971

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THE ELECTRICAL ACTIVITY OF THE OLFACTORY BULB by PETER GRAYSTONE B.Sc. , U n i v e r s i t y ' o f B r i t i s h C o l u m b i a , 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Depar tment o f PHYSIOLOGY We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1971 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of Br i t ish Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of Br i t ish Columbia Vancouver 8, Canada T H E E L E C T R I C A L A C T I V I T Y OF THE O L F A C T O R Y BULB A B S T R A C T T h e e l e c t r i c a l a c t i v i t y o f t h e o l f a c t o r y b u l b s o f m a n y v e r t e b r a t e s i s c h a r a c t e r i z e d by l a r g e a m p l i t u d e r e g u l a r b u r s t s o f w a v e s . T h e s e w a v e s , known a s i n d u c e d w a v e s , a p p e a r w i t h e a c h i n s p i r a t i o n o f o d o u r o u s a i r t h r o u g h t h e n o s t r i l s . T h e w o r k d e s c r i b e d i n t h i s t h e s i s e s t a b l i s h e s t h a t t h e i n d u c e d w a v e s a r e d e t e c t a b l e i n a v a r i e t y o f s p e c i e s i n v e s t i g a t e d , f r o m a m p h i b i a t o m a m m a l i a . T h e o c c u r r e n c e o f t h e w a v e s u n d e r s i m i l a r e x p e r i m e n t a l c o n d i t i o n s l e a d s t o t h e c o n c l u s i o n t h a t t h e m e c h a n i s m o f t h e i r g e n e s i s i s s i m i l a r i n a l l a n i m a l s . T h e g r o u p a c t i v i t y o f t h e n e u r o n e s i n t h e v a r i o u s l a y e r s o f t h e o l f a c t o r y b u l b was s t u d i e d t o g e t h e r w i t h t h e a m p l i t u d e o f t h e i n d u c e d w a v e s i g n a l . I t was f o u n d t h a t m o s t c e l l u l a r a c t i v i t y o c c u r r e d i n t h e e x t e r n a l p l e x i f o r m a n d m i t r a l c e l l l a y e r s w h e r e a s t h e maximum o f t h e i n d u c e d w a v e a c t i v i t y was i n t h e ' g r a n u l a r l a y e r . A p e a k o f c e l l u l a r a c t i v i t y was o b s e r v e d i n t h e e x t e r n a l p l e x i f o r m a n d m i t r a l c e l l l a y e r s w i t h b o t h n o s t r i l s o c c l u d e d . On o p e n i n g t h e i p s i l a t e r a l n o s t r i l t h e c e l l u l a r a c t i v i t y was e n h a n c e d i n t h e s e same l a y e r s a n d o n o p e n i n g t h e c o n t r a l a t e r a l n o s t r i l i t was d e c r e a s e d . W i t h s t r o n g o l f a c t o r y s t i m u l a t i o n a n i n c r e a s e i n t h e c e l l u l a r a c t i v i t y i n t h e g r a n u l e c e l l l a y e r was o b s e r v e d . R e c o r d i n g s f r o m s i n g l e o l f a c t o r y n e u r o n e s w e r e made and w h e r e a s t h e s e w e r e e a s i l y o b s e r v e d i n t h e e x t e r n a l p l e x i f o r m and m i t r a l c e l l l a y e r s t h e y were f o u n d o n l y r a r e l y i n t h e g r a n u l e c e l l l a y e r . I t i s p r o p o s e d t h a t t h e g r a n u l e c e l l l a y e r i s t h e s i t e o f i n t e g r a t i o n o f t h e i n h i b i t o r y i n f l u e n c e f r o m t h e c o n t r a -l a t e r a l o l f a c t o r y b u l b and f r o m h i g h e r c e n t r e s o f t h e b r a i n . I t i s f u r t h e r p o s t u l a t e d t h a t t h e c e l l s i n t h e g r a n u l e c e l l l a y e r do n o t n o r m a l l y g e n e r a t e a c t i o n p o t e n t i a l s b u t t h a t t h e y a r e c a p a b l e o f d o i n g so w i t h s t r o n g o l f a c t o r y s t i m u l a t i o n . I t i s t h o u g h t t h a t t h e s e c e l l s a r e t h e s i t e o f g e n e s i s o f t h e i n d u c e d wave a c t i v i t y w h i c h . i s p r o b a b l y g e n e r a t e d a t t h e d e n d r o -d e n d r i t i c s y n a p t i c c o n n e c t i o n s w i t h t h e s e c o n d a r y n e u r o n e s . E v i d e n c e i n f a v o u r o f t h e s e d e n d r o d e n d r i t i c s y n a p s e s b e i n g t h e norma 1. p a t h w a y b e t w e e n t h e m i t r a l and g r a n u l e c e l l s i s p r e s e n t e d . T A B L E OF CONTENTS Page I n t r o d u c t i o n A Wave A c t i v i t y o f t h e O l f a c t o r y B u l b a n d M u c o s a 1 B P e r i p h e r a l and C e n t r a l F a c t o r s A f f e c t i n g t h e I n d u c e d Waves o f t h e O l f a c t o r y B u l b 6 C A C o m p a r a t i v e S t u d y o f t h e F r e q u e n c y o f t h e I n d u c e d Waves i n O l f a c t o r y B u l b s o f V e r t e b r a t e s 10 D The C e l l u l a r S t r u c t u r e and t h e E l e c t r i c a l A c t i v i t y o f O l f a c t o r y B u l b N e u r o n e s . . . . 24 P r e s e n t I n v e s t i g a t i o n E x p e r i m e n t a l P r o c e d u r e 45 R e s u l t s A I n d u c e d Wave A c t i v i t y i n P o i k i 1 o t h e r m s 51 B I n d u c e d Wave and S p o n t a n e o u s F i r i n g P r o f i l e s 52 C S i n g l e C e l l R e c o r d i n g s . . 86 D i s c u s s i o n 1. Wave A c t i v i t y 96 2. N e u r o n a l A c t i v i t y 100 3. T h e P a t h w a y s o f t h e O l f a c t o r y B u l b 101 B i b l i o g r a p h y 1 03 A p p e n d i c e s I A S i m p l e B a t t e r y O p e r a t e d T e m p e r a t u r e C o n t r o l U n i t f o r S m a l l A n i m a l R e s e a r c h 116 I I A S e l f C e n t e r i n g R e s p i r a t i o n M o n i t o r f o r S m a l l A n i m a l R e s e a r c h 120 i i . P a g e A p p e n d i c e s C o n t ' d I I I A R a t e m e t e r f o r R e c o r d i n g t h e R a t e o f F i r i n g o f N e r v e C e l l s i n t h e B r a i n s o f A n i m a l s . . . 124 IV A Two C h a n n e l B a n d P a s s F i l t e r w i t h E n v e l o p e D e t e c t i o n 1 2 9 i i i . LIST OF TABLES Page I Homeotherms 12-16 II Poi ki 1 o therms 17-18 III Effect of odour on the frequency of the induced waves in the olfactory bulb of a coachwhip snake 60 IV The relationships between the frequency of olfactory induced waves (f) and temperature (T) 61 V Temperature c o e f f i c i e n t s (Qio) f ° r olfactory induced waves 99 \ i v. L I S T OF FIGURES F i g u r e Page l a . T e m p e r a t u r e d e p e n d e n c e o f t h e f r e q u e n c y o f t h e i n d u c e d waves - l i n e a r 54 l b . T e m p e r a . t u r e d e p e n d e n c e o f t h e f r e q u e n c y o f t h e i n d u c e d waves - s e m i 1 o g a r i t h m i c 55 2. I n d u c e d waves i n I g u a n a - e f f e c t o f p l u g g i n g n o s t r i l s . . . . 56 3. I n d u c e d waves f r o m c o a c h w h i p s n a k e 57 4. S e c o n d h a r m o n i c i n i n d u c e d wave f r e q u e n c y i n c o a c h w h i p s n a k e s 58 5. E f f e c t o f o l f a c t o r y s t i m u l u s on i n d u c e d w aves f r o m c o a c h w h i p s n a k e 59 6. I n d u c e d wave and c e l l f i r i n g p r o f i l e s ( i ) . . . . 63 7. I n d u c e d wave and c e l l f i r i n g p r o f i l e s ( i i ) . . . 64 8. I n d u c e d w a v e s , c e l l u l a r a c t i v i t y and r e s p i r a t i o n a t 200 urn s t e p s , u o p e r h a l f o f b u l b ' '. 66 9. I n d u c e d w a v e s , c e l l u l a r a c t i v i t y and r e s p i r a t i o n a t 200 y s t e p s , l o w e r h a l f o f b u l b 67 10. C e l l u l a r a c t i v i t y p r o f i l e ( i ) . '.. 70 1 1 . C e l l u l a r a c t i v i t y p r o f i l e ( i i ) 71 12. , P r o f i l e o f i n c r e a s e i n c e l l u l a r a c t i v i t y w i t h i p s i l a t e r a l n o s t r i l o p e n ( i ) 72 13. P r o f i l e ' o f i n c r e a s e i n c e l l u l a r a c t i v i t y w i t h i p s i l a t e r a l n o s t r i l o p e n ( i i ) 73 14. P r o f i l e o f d e c r e a s e i n c e l l u l a r a c t i v i t y w i t h c o n t r a l a t e r a l n o s t r i l o p e n ( i ) . 74 , 15. P r o f i l e o f d e c r e a s e i n c e l l u l a r a c t i v i t y w i t h c o n t r a l a t e r a l n o s t r i l o p e n ( i i ) 75 16. E f f e c t o f o l f a c t o r y s t i m u l u s on c e l l f i r i n g p r o f i l e 77 V . F i g u r e Page 17. E f f e c t o f s t r o n g o l f a c t o r y s t i m u l u s on c e l l f i r i n g p r o f i l e . . 78 18. E f f e c t o f b l o w i n g smoke a t a n i m a l on c e l l f i r i n g p r o f i l e 81 19. I n c r e a s e i n i n d u c e d wave a m p l i t u d e and c e l l f i r i n g p r o f i l e w i t h smoke b l o w n d i r e c t l y a t a n i m a l 82 20. E f f e c t o f a r o u s a l on c e l l f i r i n g p r o f i l e . . . . 83 2 1 . I n c r e a s e o f c e l l f i r i n g w i t h i p s i l a t e r a l n o s t r i l o p e n 84 22. H i s t o l o g i c a l s e c t i o n o f o l f a c t o r y b u l b s h o w i n g e l e c t r o d e m a r k s 85 23 . C e l l u l a r a c t i v i t y o f c e l l s i n m i t r a l c e l l 1 a y e r 90 24. C e l l u l a r r e s p o n s e t o smoke, g r a n u l e 1 a y e r . 91 25. C e l l u l a r r e s p o n s e t o v a r i o u s o l f a c t o r y s t i m u l i 92 26. R e s p o n s e t o smoke m i t r a l c e l l l a y e r . . . . 93 27. R e s p o n s e t o p e p p e r m i n t m i t r a l c e l l l a y e r . . . . 94 28. R e s p o n s e t o smoke e x t e r n a l p l e x i f o r m l a y e r 95 ACKNOWLEDGEMENT The h e l p f u l g u i d a n c e o f D r . McLennan i n a l l phases of t h i s work i s g r a t e f u l l y a c k n o w l e d g e d . The h e l p o f D r . Bobb i Low and Joan Rogers w i t h the p o i k i 1 o t h e r m i c s t u d i e s d e s e r v e s s p e c i a l men t i on and the t e c h n i c a l h e l p o f M r . A s s i n n a , Mr . H e n z e , Mr . Wa lke r and M r s . Heap i s a l s o g r e a t l y a p p r e c i a t e d . The c o n s t r u c t i v e c r i t i c i s m of the cand ida te ' s commi t tee D r . D. H. C o p p , D r . F . D. G a r r e t t , D r . J . R. Ledsome, D r . J . A . P e a r s o n , D r . A . G . P h i l l i p s i s g r a t e f u l l y a c k n o w l e d g e d . I would a l s o l i k e to thank M r s . Heck l who comp le ted a l l the rough d r a f t s and r e t y p e s . o f t h i s t h e s i s and my w i f e f o r her f c r e b e a r a n e e d u r i n g the whole p r o j e c t . I N T R 0 D U C T I 0 N In t h i s t h e s i s the very pronounced e l e c t r i c a l wave a c t i v i t y c h a r a c -t e r i s t i c o f the o l f a c t o r y bulbs o f many s p e c i e s w i l l be d i s c u s s e d . The s i m i l a r i t i e s o f these waves i n the d i f f e r e n t s p e c i e s w i l l be d e s c r i b e d with p a r t i c u l a r emphasis on the waves induced by an odour. The dependence o f the frequency o f these induced waves on the temperature o f the animal w i l l be d e s c r i b e d i n d e t a i l and t h e i r o c currence w i l l be r e l a t e d to the e l e c -t r i c a l a c t i v i t y o f the o l f a c t o r y bulb neurones. A) Wave A c t i v i t y o f the O l f a c t o r y Bulb and Mucosa Gerard and Young (1937) r e p o r t e d f i n d i n g " d r a m a t i c a l l y o u t s t a n d i n g p o t e n t i a l s " i n the o l f a c t o r y bulbs o f d e c a p i t a t e d f r o g s . These p o t e n t i a l s were o f the form o f "extremely r e g u l a r " waves a t 3 - 8 Hz with the second harmonic a l s o sometimes p r e s e n t . The temperature o f the p r e p a r a t i o n was not g i v e n . A d r i a n and Ludwig (1938) recorded from the o l f a c t o r y s t a l k o f d e c a p i t a t e d c a t f i s h and expressed s u r p r i s e t h a t they c o u l d not d e t e c t any kind o f synchronous a c t i v i t y i n the s t a l k . A d r i a n (1942) r e p o r t e d s t u d i e s on the o l f a c t o r y bulbs o f hedgehogs and c a t s under Nembutal and c h l o r a l o s e a n a e s t h e s i a . He found "synchronized v o l l e y s " when r e c o r d i n g from c e l l s i n the m i t r a l c e l l l a y e r . He r e p o r t e d a frequency (produced by o l f a c t o r y s t i m u l a t i o n ) o f 40 Hz i n the hedgehog under c h l o r a l o s e and a lower frequency o f 20 - 30 Hz under Nembutal. The b u r s t s o f synchronous a c t i v i t y appeared when a i r passed inward over the o l f a c t o r y mucosa. In the c a t he found waves o f 15 Hz under D i a l and 10 Hz under Nembutal. A d r i a n s t a t e d t h a t a i r f low through the nose produced l a r g e r e g u l a r waves and t h a t a s t r o n g smell converted these to small i r r e g u l a r waves. He l a t e r r e t r a c t e d t h i s o p i n i o n ( A d r i a n 1951a and 1951b) and s t a t e d t h a t mechanical s t i m u l a t i o n d i d d i d not p l a y a p a r t and t h a t the induced waves were due to odour. 2 A d r i a n (1950) p u b l i s h e d h i s f i n d i n g s on r e c o r d i n g s from the r a b b i t o l f a c t o r y bulb. In t h i s paper he i n t r o d u c e d the terminology o f " i n t r i n s i c " o r r e s t i n g waves and "induced" or odour-produced waves, and s e t the b a s i s f o r most of what i s known today about them. He was the f i r s t to note t h a t a b u r s t o f these waves appeared a t each i n s p i r a t i o n . In the r a b b i t under urethane a n a e s t h e s i a the frequency of the waves was 50 - 60 Hz and was c o n s t a n t throughout the b u r s t , w h i l e under Nembutal or D i a l the frequency was reduced to 10 - 15 Hz. He a l s o noted t h a t the induced waves appeared l a t e r than the i n c r e a s e o f m i t r a l c e l l f i r i n g , and i t i s apparent on s t u d y i n g h i s r e s u l t t h a t the "synchronous v o l l e y s " o f m i t r a l c e l l a c t i v i t y r e p o r t e d i n h i s e a r l i e r work probably d i d not r e p r e s e n t a synchrony i n the f i r i n g p a t t e r n o f the m i t r a l c e l l s . I t i s l i k e l y t h a t these v o l l e y s were merely the a d d i t i o n , due to h i s broadband r e c o r d i n g method, o f the m i t r a l c e l l f i r i n g and the induced wave a c t i v i t y . Had he f i l t e r e d out the lower frequency induced waves, the f i r i n g o f the m i t r a l c e l l s would have been found to be r e l a t i v e l y c o n s t a n t . As a l r e a d y mentioned, A d r i a n was unable to r e c o r d synchronous f i r i n g from the o l f a c t o r y t r a c t o f c a t f i s h . S i n c e the m a j o r i t y o f the f i b r e s i n the o l f a c t o r y t r a c t are the axons o f the m i t r a l c e l l s , synchronous a c t i v i t y would be expected to be found i n the t r a c t i f i n f a c t the m i t r a l c e l l s were f i r i n g synchronously. A d r i a n h i m s e l f i n h i s 1950 paper p o s t u l a t e d t h a t the induced waves d i d not o r i g i n a t e i n the m i t r a l c e l l s but i n the s h o r t axon c e l l s o f C a j a l (granule c e l l s ) l o c a t e d below the m i t r a l c e l l l a y e r . A l l i s o n (1953) s t r e s s e d the behavioural importance o f the o l f a c t o r y system i n f i s h , amphibia, r e p t i l e s and most mammals. He a l s o pointed out the v a l u e of the o l f a c t o r y system i n comparative r e s e a r c h due to i t s r e l a t i v e l y c o n s t a n t morphology from cylostomata to mammalia. I t r e p r e s e n t s a p r i m i t i v e p a r t o f the b r a i n t h a t has remained l a r g e l y unchanged d u r i n g 3 e v o l u t i o n , the remainder o f the f o r e b r a i n having developed l a t e r . He commented on the f a c t t h a t past work had been done with mammals and t h a t t h e r e was l i t t l e i n f o r m a t i o n a v a i l a b l e on lower v e r t e b r a t e s . A l l i s o n a l s o noted t h a t i n amphibia and lower orders the m i t r a l and t u f t e d c e l l s are not d i f f e r e n t i a t e d . T h i s d i f f e r e n t a t i o n i s f i r s t e v i d e n t i n r e p t i l e s and i s complete i n mammals. In h i s paper A l l i s o n demonstrated t h a t i n mammals the m i t r a l and t u f t e d c e l l s have o n l y one main or a p i c a l d e n d r i t e and each connects with one glomerulus. In c o n t r a s t i n f i s h e s t here i s more than one d e n d r i t e from each u n d i f f e r e n t i a t e d c e l l making c o n t a c t with two or more g l o m e r u l i . Sem Jacobsen e t . a l . (1953 a & b, 1955, 1956) r e p o r t e d f i n d i n g induced waves with a frequency o f 30 Hz (24 - 36 Hz) when r e c o r d i n g from e l e c t r o d e s implanted i n human p a t i e n t s . Ottoson (1954) f i r s t demonstrated t h a t l a r g e slow p o t e n t i a l s could be recorded from the o l f a c t o r y bulb when od o u r i z e d a i r was blown i n t o the n o s t r i l o f a r a b b i t under l i g h t urethane a n a e s t h e s i a . He i l l u s t r a t e d h i s paper w i t h a slow p o t e n t i a l having an induced wave with a frequency o f approximately 50 Hz superimposed upon i t . The induced waves were a b o l i s h e d with deep urethane a n a e s t h e s i a l e a v i n g the slow wave r e l a t i v e l y u n a f f e c t e d . Deeper a n a e s t h e s i a blocked the slow wave a l s o . Ottoson f u r t h e r demonstrated t h a t these waves were not e l i c i t e d by s t i m u l a t i o n with pure a i r , s u b s t a n t i a t i n g A d r i a n ' s f i n d i n g (1951) t h a t the p r o d u c t i o n o f the induced waves was not due to the mechanical e f f e c t o f movement of a i r over the mucosa. Ottoson (1956) extended t h i s work u s i n g d e c a p i t a t e d f r o g s , and again found a rhythmical o s c i l l a t i o n superimposed on the slow wave, t h i s time w h i l e r e c o r d i n g from the o l f a c t o r y e p i t h e l i u m . The frequency was approximately 18 Hz and the temperature was not g i v e n . On removal o f the bulb the slow p o t e n t i a l could be recorded from the c u t ends o f o l f a c t o r y nerves and a p p l i c a t i o n o f c o c a i n e s u f f i c i e n t to b lock nerve conduction d i d not a b o l i s h the slow p o t e n t i a l . T h i s was taken 4 t o i m p l y t h a t the slow p o t e n t i a l r e c o r d e d f r o m t h e o l f a c t o r y b u l b was p r o p a g a t e d e l e c t r o n i c a l l y a l o n g t h e v e r y s h o r t o l f a c t o r y n e r v e s . O t t o s o n (1959a) r e c o r d i n g f r o m t h e o l f a c t o r y b u l b s o f r a b b i t s under u r e t h a n e a n a e s t h e s i a , c o n f i r m e d t h a t i n t h e a b s e n c e o f an o l f a c t o r y s t i m u l u s t h e r e was a background o f i n t r i n s i c a c t i v i t y . He a g a i n f o u n d t h a t p u r i f i e d a i r p r o d u c e d no i n d u c e d wave a c t i v i t y and t h a t o d o u r i z e d a i r p r o d u c e d a s l o w wave w i t h a s u p e r i m p o s e d i n d u c e d wave. He s t u d i e d t h e e f f e c t s o f i n c r e a s i n g d e p t h o f u r e t h a n e a n a e s t h e s i a and o f a s p h y x i a on t h i s e l e c t r i c a l a c t i v i t y and f o u n d t h a t i n both c a s e s t h e i n t r i n s i c a c t i v i t y was b l o c k e d more r e a d i l y t h a n the i n d u c e d a c t i v i t y . The slow r e s p o n s e was t h e most r e s i s t a n t and o n l y d i s a p p e a r e d a f t e r 20 - 30 m i n u t e s o f a s p h y x i a . He f o u n d t h e same sequence o f e v e n t s w i t h c o c a i n e a p p l i e d t o p i c a l l y t o t h e b u l b . I t i s i n t e r e s t i n g t o note t h a t a f t e r t h e s l o w wave i n t h e o l f a c t o r y b u l b had been b l o c k e d by a l o n g p e r i o d o f a s p h y x i a i t c o u l d s t i l l be r e c o r d e d from the o l f a c t o r y n e r v e b u n d l e s and c o u l d be evoked i n t h e s e b u n d l e s by odourous a i r f o r hours a f t e r t h e a r r e s t o f c i r c u l a t i o n . The i m p l i c a t i o n o f t h i s f i n d i n g i s t h a t t h e slow wave a l t h o u g h i n i t i a t e d i n t h e o l f a c t o r y mucosa i s somehow r e g e n e r a t e d i n t h e o l f a c t o r y b u l b . T h i s was i n v e s t i g a t e d by O t t o s o n (1959) i n c u r a r i z e d . f r o g s . He f o u n d t h a t t h e s l o w waves r e c o r d e d from d i f f e r e n t p a r t s o f t h e b u l b v a r i e d c o n s i d e r a b l y i n shape. O t t o s o n i n t h i s paper i n t r o d u c e d t h e t e r m i n o l o g y o f e l e c t r o - o l f a c t o g r a m (EOG) f o r t h e slow wave p o t e n t i a l r e c o r d e d f r o m t h e n a s a l mucosa. He f o u n d t h a t t h e s l o w p o t e n t i a l c o u l d a l s o be r e c o r d e d f r o m t h e o l f a c t o r y n e r v e b u n d l e s . He c o n c l u d e d t h a t t h e p o t e n t i a l was e l e c t r o t o n i c a l l y c o n d u c t e d from t h e mucosa a l o n g t h e o l f a c t o r y n e r v e s . O t t o s o n (1959b) f o u n d t h a t w i t h s t r o n g odourous s t i m u l a t i o n he r e c o r d e d an " i n d u c e d " wave sup e r i m p o s e d on t h e EOG and t h a t s i m u l t a n e o u s r e c o r d i n g f r o m t h e o l f a c t o r y b u l b and t h e mucosa i n d i c a t e d t h a t t h e two waves were o f 5 d i f f e r e n t o r i g i n . The induced wave i n the bulb had a frequency o f about 6 Hz and t h a t i n the mucosa had a frequency o f about 15 Hz (estimated by the present a u t h o r ) . No temperature was g i v e n . Ottoson noted t h a t the mucosal "induced" waves occured l a t e r than the o l f a c t o r y induced waves and o u t l a s t e d them. These f i n d i n g s o f Ottoson's e x p l a i n the d i f f i c u l t y en-countered by A d r i a n i n t e r p r e t i n g h i s r e s u l t s as e a r l i e r d e s c r i b e d . I t w i l l be remembered t h a t as e a r l y as 1937 Gerard and Young had r e p o r t e d f i n d i n g two f r e q u e n c i e s i n the o l f a c t o r y bulbs of d e c a p i t a t e d f r o g s , one a t 3 - 8 Hz and one a t double t h i s frequency. I t seems l i k e l y t h a t the higher frequency which was seen l e s s o f t e n was generated i n the mucosa wh i l e the lower was produced i n the bulb. T h i s s i t u a t i o n was v e r i f i e d by Takagi and Shibuya (1960b) who c o i n e d the term " p o t e n t i a l o s c i l l a t i o n s " f o r the o s c i l l a t o r y s i g n a l recorded from the o l f a c t o r y mucosa to d i s t i n g u i s h i t from the "induced waves" o f the o l f a c t o r y bulb. They noted t h a t the p o t e n t i a l o s c i l l a t i o n o f t e n appeared a f t e r the o l f a c t o r y nerve impulses and induced waves had begun to d i s a p p e a r or decrease. T h e i r p r e p a r a t i o n c o n s i s t e d o f toads d e c a p i t a t e d under e t h e r . . They measured the frequency o f the p o t e n t i a l o s c i l l a t i o n s and the induced waves but u n f o r t u n a t e l y d i d not r e p o r t the temperature o f t h e i r p r e p a r a t i o n s . They d e s c r i b e d a s l i d i n g frequency f o r both p o t e n t i a l o s c i l l a t i o n and induced waves with the i n i t i a l frequency h i g h e s t . The f i g u r e s given f o r two p r e p a r a t i o n s are: p o t e n t i a l o s c i l l a t i o n , i n i t i a l l y 24 - 16 Hz s l i d i n g to 17 - 14 Hz and f o r the induced waves of the bulb: i n i t i a l l y 14 - 12 Hz s l i d i n g to 9 - 7 Hz. These f i g u r e s c o n f i r m the approximate 2/1 r a t i o n between the frequency of the p o t e n t i a l o s c i l l a t i o n s and the induced waves i n the main phase o f the b u r s t . S i m i l a r r e s u l t s were r e p o r t e d i n the same paper f o r f r o g s . In a l a t e r paper Takagi and Shibuya (1966) f u r t h e r s t u d i e d the p o t e n t i a l o s c i l l a t i o n s and showed t h a t the shapes and magnitude of the bursts v a r i e d when recorded from d i f f e r e n t p a r t s o f the o l f a c t o r y e p i t h e l i u m o f d e c a p i t a t e d toads. They found no r e l a t i o n s h i p between types o f odour and the v a r i a t i o n s i n magnitude, shape and d u r a t i o n o f the p o t e n t i a l o s c i l l a t i o n . The authors commented t h a t s i n c e there appeared to be no r e l a t i o n s h i p between the f i r i n g p a t t e r n o f the o l f a c t o r y nerves and the p o t e n t i a l o s c i l l a t i o n s : "These f i n d i n g s s t r o n g l y suggest t h a t the p o t e n t i a l o s c i l l a t i o n s o r i g i n a t e i n o t h e r elements than the o l f a c t o r y c e l l s . " I t should be remembered t h a t A d r i a n i n 1950 had noted t h a t the induced waves appeared l a t e r than the i n c r e a s e i n m i t r a l c e l l a c t i v i t y recorded from the o l f a c t o r y bulb. A s i m i l a r c o n c l u s i o n t h e r e f o r e c o u l d be made, i . e . t h a t the induced waves o r i g i n a t e i n elements o t h e r than the m i t r a l c e l l s . B) P e r i p h e r a l and C e n t r a l F a c t o r s A f f e c t i n g the Induced Waves of the  O l f a c t o r y Bulb Domino and Ueki (1960) and Ueki and Domino (1961) r e p o r t e d 40 Hz induced waves i n monkeys wi t h the b u r s t s e x a c t l y l o c k e d to i n s p i r a t i o n . In the o l f a c t o r y bulb o f dogs they found b u r s t s o f 40 - 46 Hz with a second frequency o f 20 - 23 Hz. A l l induced a c t i v i t y was removed by o c c l u s i o n o f the i p s i l a t e r a l n o s t r i l o r by s p r a y i n g the n o s t r i l with l o c a l a n a e s t h e t i c . They r e p o r t e d an enchancement o f the induced waves with o c c l u s i o n o f the mouth and c o n t r a l a t e r a l n o s t r i l . I t i s i n t e r e s t i n g to note t h a t whereas most authors agree t h a t the induced waves are l o c k e d to the i n s p i r a t o r y phase o f r e s p i r a t o r y c y c l e , these workers, using dogs, r e p o r t e d bursts a l s o on the e x p i r a t o r y phase (Domino and Ueki, 1960; Ueki and Domino, 1961). However they were measuring r e s p i r a t i o n by t h o r a c i c movement, which may not be synchronous with nasal a i r flow. In a comprehensive study of a r e p t i l e , 7 the caiman, Huggins, Parsons and Pena (1968) recorded induced waves from the o l f a c t o r y bulbs of c h r o n i c animals. They found t h a t the induced waves occured on i n s p i r a t i o n o r s n i f f i n g . Plugging one n o s t r i l u s u a l l y removed the induced waves on t h a t s i d e with no e f f e c t on the c o n t r a l a t e r a l s i d e . With both n o s t r i l s plugged no induced waves were seen on e i t h e r s i d e . Ether vapour i n s t i l l e d i n t o one n o s t r i l removed the induced waves from the bulb on t h a t s i d e f o r 30 - 45 minutes and on the c o n t r a l a t e r a l s i d e f o r 10 minutes. F o r c i n g a i r i n t o one n o s t r i l produced induced waves on the i p s i l a t e r a l s i d e with reduced and l e s s r e g u l a r waves on the c o n t r a l a t e r a l s i d e . Von Baumgarten e t . a l . (1962a) showed t h a t induced waves were not produced i n the c o n t r a l a t e r a l bulb when odour was i n t r o d u c e d i n t o the i p s i l a t e r a l nare. They a l s o demonstrated t h a t induced waves occured i n an o l f a c t o r y bulb which had been severed from the b r a i n by t r a n s e c t i o n o f the o l f a c t o r y peduncle. These workers i n v e s t i g a t e d the r e l a t i o n s h i p of the a c t i v i t y o f nerones i n the o l f a c t o r y bulb to the occurence o f the induced waves. T h e i r f i n d i n g s w i l l be d i s c u s s e d f u l l y i n s e c t i o n D. G a u l t and Leaton (1963) using c h r o n i c c a t s showed t h a t the 40 Hz a c t i v i t y appeared i n the o l f a c t o r y bulb o n l y when the slow p o t e n t i a l (Ottoson Wave) was present and t h a t b l o c k i n g o f the nasal passage caused the disappearance o f the induced wave a c t i v i t y . They concluded t h a t the g r e a t l y i n c r e a s e d 40 Hz a c t i v i t y observed i n the o l f a c t o r y bulb and amygdala d u r i n g arousal r e q u i r e d the i n s p i r a t i o n o f a i r f o r i t s g e n e s i s . In agreement with t h i s f i n d i n g i s t h a t of Mechelse and Lieuwens (1963) who showed t h a t i n a c h r o n i c c a t w i t h one o l f a c t o r y bulb i s o l a t e d , the 40 Hz a c t i v i t y appeared and disappeared i n both bulbs s i m u l t a n e o u s l y with awakening and onset o f s l e e p r e s p e c t i v e l y . G a u l t and Leaton's work showed t h a t although the movement of a i r over the nasal mucosa i s necessary f o r the genesis o f the 40 Hz a c t i v i t y the amplitude i s 8 m o d i f i e d by c e n t r a l c o n t r o l . This was confirmed by G a u l t and Coustan (1965) using acute and c h r o n i c c a t s . The mechanism o f enhancement of 40 Hz induced a c t i v i t y by c e n t r a l f a c t o r s was i n v e s t i g a t e d by a number of workers. Lavfn, Alcocer-Cuaron and Hernandez-Peon (1959) noted t h a t almost any stimulus would i n c r e a s e the a c t i v i t y i n the o l f a c t o r y bulbs o f c h r o n i c c a t s . Domino and Ueki (1960) confirmed t h i s and showed t h a t i n c h r o n i c dogs and monkeys the amplitude o f the c h a r a c t e r i s t i c b u r s t s o f induced waves i n the o l f a c t o r y b u l b s , o l f a c t o r y s t r i a e , p y r i f o r m and p r e p y r i f o r m c o r t i c e s and medial amygdalae depended on the degree o f gross behavioural a r o u s a l and r e s p i r a t o r y s t i m u l a t i o n . Hernandez-Peon, Lavfn, Alcocer-Cuaron and Marcel i n (1960) s t i m u l a t e d the mesencephalic r e t i c u l a r f o r m a t i o n o f c h r o n i c c a t s and showed an enhancement of the induced waves i n the o l f a c t o r y bulb. Yamamato and Iwama (1961) i n c o n t r a s t d e s c r i b e d a d e p r e s s i o n o f the induced wave a c t i v i t y i n unanaesthetized r a b b i t s f o l l o w i n g s t i m u l a t i o n o f mesencephalic r e t i c u l a r f o r m a t i o n . With a c h r o n i c tracheotomized r a b b i t the b u r s t a c t i v i t y was not apparent. They concluded t h a t : "the arousal d i s c h a r g e s are not newly produced a c t i v i t y but simply r e p r e s e n t the p r e x i s t i n g induced waves which are enchanced by augmented r e s p i r a t i o n and general a r o u s a l r e a c t i o n s . " The q u e s t i o n o f whether the enhancement o f induced waves upon arousal was due to i n c r e a s e d a i r f low over the o l f a c t o r y mucosa was c l a r i f i e d by Pagano (1966). He showed t h a t with a c o n s t a n t a i r flow over the mucosa i n c u r a r i z e d tracheotomized c a t s , s t i m u l a t i o n o f the mesencephalic r e t i c u l a r f o r m a t i o n , medullary r e t i c u l a r f o r m ation and the mesencephalic c e n t r a l grey matter produced a t w o - f o l d i n c r e a s e i n induced a c t i v i t y recorded from the o l f a c t o r y b u l b s . On c u t t i n g the o l f a c t o r y peduncle the e f f e c t was removed. T h i s l a t t e r p a r t o f the experiment e l i m i n a t e s the p o s s i b l e argument t h a t even though the a i r flow 9 were held constant the amount of air reaching the mucosa was being modified by a change in the shape of the nostril. It is safe to assume on the basis of this finding that the increase in induced wave activity with behavioural arousal is mediated at least in part by a centrifugal activation system which has its fibres running in the olfactory peduncle. All authors with the exception of one group agree that the flow of air over the nasal mucosa is a requirement for the induced waves in the olfactory system. Centrally induced enhancement of these waves may be produced by various means but only in the presence of nasal air flow. The lone dissenters on this subject are Penaloza—Rojas and Alcocer-Cuaron in (1967). They reported that in chronic tracheotomized cats with the nares blocked with saline soaked cotton or taped with adhesive, 43 Hz bursts were seen in the olfactory bulbs during alerting of the animal. No explanation can be given for this finding and adequate precautions to avoid air flow through the nostrils seem to have been taken. In a more recent paper by Hobson (1967) the requirement for air flow over the mucosa for the genesis of the induced wave was deemed "the necessary ingredient." Kerr & Hagbarth (1955) commented on the centrifugal fibres running in the anterior limb of the anterior commissure. These fibres are apparently derived from basal rhinencephal ic areas and end on the granule cells in the internal and external plexiform layers and in the periventricular layer. (See section D). They also describe fibres from the opposite olfactory bulb which run in the same tract and similarly end on the dendrites of these cells. The dominant effect of stimulating the region of the olfactory tract projection was to depress the induced waves in the contralateral bulb. The ipsilateral induced wave activity was unchanged or slightly enchanced. If 10 the l a t e r a l o l f a c t o r y t r a c t and a n t e r i o r commissure were s e c t i o n e d on the s i d e i p s i l a t e r a l to the s t i m u l a t i o n , the c o n t r a l a t e r a l d e p r e s s i o n was not removed. S e c t i o n o f the a n t e r i o r commissure c o n t r a l a t e r a l to the stimulus completely removed the c o n t r a l a t e r a l d e p r e s s i o n o f induced wave a c t i v i t y . These authors a l s o demonstrated the e f f e c t o f l a t e r a l o l f a c t o r y t r a c t and a n t e r i o r commissure s t i m u l a t i o n on the induced waves o f the bulb. They showed t h a t low frequency s t i m u l a t i o n (30/sec) to the l a t e r a l o l f a c t o r y t r a c t enhanced induced wave a c t i v i t y whereas high frequency s t i m u l a t i o n (100/sec) d i d not a f f e c t i t a p p r e c i a b l y . Low frequency s t i m u l a t i o n (30/sec) to the a n t e r i o r commissure i n c r e a s e d the induced wave amplitude i n the bulb and high frequency s t i m u l a t i o n 100/sec.abolished i t . C u t t i n g the a n t e r i o r commissure caused an i n c r e a s e i n the induced wave a c t i v i t y . C) A Comparative Study o f the Frequency o f the Induced Waves i n O l f a c t o r y  Bulbs o f V e r t e b r a t e s In view o f A l l i s o n ' s comments on the s i m i l a r i t y o f the o l f a c t o r y bulb c y t o s t r u c t u r e i n the v a r i o u s s p e c i e s , i t i s o b v i o u s l y o f i n t e r e s t to compare the induced waves from these s p e c i e s with a view to e s t a b l i s h i n g the s i m i -l a r i t i e s and d i f f e r e n c e s and to determine i f such d i f f e r e n c e s can be c o r r e l a t e d to the e v o l u t i o n a r y changes i n the c e l l u l a r o r g a n i z a t i o n o f the b u l b s . To date approximately 40 papers have been p u b l i s h e d c o n t a i n i n g work on induced waves o f the o l f a c t o r y bulbs o f mammals and o f these 15 were on r a b b i t s and 13 on c a t s . The remainder were r e s t r i c t e d to 5 other s p e c i e s (man 5, monkey 3, dog 2, hedgehog 2, r a t 2, and opossum 2.). One b i r d , the pigeon, (1 paper) has a l s o been s t u d i e d . Amongst the p o i k i l o t h e r m s a t o t a l o f 13 papers o n l y have been p u b l i s h e d ; 7 on amphibia (3 on f r o g s , 4 on toads) 5 on f i s h and one on a r e p t i l e , the caiman. Much of the work on p o i k i l o t h e r m s has been p u b l i s h e d w i t h l i t t l e o r no mention o f the temperature o f the p r e p a r a t i o n . Added c o m p l i c a t i o n s are the e f f e c t s o f the s u r g i c a l p r e p a r a t i o n and a n a e s t h e t i c s ( e s p e c i a l l y b a r b i t u r a t e s ) on the frequency o f the induced waves. A summary o f the r e s u l t s o f induced wave r e c o r d i n g s from these animals i s gi v e n i n Tables I and I I . 12 H O M E O T H E R M S TABLE I 13 AUTHORS DATE PREPARATION OLFACTORY BULB INDUCED WAVE FREQUENCY (Hz) R A B B I T Adr i a n 1950 Ottoson 1954 Mozell & Pfaffman 1954 Ad r i a n 1956 Ottoson 1959 Moulton 1961 Yamamoto & Iwama 1961 Baumgarten, Green 1962 Mancia Moulton 1963 Faure & V i n c e n t 1964 Khazan, K a n d a l a f f 1967 Stone, W i l l i a m s 1968 Carr e g a l Putkonen & Sa r a j a s 1968 urethane D i a l urethane urethane Nembutal urethane c h r o n i c ether & c u r a r e ether c h r o n i c c h r o n i c c h r o n i c c h r o n i c 55 - 65 10 - 15 48 1 0 - 5 0 16*. 41 22 plus 44 55 - 75 30 - 50 36 - 47 50 40 50 50 70 60 60 70% c h l o r a l hydrate 50 @ 37° C 30% P e n t o b a r b i t a l 8 @ 20° C N i c o l l 1969 e t h e r and pento- 37 - 39 b a r b i t a l AUTHORS 14 PREPARATION OLFACTORY BULB INDUCED WAVE.FREQUENCY (Hz) Adr i a n A r d u i n i & Moruzzi L a v u i e t . a l . Hernandez-Peon e t . a l . Gault & Leaton Pagano & G a u l t Ueki, Tanaki & Sugano Boudreau Gaul t & Coustan Penaloza-Rojas & Zeidenweber McLennan & Graystone McLennan, Graystone & Desautels Penaloza-Rojas Alcocer-Cuaron Mechelese & Lieuwens C A T 1942 D i a l 15 Nembutal 1953 Cerveau . 30 - 35 ence-phale l s o 1 e 1959 c h r o n i c 34 - 48 1960 c h r o n i c 34 - 43 1963 c h r o n i c 40 1964 c h r o n i c 40 1964 decamethonium 35 - 36 1964 c h r o n i c 40 1965 ether then c u r a r e or d e c e r e b r a t e 1965 c h r o n i c 37 - 41 1965 c h r o n i c 40 1967 c h r o n i c 42.4 + 2.6 SD 39.8 + 1.7 SD 1967 c h r o n i c 43 1969 c h r o n i c 20 - 60 AUTHORS 1 5 PREPARATION OLFACTORY BULB INDUCED WAVE.FREQUENCY (Hz) M A N Sem Jacobsen e t . a l . 1953 Sem Jacobsen e t . a l . 1953 Sem Jacobsen e t . a l . 1955 Sem Jacobsen e t . a l . 1956 c h r o n i c c h r o n i c c h r o n i c e t h e r t h i o p e n t a l c h r o n i c 30 28 24 26 40 17 30 32 28 25 32 36 38 48 25 34 36 39 35 M 0 N K E Y Domino & Ueki Ueki & Domino Hughes & Mazurowski 1960 1961 1962 c h r o n i c 40 Acute,decamethoniurn 40 c h r o n i c 30 - 50 (43) A d r i a n Putkonen e t . a l . 1942 1964 H E D G E H 0 G c h l o r a l o s e c h r o n i c 40 and 20 8 - 9 Hz, 16 25 - 30 Hz, 3 0 u C 19° C 16 AUTHORS "DATT PREPARATION OLFACTORY BULB INDUCED WAVE FREQUENCY (Hz) D O G Domino & Ueki 1960 c h r o n i c 40 - 46 20 - 23 Ueki & Domino 1961 c h r o n i c 40 - 44 R A T Kl i n g b e r g & Pickenhain 1965 c h r o n i c 50 - 90 O P O S S D M Vaccarezza & A f f a n n i 1964 c h r o n i c 40 - 50 Vaccarezza & Affanni 1966 c h r o n i c 40 - 60 P I G E O N Si e c k & Wenzel 1969 c h r o n i c 25 40 (one animal) P 0 I K I L P T H E R M S TABLE II AUTHOR'S 18 DATE PREPARATION OLFACTORY BULB TEMPERATURE INDUCED WAVE FREQUENCY (Hz) F R O G Gerard & Young 1937 Takagi & Shibuya 1960b d e c a p i t a t e d and 3 - 8 i s o l a t e d a l s o 9 d e c a p i t a t e d under 8 ~* 4 e i t h e r 16 -* 10 Hobson B U L L F R O G 1967 c h r o n i c 12 ^ 18 Takagi & Shibuya 1960a Takagi & Shibuya 1960b Takagi & Shibuya 1961 Segura & De Juan 1966 T O A D i n s i t u decap under e t h e r e t h e r then f l a x e d i l c h r o n i c 18-* 9 14 -» 9 12^-7 10 - 5.7 7.7-*5.0 7 - 11 25 +. 7 25 +. 7 Be r t & Godet Hara, Ueda, Gorbman Hara & Gorbman Dupe & Godet F I S H 1963 Lung F i s h 1965 Salmon c h r o n i c 15 MS222 7 -& f l a x e d i l 8 - 9 10 MS222 & f l a x e d i l 1967 G o l d f i s h 1969 L u n g f i s h c h r o n i c Huggins, Parsons 1968 and Pena C A I M A N c h r o n i c 100 22 10 - 13 f = 0.63T - 4.15 20 31 19 Many of the papers l i s t e d i n Tables I and II have been d i s c u s s e d e a r l i e r . Most authors mentioned the induced wave frequency only i n passing and some d i d not r e p o r t the frequency a t a l l . In these cases i t was estimated where p o s s i b l e by the prese n t author by measurement from the p u b l i s h e d r e c o r d s . HOMEOTHERMS (1) MAMMALS RABBIT I t can be seen t h a t the average induced wave frequency i n the un-a n a e s t h e t i z e d r a b b i t l i e s between 50 and 60 Hz. D i a l , Nembutal, urethane and ether a l l depress the frequency, although o f the f o u r , urethane seems to have the l e a s t e f f e c t on the induced waves. One paper i s of p a r t i c u l a r i n t e r e s t , t h a t o f Putkonen & S a r a j a s (1968). These authors c o o l e d r a b b i t s to 20 - 30° C u s i n g e t h e r i n i t i a l l y to depress t h e r m o r e g u l a t i o n . P r e v i o u s l y implanted e l e c t r o d e s were pre s e n t i n the o l f a c t o r y bulbs. The animals were allowed to rewarm spontaneously over a p e r i o d o f 6 - 9 hours. O l f a c t o r y s t i m u l i were i n t r o d u c e d d u r i n g t h i s p e r i o d and the f r e q u e n c i e s of the induced waves t o g e t h e r with b r a i n temperatures were rec o r d e d . A g r a p h i c a l r e p r e s e n t a t i o n o f induced wave frequency a g a i n s t b r a i n temperature was i n c l u d e d . The frequency/temperature r e l a t i o n s h i p was shown to be l i n e a r , a t l e a s t over the range 25° - 38.5° C. The authors d i d not c a l c u l a t e the equation o f the l i n e and t h i s has been estimated by the present author to be f = 2.6T-47. CAT In the c a t the m a j o r i t y o f work has been done using c h r o n i c p r e p a r a t i o n s 20 thereby y i e l d i n g l e s s v a r i a b l e r e s u l t s f o r the induced wave frequency. Most authors agree on a frequency c l o s e to 40 Hz. McLennan & Graystone (1967) i n a s t a t i s t i c a l study on the v a r i a t i o n o f frequency f o r a g i v e n animal quoted the f o l l o w i n g r e p r e s e n t a t i v e f i g u r e s : CAT 1 42.4 + 2.6 (S'.D.) N = 34 where N i s the number o f o b s e r v a t i o n s and S.D. i s the standard d e v i a t i o n CAT 2 39.8 + 1.7 (S.D.) N = 13 I t should be p o i n t e d out t h a t the v a r i a t i o n found was a r e a l p h y s i o l o g i c a l e f f e c t and not due to experimental e r r o r . A h i g h l y a c c u r a t e computer method o f a u t o c o r r e l a t i o n was used to c a l c u l a t e the induced wave f r e q u e n c i e s . The phase r e l a t i o n s h i p o f the induced wave w i t h i n each i n s p i r a t o r y b u r s t was a l s o computed and the frequency shown to be c o n s t a n t w i t h i n a b u r s t . MAN One group o f workers has c o n t r i b u t e d a l l the papers on the induced waves i n the o l f a c t o r y bulbs of man. Sem Jacobsen e t . a l . (1953, 1953, 1955, 1956) quoted f i g u r e s around 30 Hz; however i t i s not c l e a r whether t h i s ' frequency might have been depressed by b a r b i t u r a t e therapy i n these p a t i e n t s . They showed t h a t the e f f e c t o f ether was to r a i s e the frequency and t h i o p e n t a l to depress i t . MONKEY Two groups o f workers have r e p o r t e d induced waves i n the o l f a c t o r y bulb o f monkeys and agree t h a t the frequency i s c l o s e to 40 Hz i n c h r o n i c animals. HEDGEHOG Two groups o f workers have i n v e s t i g a t e d the induced waves of hedgehogs .Adrian (1942) found 40 Hz and a second frequency o f 20 Hz and Putkonen, S a r a j a s and Suomalainen (1964) i n animals a r o u s i n g from h i b e r n a t i o n gave f r e q u e n c i e s o f 8 - 9 Hz a t 16 - 19° C and 25 - 30 Hz a t 30° C. They quoted o n l y these approximate f i g u r e s and on the b a s i s o f these and from measurement o f t h e i r p u b l i s h e d r e c o r d s the present author estimated the equation f o r the frequency/temperature dependence as f = 1.4T - 14. T h i s y i e l d s a frequency o f 38 Hz a t 37° C which i s comparable to the f i g u r e f o r c a t and monkey. DOG One group o f workers o n l y has recorded induced waves i n c h r o n i c dogs. Domino and Ueki 1960 (23) and Ueki and Domino 1961 (24) quoted f r e q u e n c i e s o f 40 - 46 and 40 - 44 Hz f o r these waves. I t i s o f i n t e r e s t to note t h a t a second o l f a c t o r y bulb frequency o f 20 - 23 Hz was r e p o r t e d i n the f i r s t paper and i n both papers t h i s lower frequency was a l s o recorded from the amygdala. RAT One paper o n l y , t h a t o f K l i n g b e r g and Pickenhain (1965), r e p o r t e d on the induced waves i n the o l f a c t o r y bulbs o f c h r o n i c a l b i n o r a t s . The authors r e p o r t e d f r e q u e n c i e s o f 50 - 90 Hz s t a r t i n g with maximum frequency then slowing d u r i n g b u r s t . OPOSSUM One group o f workers has s t u d i e d one marsupial - the opossum, and has pu b l i s h e d the r e s u l t s i n two papers (Vaccarezza and Affanni (1964, 1966). They quote a f i g u r e o f 40 - 50 Hz i n t h e i r f i r s t paper and 40 - 60 Hz i n the second f o r the frequency o f induced waves i n an a d u l t opossum. S e c t i o n o f the o l f a c t o r y peduncle produced no change. (2) BIRDS PIGEON One paper has been p u b l i s h e d on the induced waves i n the o l f a c t o r y bulb o f b i r d s . S i e c k and Wenzel (1969) obtained r e c o r d s from c h r o n i c a l l y implanted e l e c t r o d e s i n the o l f a c t o r y bulbs o f pigeons. They r e p o r t e d induced wave f r e q u e n c i e s o f 15 - 25 Hz and i n one b i r d o n l y , a frequency o f 40 Hz. I t i s perhaps s i g n i f i c a n t t h a t the e l e c t r o d e s i n t h i s b i r d were s i t u a t e d i n the o u t e r g l o m e r u l a r zone o f the o l f a c t o r y bulb. They noted t h a t the frequency o f the waves was not c o n s t a n t but s t a r t e d higher and then slowed d u r i n g the wave t r a i n . P0IKIL0THERMS LUNGFISH Bert and Godet (1963) r e p o r t e d the occurrence o f 15 Hz induced waves i n the o l f a c t o r y bulbs o f the A f r i c a n l u n g f i s h ( P r o t e r o p t e r u s annectens) upon o l f a c t o r y s t i m u l a t i o n . They d i d not r e p o r t the temperature o f the experiment. In a r e c e n t paper Dupe /and Godet (1969), r e c o r d i n g from c h r o n i c u n r e s t r a i n e d l u n g f i s h with c o n t r o l l e d nasal a i r flow, showed reco r d s o f induced waves a t d i f f e r e n t temperatures. They d i d not quote i n d i v i d u a l f r e q u e n c i e s but gave the equation f o r the frequency/temperature r e l a t i o n s h i p from e a r l i e r work as f = 0.63T - 4.15. Examination o f 23 the 1969 p u b l i s h e d records y i e l d e d the f o l l o w i n g approximate v a l u e s : 7 Hz a t 20° C, 10 Hz a t 26° C, 14 Hz a t 30° C, which are i n c l o s e agreement with the equation given f o r the e a r l i e r work. SALMON AND GOLDFISH Hara and Gorbman (1967) recorded induced waves from the o l f a c t o r y bulb o f g o l d f i s h ( C a r a s s i u s auratus L.) but d i d not r e p o r t the frequency o f t h i s a c t i v i t y . Hara, Ueda and Gorbman (1965) r e p o r t e d f r e q u e n c i e s o f 7 - 9 Hz i n young salmon and 8 - 10 Hz i n a d u l t salmon a t a temperature o f 10 - 13° C. The f i s h were a n a e s t h e t i z e d w i t h t r i c a i n e methanesulphonate (MS222) and immobilized with d - t u b o c u r a r i n e c h l o r i d e . AMPHIBIA FROGS AND TOADS In the seven papers d e a l i n g with induced waves i n these animals, one o n l y r e p o r t e d the temperature a t which the induced waves were reco r d e d . Segura & DeJuan (1966) quoted a f i g u r e o f 7 - 11 Hz a t 25 + 0.7° C i n acute p r e p a r a t i o n s prepared under ether and with deep c u r a r i z a t i o n . They d i d not g i v e a frequency f o r t h e i r c h r o n i c prepara-t i o n s , however they d i d i n c l u d e a r e c o r d from which the present author was a b l e to estimate the frequency f a i r l y a c c u r a t e l y . I t was found to be between 8| and 9^Hz a t the same experimental temperature. 24 REPTILES CAIMAN One group o f authors have r e p o r t e d on the caiman. In a study o f the e f f e c t o f temperature on the o l f a c t o r y induced waves, Huggins e t . a l . (1968) showed a l i n e a r r e l a t i o n s h i p between frequency and temperature over the range 16° C - 30° C; above 30° the frequency approached a l i m i t o f 22 Hz.. They d i d not r e p o r t the equation o f the l i n e , however the present author has estimated i t to be g i v e n by f = 1.1T - 12. D) The C e l l u l a r S t r u c t u r e and the E l e c t r i c a l A c t i v i t y of O l f a c t o r y Bulb  Neurones i ) C e l l u l a r o r g a n i z a t i o n of the o l f a c t o r y bulb. The o l f a c t o r y bulb i s a l a y e r e d s t r u c t u r e which i s approximately symmetrical d o r s a l and v e n t r a l to the small c e n t r a l lumen. The b i p o l a r r e c e p t o r c e l l s o f the o l f a c t o r y mucosa send t h e i r s h o r t f i n e unbranched, unmyelinated f i b r e s i n bundles through the ethmoid bone to the o l f a c t o r y bulb, where they enter the g l o m e r u l i and synapse with the a p i c a l d e n d r i t e s o f the secondary neurones o f the o l f a c t o r y pathway. These secondary neurones c o n s i s t o f two types i n the higher c l a s s e s o f v e r t e b r a t e s - m i t r a l c e l l s and t u f t e d c e l l s . A l e s s d i f f e r e n t i a t e d c e l l r e p l a c e s these two types i n c l a s s e s below the e l a s m o b r a n c h i i . The m i t r a l c e l l s become grouped i n t o a d e f i n i t e narrow l a y e r f i r s t i n the r e p t i l e s , and t r u e t u f t e d c e l l s o ccur o n l y i n the mammals, l o c a t e d on the o u t s i d e o f the m i t r a l c e l l l a y e r and below the g l o m e r u l i . C e l l s thought to be p r e c u r s o r s of the t u f t e d c e l l s l i e on the i n s i d e o f the m i t r a l c e l l l a y e r i n the r e p t i l e s . Short axon p e r i g l o m e r u l a r c e l l s and g r a n u l e c e l l s are a l s o present and the secondary 25 d e n d r i t e s o f the m i t r a l and t u f t e d c e l l s t o g e t h e r with processes o f these s m a l l e r c e l l s i n t e r c o n n e c t between the g l o m e r u l i and synapse i n v a r y i n g f a s h i o n with each o t h e r . For a complete comparative d e s c r i p t i o n o f the c e l l u l a r s t r u c t u r e see A l l i s o n (1953). A l l i s o n (1949) i n a q u a n t i t a t i v e h i s t o l o g i c a l study o f the r a b b i t o l f a c t o r y bulb estimated t h a t t h e r e were 50 m i l l i o n r e c e p t o r c e l l s i n each mucosa. These connect with 1,900 g l o m e r u l i so t h a t each glomerulus r e c e i v e s i n f o r m a t i o n from approximately 26,000 r e c e p t o r s . He estimated t h a t each bulb c o n t a i n e d 45,000 m i t r a l c e l l s and 150,000 t u f t e d c e l l s , so t h a t each glomerulus r e l a y s i n f o r m a t i o n to approximately 24 m i t r a l c e l l s and 68 t u f t e d c e l l s . He estimated a l s o t h a t t h e r e were 60,000 f i b r e s i n each o l f a c t o r y t r a c t . a n d t h a t these were l a r g e l y axons o f the m i t r a l c e l l s . T h i s leaves t h e a n t e r i o r commissure as the o n l y pathway f o r the t u f t e d c e l l axons. I t has been now shown t h a t these axons do not a c t u a l l y run i n the a n t e r i o r commissure but synapse i n the a n t e r i o r o l f a c t o r y nucleus a t the caudal end o f the bulb and the axons o f some o f these t e r t i a r y c e l l s run i n the a n t e r i o r commissure as w i l l be d i s c u s s e d l a t e r . In a d d i t i o n to the a f f e r e n t system, a system o f e f f e r e n t f i b r e s to the o l f a c t o r y bulb from the cerebrum and the c o n t r a l a t e r a l bulb a l s o e x i s t s , as w i l l a l s o be d i s c u s s e d l a t e r . The l a y e r e d s t r u c t u r e o f the o l f a c t o r y bulb lends i t s e l f to e l e c t r o -p h y s i o l o g i c a l i n v e s t i g a t i o n . The c e l l u l a r s t r u c t u r e o f the l a y e r s was d e s c r i b e d i n C a j a l (1911) and i s summarized f o r the v a r i o u s s p e c i e s by Andres (1970). In the mammal the 6 l a y e r s are as f o l l o w s : 1) An o u t e r l a y e r (SN) c o n t a i n i n g the o l f a c t o r y nerves f i b r e s . 2) Immediately below t h i s i s the g l o m e r u l a r l a y e r (SG), c o n t a i n i n g the main s y n a p t i c connections between the incoming o l f a c t o r y nerves and the main, or a p i c a l , d e n d r i t e s o f the m i t r a l and t u f t e d c e l l s . C l u s t e r e d around the g l o m e r u l i are the p e r i glomerular c e l l s with s h o r t axons. 3) Below the glome r u l a r l a y e r i s the e x t e r n a l p l e x i f o r m l a y e r (EPL) which i s the r e g i o n o f i n t e r a c t i o n o f the granule c e l l d e n d r i t e s and the secondary d e n d r i t e s o f the m i t r a l c e l l s . A l s o i n t h i s l a y e r are l o c a t e d the somata of the t u f t e d c e l l s . 4) The m i t r a l c e l l l a y e r i s l o c a t e d approximately 700 - 900 y below the b u l b a r s u r f a c e i n the r a b b i t . I t i s a very t h i n l a y e r o f c e l l s , some 50 yi t h i c k , and serves as a r e l i a b l e landmark both h i s t o l o g i c a l l y and e l e c t r o p h y s i o l o g i c a l l y , as w i l l be d i s c u s s e d l a t e r . 5) Below the m i t r a l c e l l l a y e r i s the r e g i o n c o n t a i n i n g the c e l l bodies o f the granule c e l l s t o g e t h e r with some o f t h e i r d e n d r i t i c p r o c e s s e s . These c e l l s have very s h o r t axons and a r e the short-axon c e l l s d e s c r i b e d by C a j a l (1911). In t h i s l a y e r there are a l s o the axon c o l l a t e r a l s o f the m i t r a l and t u f t e d c e l l s about which more w i l l be s a i d l a t e r . This l a y e r i s named the i n t e r n a l p l e x i f o r m l a y e r (IPL) by Andres (1970) and r e p r e s e n t s the gr a n u l e c e l l l a y e r d e s c r i b e d by C a j a l (1911). 6) F i n a l l y t h e r e i s the innermost l a y e r o r p e r i v e n t r i c u l a r l a y e r (VL) next to the lumen. The l a y e r s r e p e a t on the ot h e r s i d e o f the lumen, the bulb being approximately symmetrical i n a v e r t i c a l plan with a f l a t t e n i n g o f the d o r s a l s u r f a c e l a y e r s . This f l a t t e n i n g i s advantageous i n t h a t i s minimizes the e f f e c t o f a l a t e r a l e r r o r i n e l e c t r o d e placement on the measured e l e c t r o d e depth; the e l e c t r o d e always c r o s s i n g a t r i g h t angles to the c e l l u l a r l a y e r s a t l e a s t i n the upper h a l f o f the bulb. 27 The m i t r a l c e l l s The l a r g e s t c e l l s i n the o l f a c t o r y bulb are the m i t r a l c e l l s and the axons o f these c e l l s form the m a j o r i t y of the f i b r e s i n the l a t e r a l o l f a c t o r y t r a c t (LOT). I t must be presumed t h a t these f i b r e s r e p r e s e n t the major a f f e r e n t pathway f o r the o l f a c t o r y i n f o r m a t i o n . I t i s not s u p r i s i n g t h e r e f o r e t h a t these c e l l s have been the most thoroughly i n v e s t i g a t e d c e l l s i n the o l f a c t o r y bulb. T h e i r l o c a l i z e d p o s i t i o n and r e l a t i v e l y easy access to the t r a c t c o n t a i n i n g t h e i r axons, makes them r e a d i l y i d e n t i f i a b l e e l e c t r o p h y s i o l o g i c a l l y . A d r i a n (1951, 1953, 1956) showed t h a t i n the r a b b i t t h e r e was a s p a t i a l o r g a n i z a t i o n o f the f i r i n g p a t t e r n o f c e l l s i n the m i t r a l c e l l l a y e r e x c i t e d with d i f f e r e n t odours. I t i s worth n o t i n g t h a t Leveteau & MacLeod (1966) and Leveteau (1967) found t h a t the o l f a c t o r y g l o m e r u l i a l s o showed t h i s s p e c i f i c i t y to odours. Mancia e t . a l . (1962a).showed t h a t the response o f secondary neurones to smell was v a r i a b l e and t h a t some were e x c i t e d and some i n h i b i t e d . They found t h i s to be t r u e f o r other neurones i n the bulb as d i d Shibuya and Takagi (1962). Doving (1964) repeated t h i s work using f r o g s , and f u r t h e r noted t h a t most o f the u n i t s were spontaneously a c t i v e and t h a t e i t h e r an a c t i v a t i o n or d e p r e s s i o n c o u l d be observed with odour p r e s e n t a t i o n . The most f r e q u e n t l y observed phenomenon was a d e p r e s s i o n f o l l o w e d by a rebound o f i n c r e a s e d a c t i v i t y when s t i m u l a t i o n was d i s c o n t i n u e d . These e f f e c t s were c l o s e l y r e l a t e d to Ottoson's EOG recorded from the o l f a c t o r y mucosa. One example i s g i v e n i n which the response was changed from an e x c i t a t i o n f o r low c o n c e n t r a t i o n o f odour to an i n h i b i t i o n with p o s t - s t i m u l u s rebound f o r a higher c o n c e n t r a t i o n . The m i t r a l c e l l s have been i n v e s t i g a t e d by a number o f workers by e l e c t r i c a l s t i m u l i o n o f the l a t e r a l o l f a c t o r y t r a c t ( a n t i d r o m i c ) or o f 28 the mucosa or o l f a c t o r y nerve bundles ( o r t h o d r o m i c ) . Von Baumgarten, Green and Marcia (1961), P h i l l i p s , Powell and Shepherd (1961), Yamamoto (1961) , Yamamoto and Iwama (1962), Green, Mancia and von Baumgarten (1962) , von Baumgarten, Green and Mancia (1962b), P h i l l i p s Powell and Shepherd (1963), Ochi (1963), and Shepherd (1963a). In a l l cases the r e s u l t o f the stimulus was to f i r e the m i t r a l c e l l under o b s e r v a t i o n . T h i s a c t i v a t i o n was immediately f o l l o w e d by an i n h i b i t i o n both o f spontaneous f i r i n g o f the c e l l and o f the response to a second stimulus a p p l i e d to the l a t e r a l o l f a c t o r y t r a c t . The p e r i o d o f the i n h i b i t i o n was 50 msec, or lon g e r and oc c u r r e d with a s h o r t l a t e n c y a f t e r the i n i t i a l s t i m u l u s . These workers were d i v i d e d i n t h e i r i n t e r p r e t a t i o n o f t h i s . Some thought t h a t i n h i b i t o r y interneurones (probably g r a n u l e c e l l s ) were a c t i v a t e d by the m i t r a l c e l l axon c o l l a t e r a l s d e s c r i b e d by C a j a l (1911) and o t h e r s suggested a d i r e c t i n h i b i t i o n by these c o l l a t e r a l s . Yamamoto e t . a l . (1963) working with the r a b b i t recorded i n t r a -c e l l u l a r ^ from m i t r a l c e l l s w i t h i n the granule l a y e r d u r i n g s t i m u l a t i o n o f the l a t e r a l o l f a c t o r y t r a c t . They noted t h a t the i n h i b i t o r y post s y n a p t i c p o t e n t i a l (IPSP) observed i n the m i t r a l c e l l s might be a t t r i b u t e d to a c t i v i t y i n interneurones which f i r i n g r e p e t i t i v e l y to a l a t e r a l o l f a c t o r y t r a c t (LOT) shock. They found response p a t t e r n s o f t h i s l a t t e r type o n l y i n the deep l a y e r c e l l s and concluded t h a t a t l e a s t some o f the deep l a y e r c e l l s p a r t i c i p a t e i n g e n e r a t i o n o f the r e c u r r e n t i n h i b i t i o n . These workers a l s o s t i m u l a t e d the o l f a c t o r y mucosa e l e c t r i c a l l y and found an IPSP again i n the m i t r a l c e l l s . They concluded t h a t orthodromic s t i m u l a t i o n o f a m i t r a l c e l l causes i t to a c t i v a t e interneurones which i n t u r n send i n h i b i t o r y bombardments to nearby m i t r a l c e l l s . By s t i m u l a t i n g the a n t e r i o r commissure (AC) they were a b l e to produce an IPSP i n a m i t r a l 29 c e l l , however t h i s AC s t i m u l a t i o n a b o l i s h e d the IPSP normally produced i n the m i t r a l c e l l by LOT s t i m u l a t i o n . They a l s o found t h a t most o f the deep l a y e r c e l l s were suppressed by a c t i v a t i o n o f the AC. They summarized t h e i r f i n d i n g s by p o s t u l a t i n g two interneurones i n a d d i t i o n to the proposed i n t e r n e u r o n d i r e c t l y i n h i b i t o r y to the m i t r a l c e l l ; one o f these i n h i b i t o r y to the m i t r a l c e l l and r e l a y i n g i n f o r m a t i o n from the AC and the o t h e r i n h i b i t o r y to the m i t r a l c e l l i n t e r n e u r o n . Reese and Brightman (1965) showed h i s t o l o g i c a l evidence f o r the occurence o f a d e n d r o d e n d r i t i c synapse between the m i t r a l and g r a n u l e c e l l d e n d r i t e s , which was supported by the e l e c t r o n m i c r o s c o p i c s t u d i e s o f R a i l e t . a l . (1966). The l a t t e r i n v e s t i g a t o r s found two s y n a p t i c c o n t a c t s with o p p o s i t e p o l a r i t i e s o f t e n s i d e by s i d e . They p r e d i c t e d t h a t through these r e c i p r o c a l synapses the m i t r a l c e l l s e x c i t e d the granule c e l l s , which i n t u r n i n h i b i t e d the m i t r a l c e l l s , and suggested t h a t s i n c e the granule c e l l s have no t y p i c a l axons, they might f u n c t i o n without g e n e r a t i n g a c t i o n p o t e n t i a l s . They d i d not exclude the p o s s i b i l i t y t h a t o c c a s i o n a l f i r i n g o f these c e l l s c o u l d a l s o o c c u r . They p r e d i c t e d a l s o t h a t the granule c e l l s c o u l d serve as i n h i b i t o r y interneurones i n the more general sense by i n t e g r a t i n g the i n p u t s to t h e i r deeper l y i n g d e n d r i t e s . They commented t h a t "the g r a n u l e c e l l i s s t r a t e g i c a l l y s i t u a t e d to enable i t s i n h i b i t o r y a c t i v i t y to r e p r e s e n t an i n t e g r a t i o n o f s e v e r a l i n p u t s . " These workers a l s o commented on the p o s s i b i l i t y t h a t i n t e r a c t i o n o f the m i t r a l and g r a n u l e c e l l p o p u l a t i o n s c o u l d p r o v i d e the b a s i s f o r rhythmical a c t i v i t y . R a i l & Shepherd (1968) i n a t h e o r e t i c a l treatment o f the dendro-d e n d r i t i c synapse f u r t h e r a s s e r t e d t h a t s y n a p t i c a c t i v a t i o n would not r e q u i r e a p r e s y n a p t i c a c t i o n p o t e n t i a l i n e i t h e r the m i t r a l or g r a n u l e 30 c e l l d e n d r i t e s . They noted t h a t r e p o r t s o f u n i t a r y a c t i v i t y i n g r a n u l e c e l l s are r a r e . They again put forward the proposal t h a t the dendroden-d r i t i c s y n a p t i c i n t e r a c t i o n s are well s u i t e d f o r the development o f rhythmical a c t i v i t y such as the induced waves of the o l f a c t o r y b u l b . P r i c e (1968) has shown i n e l e c t r o n m i c r o s c o p i c s t u d i e s o f the r a t o l f a c t o r y bulb, t h a t two types o f synapses are found between the m i t r a l and t u f t e d c e l l d e n d r i t e s and those o f the g r a n u l e c e l l s . In a second paper P r i c e (1969b) proposed t h a t the synapses with a s y m e t r i c a l s y n a p t i c t h i c k e n i n g s and s p h e r i c a l v e s i c l e s are e x c i t a t o r y and the synapses with symmetrical t h i c k e n i n g s and s m a l l e r f l a t t e n e d v e s i c l e s are i n h i b i t o r y . S i m i l a r synapses have been found by C o l o n n i e r (1968) i n the c a t v i s u a l c o r t e x . Recently a number o f workers have i n v e s t i g a t e d the d e n d r o d e n d r i t i c mechanism i n the o l f a c t o r y bulb and t h e i r r e s u l t s support the e x p l a n a t i o n o f the m i t r a l c e l l i n h i b i t i o n i n v o l v i n g t h i s mechanism as opposed to the axon c o l l a t e r a l approach. Westecker (1969, 1970a, 1970b), Shepherd (1969) and N i c o l l (1969, 1970). In two r e c e n t papers P i n c h i n g (1970) and Hinds (1970) r e p o r t f i n d i n g d e n d r o d e n d r i t i c synapses i n the g l o m e r u l a r r e g i o n o f the o l f a c t o r y bulb. These may be between the d e n d r i t e s o f the m i t r a l and t u f t e d c e l l s and those o f the p e r i g l o m e r u l a r and short-axon c e l l s . Hinds (1970) a l s o suggests the p o s s i b i l i t y t h a t t u f t e d c e l l s , which have been c o n s i d e r e d to be m o d i f i e d p e r i g l o m e r u l a r c e l l s , might form d e n d r o d e n d r i t i c synapses with the m i t r a l c e l l s . 31 The t u f t e d c e l l s The p r o p e r t i e s o f the t u f t e d c e l l s a re l e s s well understood than those of the m i t r a l c e l l s . H i s t o l o g i c a l l y they appear to have s i m i l a r connections to those o f the m i t r a l c e l l s . C a j a l (1911) s t a t e d t h a t the t u f t e d c e l l s u s u a l l y send primary d e n d r i t e s to the g l o m e r u l i and t h a t t h e i r axon c o l l a t e r a l s are g i v e n o f f as h o r i z o n t a l branches deep to the m i t r a l c e l l bodies. Some workers c o n s i d e r t h a t d e n d r o d e n d r i t i c synapses s i m i l a r to those between the m i t r a l and granule c e l l d e n d r i t e s a l s o occur between the t u f t e d c e l l s and the gr a n u l e c e l l s , ( P r i c e 1968, 1969; P i n c h i n g , 1970). C a j a l (1911) and A l l i s o n (1953) thought the axons of the t u f t e d c e l l s ran i n the a n t e r i o r limb o f the a n t e r i o r commissure, however i t i s now knownthat t h i s i s not t r u e . Lohman (1963) i n the guinea p i g and Powell and Cowan (1963) i n the r a t showed by degeneration s t u d i e s t h a t no t u f t e d c e l l axons entered the a n t e r i o r commissure. This f i n d i n g was i n agreement with the e l e c t r o p h y s i o l o g i c a l work o f von Baumgarten e t . a l . (1962b) and Yamamato e t . a l . (1963) who were unable to a c t i v a t e t u f t e d c e l l s a n t i -d r o m i c a l l y by s t i m u l a t i o n of the a n t e r i o r commissure. Green e t . a l . (1962) s t i m u l a t e d the l a t e r a l o l f a c t o r y t r a c t and recorded some c e l l s which were i n the e x t e r n a l p l e x i f o r m l a y e r d r i v e n by the stimulus with a l a t e n c y of 4 - 1 0 msec. They found t h a t the l a t e n c y was l e s s c o n s t a n t than f o r the an t i d r o m i c response o f m i t r a l c e l l s . The c e l l s o f t e n f i r e d 2 o r 3 times i n response to the stimulus but t h a t they o f t e n r e q u i r e d s e v e r a l repeated pulses b e f o r e they f i r e d . They i n t e r p r e t e d t h i s as i n d i c a t i v e o f a s y n a p t i c a l l y d r i v e n event. The c e l l s which they presumed were t u f t e d c e l l s would not f o l l o w r a t e s o f s t i m u l a t i o n above 50/ sec. implying t h a t the pathway of a c t i v a t i o n i n v o l v e d an i n t e r n e u r o n e . Lohman & Mentink (1969) i n the r a b b i t demonstrated axons o f the t u f t e d c e l l s i n the l a t e r a l o l f a c t o r y t r a c t . The f i b r e s o f the a n t e r i o r commissure o r i g i n a t e d i n the a n t e r i o r o l f a c t o r y nucleus and no t u f t e d c e l l axons were pr e s e n t . V a l v e r d e (1965) however, thought t h a t the t u f t e d c e l l axons d i d not l e a v e the bulb. In r e c e n t work N i c o l l (1970a) i d e n t i f i e d t u f t e d c e l l s by s t i m u l a t i n g thy l a t e r a l o l f a c t o r y t r a c t and o b t a i n i n g a n t i d r o m i c a l l y a c t i v a t e d c e l l s i n the e x t e r n a l p l e x i f o r m l a y e r . The l a t e n c y o f t h i s a c t i v a t i o n was 15 msec, compared with 2 msec, f o r the m i t r a l c e l l s . He s t a t e d t h a t these c e l l s with long l a t e n c y were t u f t e d c e l l s and t h a t a t l e a s t some t u f t e d c e l l axons ran the f u l l l e n g t h o f the l a t e r a l o l f a c t o r y t r a c t . He f u r t h e r s t a t e d t h a t s t i m u l i to the a n t e r i o r limb of the a n t e r i o r commissure f a i l e d to a c t i v a t e any c e l l s a n t i d r o m i c a l l y , a f i n d i n g r e p o r t e d e a r l i e r by von Baumgarten e t . a l . (1962b)and Yamamoto e t . a l . (1963) Yamamoto e t . a l . (1963) concluded t h a t the primary a c t i o n o f the a n t e r i o r commissure f i b r e s was to e x c i t e the interneurones o f the bul b , these i n t u r n having an i n h i b i t o r y i n f l u e n c e on the m i t r a l c e l l s . They suggested t h a t the interneurones a c t i v a t e d by the a n t e r i o r commissure f i b r e s were themselves a l s o i n h i b i t o r y to the interneurones which mediated the i n h i b i t i o n o f the m i t r a l c e l l s f o l l o w i n g a l a t e r a l o l f a c t o r y t r a c t v o l l e y . T h i s i n more r e c e n t terminology would be known as d i s i n -h i b i t i o n . I t should be remembered t h a t Kerr & Hagbarth (1955) i n the c a t r e p o r t e d d e p r e s s i o n o f the induced waves produced by an odour when the i p s i l a t e r a l a n t e r i o r commissure was s t i m u l a t e d a t 100/ pulses sec. They a l s o demonstrated an i n c r e a s e i n the amplitude o f these induced waves when the a n t e r i o r commissure was c u t . Low frequency (30/ sec) s t i m u l a t i o n o f the i p s i l a t e r a l / l a t e r a l o l f a c t o r y t r a c t produced an i n c r e a s e i n the induced wave a c t i v i t y . However these authors a l s o showed a marked i n c r e a s e i n the induced waves when s t i m u l a t i n g the a n t e r i o r commissure a t 33 30/ sec. I t should be noted t h a t Valverde (1965) r e p o r t e d c o l l a t e r a l s o f the axons o f the c e l l s o f the a n t e r i o r o l f a c t o r y nucleus which are g i v e n o f f immediately a f t e r t h e i r o r i g i n . The e f f e c t s seen by s t i m u l a t i n g the a n t e r i o r commissure w i l l undoubtedly be a l t e r e d by the a n t i d r o m i c a c t i v a t i o n o f these c o l l a t e r a l s . Orrego (1962) i n the t u r t l e demonstrated an evoked p o t e n t i a l i n the i n t e r n a l g r a n u l a r / l a y e r o f the c o n t r a l a t e r a l o l f a c t o r y bulb caused by s t i m u l a t i n g the i p s i l a t e r a l bulb. He showed t h a t s t i m u l a t i o n of the i p s i l a t e r a l bulb blocked t r a n s m i s s i o n i n the pathway from the mucosa to the p y r i f o r m c o r t e x o f the o p p o s i t e s i d e f o r p e r i o d s up to 300 msec. He i n t e r p r e t e d t h i s to i n d i c a t e t h a t the a n t e r i o r commissure f i b r e s end on the granule c e l l s , as proposed by C a j a l (1911), and t h a t these are the same c e l l s which are a c t i v a t e d by l a t e r a l o l f a c t o r y t r a c t s t i m u l a t i o n to i n h i b i t the m i t r a l c e l l s . Shepherd (1963b), using r a b b i t s a n a e s t h e t i z e d with urethane and c h l o r a l o s e , recorded the f i r i n g o f c e l l s i n the g l o m e r u l a r , e x t e r n a l p l e x i f o r m and the g r a n u l e l a y e r s . He was a b l e to r e c o r d from granule c e l l s which c o u l d be f i r e d w i t h v a r i a b l e l a t e n c y by r e p e t i t i v e s t i m u l a t i o n o f the l a t e r a l o l f a c t o r y t r a c t , and concluded t h a t these c e l l s were f i r e d by a c t i v a t i o n by m i t r a l c e l l axon c o l l a t e r a l s . He a l s o recorded from c e l l s i n the g l o m e r u l a r l a y e r which were d r i v e n by v o l l e y s to the o l f a c t o r y nerve but not by v o l l e y s to the l a t e r a l o l f a c t o r y t r a c t . He i d e n t i f i e d these as g l o m e r u l a r short-axon c e l l s . The responses ranged from s i n g l e impulses to b u r s t s o f impulses. He r e p o r t e d f i n d i n g c e l l s which were spontaneously a c t i v e and these were mostly i n the r e g i o n where the short-axon c e l l s predominate. C e n t r i p e t a l and C e n t r i f u g a l Pathways of the O l f a c t o r y Bulb There i s reasonable agreement amongst i n v e s t i g a t o r s on the d i s t r i b u t i o n 34 of the c e n t r i p e t a l o l f a c t o r y c o n n e c t i o n s . A r e c e n t review, ( S c a l i a , 1968) and a more r e c e n t paper ( S c a l i a , 1970), summarize these c o n n e c t i o n s . The areas to which f i b r e s p r o j e c t c o n s i s t o f the p r e p y r i f o r m c o r t e x and p e r i a m y d a l o i d a r e a , the c o r t i c a l amygdaloid nucleus, the l a t e r a l p a r t of the o l f a c t o r y t u b e r c l e , the nucleus o f the l a t e r a l o l f a c t o r y t r a c t and the parahippocampal a r e a . This d i s t r i b u t i o n agrees with the work o f Dennis & Kerr (1968) i n the c a t . Some c o n f u s i o n e x i s t s i n the e a r l i e r work due to the p o s s i b l e i n c l u s i o n o f the a n t e r i o r o l f a c t o r y nucleus i n the o l f a c t o r y bulb l e s i o n s ( S c a l i a , 1970). F e r r e r (1969) placed l e s i o n s i n the r o s t r a l p a r t o f the a n t e r i o r o l f a c t o r y nucleus of the hamster and t r a c e d the degenerating f i b r e s . Degeneration was found i n the l a t e r a l 2/3 of the o l f a c t o r y t u b e r c l e , p r e p y r i f o r m , p y r i f o r m and periamygdaloid c o r t i c e s , c o r t i c o m e d i a l amygdala, dorsomedial nucleus o f the thalmus, l a t e r a l habenular n u c l e u s , T a t e r a l hypothalamic area and the s u p r a - o p t i c a r e a . I f the medial p a r t o f the a n t e r i o r o l f a c t o r y nucleus was l e s i o n e d , the degeneration c o u l d be t r a c e d to the same area but two a d d i t i o n a l areas were found to be i n v o l v e d , the b a s o l a t e r a l amygdala and the mammillary a r e a s . Whereas most workers agree on the d i s t r i b u t i o n o f the c e n t r i p e t a l pathways, the p i c t u r e r e g a r d i n g the c e n t r i f u g a l pathways i s not r e s o l v e d . Ca j a l (1911) showed evidence o f c e n t r i f u g a l pathways i n the o l f a c t o r y t r a c t . T h i s has been confirmed by Cragg (1962), Powell and Cowan (1965) and Powell, Cowan and Raisman (1965) and i s now g e n e r a l l y accepted. Doving and Gemne (1965), working w i t h the l a t e r a l o l f a c t o r y t r a c t o f the burbot, have demonstrated t h r e e components o f the compound a c t i o n p o t e n t i a l , r e p r e s e n t i n g f i b r e s w i t h conduction v e l o c i t i e s of 5.5, 2.4 and 0.25 m/sec. a t 10° C. The l a t t e r component i s found o n l y i n the l a t e r a l p o r t i o n o f the medial bundle o f the t r a c t and i s thought to correspond to f i b r e s with diameters o f l e s s than 0.5 /jm. U n f o r t u n a t e l y the f u n c t i o n a l s i g n i f i c a n c e o f the s u b d i v i s i o n s o f the t r a c t i s not known, but i t seems probable t h a t the slower components r e p r e s e n t c e n t r i f u g a l f i b r e s . There i s general agreement on the c r o s s connections between the a n t e r i o r o l f a c t o r y n u c l e i , w i t h the f i b r e s running i n the a n t e r i o r commissure as a l r e a d y d i s c u s s e d . There i s however s t i l l doubt as to the o r i g i n o f the c e n t r i f u g a l f i b r e s running i n the l a t e r a l o l f a c t o r y t r a c t . Doving and Gemne (1966) recorded from the c e n t r a l stump o f a p a r t l y c u t o l f a c t o r y t r a c t i n the burbot. The a c t i v i t y recorded was a t t r i b u t e d to the axons o f neurones c a r r y i n g impulses c e n t r i f u g a l l y toward the bulb. On c u t t i n g the remainder o f t h i s t r a c t or the c o n t r a l a t e r a l t r a c t , the spontaneous a c t i v i t y was reduced. I f the i p s i l a t e r a l o r c o n t r a l a t e r a l t r a c t was s t i m u l a t e d , c e n t r i f u g a l a c t i v i t y was i n c r e a s e d i n the c u t c e n t r a l stump o f the i p s i l a t e r a l t r a c t . C u t t i n g the a n t e r i o r commissure d i d not remove the responses produced by i p s i l a t e r a l s t i m u l a t i o n , i n d i c a t i n g an uncrossed c e n t r i f u g a l system. Uni t s were a l s o observed which c o u l d be a c t i v a t e d by both i p s i - and c o n t r a l a t e r a l s t i m u l a t i o n . On touching the s k i n o f the animal a few spontaneously a c t i v e f i b r e s were i n h i b i t e d . In a second paper Doving (1966) showed t h a t c u t t i n g the o l -f a c t o r y t r a c t changed the frequency d i s t r i b u t i o n o f the r a t e o f f i r i n g o f the secondary o l f a c t o r y neurones, but t h a t the mean frequency however was not s t a t i s t i c a l l y d i f f e r e n t . S t i m u l a t i n g the c e n t r a l end o f the p a r t l y c u t o l f a c t o r y t r a c t caused e i t h e r an i n h i b i t i o n or an e x c i t a t i o n o f the o l f a c t o r y neurones, although 60% o f the u n i t s observed showed an i n h i b i t i o n . Dennis & Kerr (1968) from the r e s u l t s o f evoked p o t e n t i a l s t u d i e s i n c a t s , claimed t h a t c e n t r i f u g a l pathways e x i s t i n the l a t e r a l o l f a c t o r y t r a c t emanating from " a l l r e g i o n s r e c e i v i n g l a t e r a l o l f a c t o r y t r a c t e f f e r e n t s " . These pathways were not i n t e r r u p t e d by s e c t i o n o f the i p s i -l a t e r a l o l f a c t o r y t r a c t i n the peduncle. From degeneration s t u d i e s i n r a t s , P r i c e (1969a) s t a t e d : "These r e s u l t s i n d i c a t e t h a t the c e n t r i f u g a l f i b r e s to the o l f a c t o r y bulb a r i s e from a d i s c r e t e nucleus i n the basal f o r b r a i n , the nucleus o f the h o r i z o n t a l limb o f the diagonal band, and not from any o f the s t r u c t u r e s which r e c e i v e a primary p r o j e c t i o n from the o l f a c t o r y bulb. Furthermore although t h i s nucleus does not have d i r e c t a f f e r e n t connections from any o f these primary o l f a c t o r y s t r u c t u r e s , i t does r e c e i v e a f f e r e n t f i b r e s from the l a t e r a l hypothalamus, which r e c e i v e s c o nnections from the o l f a c t o r y pathways, and from the m i d b r a i n . There i s a l s o another i p s i l a t e r a l , b u l b o p e t a l system, but i t i s m u l t i s y n a p t i c and i s c o n f i n e d to the primary o l f a c t o r y s t r u c t u r e s ; i t c o n s i s t s o f f i b r e s which pass forwards from the p y r i f o r m c o r t e x to synapse i n the a n t e r i o r o l f a c t o r y n u c l e u s , from which, i n t u r n , axon c o l l a t e r a l s pass i n t o the o l f a c t o r y bulb along with the f i b r e s from the a n t e r i o r commissure." The m u l t i s y n a p t i c pathway d e s c r i b e d by P r i c e (1969) presumeably corresponds to t h a t o f Dennis and Kerr (1968) s i n c e the l a t t e r workers found t h a t s e c t i o n i n g the l a t e r a l o l f a c t o r y t r a c t i n the peduncle d i d not a f f e c t the pathway, implying a synapse i n the a n t e r i o r o l f a c t o r y n ucleus. I t would seem from the r e s u l t s o f these r e c e n t workers t h a t t h e r e are two c e n t r i f u g a l systems to the o l f a c t o r y b u l b s . One r e p r e s e n t s a m u l t i s y n a p t i n e g a t i v e feedback system i n v o l v i n g a l l the o l f a c t o r y t r a c t p r o j e c t i o n areas and the o t h e r i s a more d i r e c t pathway from the hypothalmus and m i d b r a i n . T h i s l a t t e r pathway would be the more l i k e l y one i n v o l v e d w i t h the a r o u s a l mechanism d i s c u s s e d i n s e c t i o n B. F a c i l i t a t i o n o f the d r i v e t h i s pathway by other c e n t r a l s t r u c t u r e s c o u l d account f o r the d e p r e s s i o n o f induced wave a c t i v i t y r e p o r t e d by Yamamoto and Iwama (1961). Response o f the o l f a c t o r y bulb neurones to p h y s i o l o g i c a l s t i m u l i The p a t t e r n s of f i r i n g o f the o l f a c t o r y bulb neurones i n response to p h y s i o l o g i c a l s t i m u l i has been r e p o r t e d o n l y by a few i n v e s t i g a t o r s . Walsh (1956) recorded from neurones i n the o l f a c t o r y bulbs o f r a b b i t s under deep urethane a n a e s t h e s i a . The c e l l s were not i d e n t i f i e d but were l o c a t e d i n r e g i o n s c o r r e s p o n d i n g approximately to the m i t r a l c e l l l a y e r . Three types o f c e l l were observed: C l a s s I, the most commonly encountered, d i s c h a r g e d c o n t i n u o u s l y and spontaneously with f i r i n g p a t t e r n s which bore no r e l a t i o n to the animals' r e s p i r a t o r y c y c l e . The p a t t e r n a l s o was not m o d i f i e d d u r i n g the p r e s e n t a t i o n o f an o l f a c t o r y s t i m u l u s . T h i s type o f c e l l was the o n l y one found i n animals with t r a c h e a l cannulae and i n which no a i r was passing i n t o the nares. C l a s s II c e l l s f i r e d i n b u r s t s only d u r i n g i n s p i r a t i o n though a few were found to d i s c h a r g e d u r i n g e x p i r a t i o n . The f i r i n g o f these c e l l s was a l s o found to be unmodified by an o l f a c t o r y s t i m u l u s . The average d i s c h a r g e was 20 a c t i o n p o t e n t i a l s per b u r s t and the r a t e was q u i t e c o n s t a n t during the b u r s t . C l a s s I I I neurones d i s c h a r g e d upon s t i m u l a t i o n with one or more odours. T h e i r r e s t i n g d i s c h a r g e s v a r i e d from q u i e s c e n t to a t o n i c i r r e g u l a r d i s c h a r g e . An odour s p e c i f i c i t y appeared to be i n d i c a t e d . More c e l l s responded to c i g a r e t t e smoke than any other odour. Von Baumgarten e t . a l . (1961) observed the a c t i v i t y o f c e l l s i n 3 8 the o l f a c t o r y bulb i n c u r a r i z e d and cerveau i s o l e r a b b i t s . They found c e l l s whose f i r i n g p a t t e r n s were m o d i f i e d by d i f f e r e n t odours but no s p e c i f i c i t y was seen. These workers demonstrated an i n h i b i t i o n o f c e l l f i r i n g upon s t i m u l a t i o n o f the o p p o s i t e o l f a c t o r y bulb o r a n t e r i o r commissure. T h i s i n h i b i t i o n was a b o l i s h e d by s e c t i o n o f the a n t e r i o r commissure. I n h i b i t i o n o f a few neurones was a l s o observed upon s t i m u l a t i o n o f the mid b r a i n tegmentum and upon t a c t i l e s t i m u l a t i o n . S t i m u l a t i o n o f the i n t r a l a m i n a r thalamus was without e f f e c t . Mancia e t . a l . (1962a) i n tracheotomized, a r t i f i c i a l l y v e n t i l a t e d r a b b i t s found spontaneously a c t i v e neurones i n a l l l a y e r s o f the bulb. They s t i m u l a t e d the o l f a c t o r y mucosa by d r i v i n g 80 ml o f odour i z e d a i r i n t o e i t h e r n o s t r i l by means o f a s p r i n g d r i v e n s y r i n g e . Using the same odour they found a g r e a t v a r i e t y of responses i n d i f f e r e n t c e l l s : a) an i n c r e a s e i n f i r i n g t h a t c o u l d o u t l a s t the stimulus f o r a few seconds, b) an i n c r e a s e i n f i r i n g d u r i n g the sti m u l u s f o l l o w e d by a r e d u c t i o n i n f i r i n g as the stimulus ended. c) a s h o r t i n h i b i t i o n a t the beginning o f the stimulus and then i n c r e a s e d f i r i n g , d) i n h i b i t i o n during or o u t l a s t i n g the stimulus o f t e n f o l l o w e d by a rebound. e) a s h o r t i n h i b i t i o n a t the beginning and ag a i n a t the end o f the s t i m u l u s , f ) b u r s t d i s c h a r g e s d u r i n g the stimulus f o l l o w e d by an a r r e s t o f f i r i n g when the stimulus ceased. C e l l s a few microns a p a r t o f t e n behaved q u i t e d i f f e r e n t l y i n response to an o l f a c t o r y s t i m u l u s . They r e p o r t e d odour s p e c i f i c i t y of c e l l f i r i n g i n the e x t e r n a l p l e x i f o r m , m i t r a l and g r a n u l a r l a y e r s . These workers a l s o i n v e s t i g a t e d the e f f e c t o f the c o n t r a l a t e r a l bulb on the i p s i l a t e r a l neurones and showed t h a t an o l f a c t o r y stimulus to the c o n t r a l a t e r a l n o s t r i l u s u a l l y i n h i b i t e d c e l l f i r i n g . They commented t h a t o l f a c t o r y s t i m u l a t i o n to the i p s i - and c o n t r a l a t e r a l n o s t r i l s o f t e n had a " m i r r o r image" e f f e c t . In another paper these i n v e s t i g a t o r s (Mancia e t . a l . 1962b) r e p o r t e d no r e l a t i o n s h i p between the c e l l u l a r a c t i v i t y and the gross e l e c t r i c a l a c t i v i t y recorded i n the f r o n t a l c o r t e x . S t i m u l a t i o n o f the midbrain r e t i c u l a r f o r mation caused o l f a c t o r y bulb c e l l s to stop f i r i n g f o r the p e r i o d o f the s t i m u l a t i o n , with t h i s i n h i b i t i o n remaining f o r a s h o r t p e r i o d a f t e r the s t i m u l u s ceased. An example was given o f an a n t i d r o m i -c a l l y i d e n t i f i e d m i t r a l c e l l which behaved i n t h i s manner. In a few cases o n l y an i n c r e a s e i n the f i r i n g of a c e l l was observed. On s t i m u l a t i n g the i n t r a l a m i n a r thalamus no e f f e c t was seen on the spontaneous f i r i n g o f the o l f a c t o r y bulb neurones. In encephale i s o l e p r e p a r a t i o n s , touching the limbs or touching or blowing the f u r of the animal i n h i b i t e d the spontaneous d i s c h a r g e o f " c e r t a i n neurones a t v a r i o u s l e v e l s " . Examples were given o f a t u f t e d and a m i t r a l c e l l which e x h i b i t e d these charac-t e r i s t i c s . An extremely i n t e r e s t i n g o b s e r v a t i o n was made by these workers on the e f f e c t o f mesencephalic r e t i c u l a r f o r mation (MRF) s t i m u l a t i o n on the response o f c e l l s to an i p s i l a t e r a l o l f a c t o r y s t i m u l u s . A h i s t o l o -g i c a l l y i d e n t i f i e d t u f t e d c e l l was i n h i b i t e d by an o l f a c t o r y stimulus a l o n e . C o n c u r r e n t l y there was an i n c r e a s e i n the induced wave a c t i v i t y . T h i s was commented upon by these workers and was estimated by the present author to have a frequency of approximately 30 Hz. S t i m u l a t i o n of the MRF 40 alone produced no e f f e c t ; however when the MRF s t i m u l a t i o n and the o l f a c t o r y s t i m u l u s were a p p l i e d t o g e t h e r , a marked i n c r e a s e i n the c e l l f i r i n g (and the wave a c t i v i t y ) was observed. The c e l l continued to have an i n c r e a s e d r a t e o f f i r i n g a f t e r both s t i m u l i were d i s c o n t i n u e d . An example o f an a n t i d r o m i c a l l y i d e n t i f i e d m i t r a l c e l l i s g i v e n . T h i s c e l l i n c r e a s e d i t s f i r i n g with an i p s i l a t e r a l o l f a c t o r y stimulus alone and was u n a f f e c t e d by MRF s t i m u l a t i o n a l o n e . When the two s t i m u l i were combined the e f f e c t o f the o l f a c t o r y s t i m u l u s was removed. There was a l s o an i n c r e a s e i n the amplitude o f the wave a c t i v i t y estimated by the p r e s e n t author to have a frequency o f approximately 30 Hz. Another example o f t h i s e f f e c t i s g i v e n w i t h a marked i n c r e a s e i n the wave a c t i v i t y a t 30 Hz. Toward the end o f the MRF s t i m u l a t i o n a c e l l with a s m a l l e r a c t i o n p o t e n t i a l was seen to s t a r t f i r i n g . T h i s had a very r e g u l a r f i r i n g r a t e , estimated by the present author to be approximately 60 s p i k e s / s e c . These workers r e f e r to the wave a c t i v i t y as induced waves but s i n c e the p r e p a r a t i o n was an encephale i s o l e r a b b i t , the induced wave frequency would have been expected to have been c o n s i d e r a b l y higher than t h i s and i f the e f f e c t s o f the s u r g i c a l a n a e s t h e s i a had worn o f f should have been c l o s e to 60 Hz. The authors conclude t h a t the r e s u l t s i n d i c a t e r e t i c u l a r c o n t r o l but t h a t i t i s u n l i k e l y to be d i r e c t l y to the second order neurones s i n c e they were not a f f e c t e d by MRF s t i m u l a t i o n a l o n e . They a l s o note t h a t MRF s t i m u l a t i o n d i d not produce "induced waves" unless there was co n c u r r e n t o l f a c t o r y s t i m u l a t i o n . The same workers, (Green e t . a l . 1962) s t i m u l a t e d the l a t e r a l o l f a c t o r y t r a c t and showed a pause i n the spontaneous a c t i v i t y o f m i t r a l , t u f t e d and granule c e l l s . They s t a t e t h a t they made a c a r e f u l search f o r a synap-t i c a l l y a c t i v a t e d i n t e r n e u r o n e but were unable to f i n d one. T h i s same group o f r e s e a r c h e r s , von Baumgarten e t . a l . (1962a) p u b l i s h e d the o n l y paper devoted to an i n v e s t i g a t i o n o f the r e l a t i o n s h i p between the wave a c t i v i t y and neuronal a c t i v i t y i n the o l f a c t o r y bulb. The experiments were performed on tracheotomized r a b b i t s under urethane a n a e s t h e s i a or with cerveau i s o l e or encephale i s o l e p r e p a r a t i o n s . The animals were tracheotomized, a r t i f i c a l l y v e n t i l a t e d and immobilized with d - t u b o c u r a r i n e . O l f a c t o r y s t i m u l i were d e l i v e r e d to the nares by means of a s p r i n g d r i v e n s y r i n g e . C e l l u l a r a c t i v i t y was recorded from a micro-e l e c t r o d e and they s t a t e d t h a t induced waves were recorded from the same e l e c t r o d e by f i l t e r i n g w i t h a low pass f i l t e r with a time constant o f 1 second, to a t t e n u a t e the the s p i k e s . (This i s very u n l i k e l y to be c o r r e c t , as such a f i l t e r would have a 3 db high frequency c u t o f f o f 0.16 Hz. I t i s assumed t h a t t h i s i s a m i s p r i n t i n the manuscript and should have been a time c o n s t a n t o f 1 m i l l i s e c o n d . T h i s would have given a 3 db high frequency c u t o f f of 160 Hz s u f f i c i e n t to a t t e n u a t e the s p i k e s but not to a t t e n u a t e or p h a s e - s h i f t the induced and i n t r i n s i c waves.) A macro-electrode was p l a c e d on the s u r f a c e o f the bulb c l o s e to the s i t e of i n s e r t i o n o f the m i c r o - e l e c t r o d e . T h i s second e l e c t r o d e was used as a r e f e r e n c e e l e c t r o d e f o r the wave a c t i v i t y . The induced wave a c t i v i t y recorded from the m i c r o - e l e c t r o d e was i n phase with t h a t recorded from the s u r f a c e macro-electrode when the m i c r o - e l e c t r o d e was i n the e x t e r n a l p l e x i f o r m l a y e r . When i t was moved to the granule l a y e r , the induced wave s i g n a l was found to be almost 180° out o f phase with the r e f e r e n c e s i g n a l on the s u r f a c e o f the bulb. No c l e a r n u l l was o b s e r v a b l e i n the m i t r a l c e l l l a y e r , however the amplitude o f the induced waves con t i n u e d to i n c r e a s e with the depth o f the e l e c t r o d e from the s u r f a c e , f o r a d i s t a n c e o f 2 mm. A c l e a r n u l l was observed i n the t h i r d component o f the evoked p o t e n t i a l produced by s t i m u l a t i o n o f the l a t e r a l o l f a c t o r y t r a c t . T h i s 42 n u l l occured a t the m i t r a l c e l l l a y e r . T r a n s e c t i o n o f the a n t e r i o r commissure i n c r e a s e d the amplitude o f the induced waves and complete i s o l a t i o n o f the o l f a c t o r y bulb d i d not prevent the occurrence o f induced waves or spontaneous a c t i v i t y i n the bulb. These f i n d i n g s are i n agreement with the work o f Kerr & Hagbarth (1955). Stimu-l a t i o n o f one o l f a c t o r y bulb by the passage o f an odour over the mucosa on t h a t s i d e d i d not produce induced waves i n the o t h e r o l f a c t o r y bulb. T h i s i s i n agreement wi t h the work o f A d r i a n (1942) and subsequent workers, as d e s c r i b e d i n s e c t i o n B o f the p r e s e n t t h e s i s . Von Baumgarten e t . a l . (1962a) a l s o d e s c r i b e d the r e l a t i o n s h i p o f the c e l l u l a r a c t i v i t y o f m i t r a l , t u f t e d and granule c e l l s i n r e l a t i o n to the induced wave a c t i v i t y . They noted t h a t the c o r r e l a t i o n f o r the m i t r a l c e l l s was l e s s exact than t h a t f o r the other two c e l l t y pes. They r e p o r t e d t h a t the t u f t e d c e l l s f i r e d predominantly i n the e a r l y p a r t o f the negative going phase o f the induced wave. The granule c e l l s showed a r e d u c t i o n o f f i r i n g a t approximately the same p o i n t i n the phase r e l a t i o n s h i p t h a t the t u f t e d c e l l s showed a peak. They d i d not r e p o r t any attempt to c l a s s i f y the g r a n u l e c e l l s on the b a s i s of t h e i r response to an o l f a c t o r y s t i m u l u s . They a l s o noted i n t h e i r d i s c u s s i o n t h a t an a n t i d r o m i c v o l l e y by way o f the l a t e r a l o l f a c t o r y t r a c t f a i l e d to a c t i v a t e any granule c e l l s . In view o f the anatomy of the m i t r a l and granule c e l l i n t e r a c t i o n s , s t i m u l a t i o n o f the l a t e r a l o l f a c t o r y t r a c t would have been expected to e x c i t e g r a n u l e c e l l s e i t h e r by axon c o l l a t e r a l s or d e n d r o d e n d r i t i c synapses both o f which are thought to cause e x c i t a t i o n of the g r a n u l e c e l l . One i s reminded o f the p o s t u l a t e o f R a i l e t . a l . (1966) t h a t the granule c e l l s may not normally produce an a c t i o n p o t e n t i a l . The s i g n i f i c a n c e of the f i n d i n g s o f von Baumgarten e t . a l . (1962a,)can best be e v a l u a t e d by quoting from t h e i r own d i s c u s s i o n o f the r e s u l t s . "As to the r e l a t i o n s h i p between the induced waves and u n i t a r y d i s c h a r g e s , we cannot assume t h a t the induced waves are merely envelopes o f a c t i o n p o t e n t i a l s because high s y n c h r o n i z a t i o n i n the bulb has been r e p o r t e d a f t e r clamping the c a r o t i d a r t e r i e s ( A r d u i n i and Moruzzi 1953). Under these c o n d i t i o n s presumably c e l l s do not f i r e i n the granule l a y e r . ... ... In some e a r l y experiments i n which we f a i l e d to a c h i e v e maximum amplitude o f the induced wave, the r e l a t i o n s h i p between u n i t s and slow waves was much l e s s exact and the c e l l c o u l d f i r e a t v a r i o u s p o i n t s ... ...On the o t h e r hand when a high degree o f r e g u l a r s i n u s o i d a l a c t i v i t y was achieved, as a f t e r the i n j e c t i o n o f s t r y c h n i n e a very p r e c i s e phase r e l a t i o n was observed between the slow waves and the u n i t a r y d i s c h a r g e s . " These workers summarized t h e i r f i n d i n g s i n the f o l l o w i n g way: "the p o s i t i v e component o f the evoked p o t e n t i a l recorded i n the granule l a y e r and the induced wave recorded a t the same l e v e l are most e a s i l y a t t r i b u t e d to s y n a p t i c and p o s t - s y n a p t i c events, s i n c e n e i t h e r o f them c o u l d be c o r r e l a t e d to a c t i o n p o t e n t i a l s a t the same l e v e l . " The most r e c e n t work r e l a t i n g the a c t i v i t y of the o l f a c t o r y bulb neurones to o l f a c t o r y s t i m u l a t i o n w i t h chemical substances i s t h a t o f Doving and Hyvarinen (1969) i n the burbot. From i n t e r v a l histograms and s e r i a l c o r r e l a t i o n s o f the f i r i n g p a t t e r n s of secondary o l f a c t o r y neurones they concluded " i t seems l i k e l y t h a t d i f f e r e n c e s i n e x t e r n a l c o n d i t i o n s , f o r example d i f f e r e n c e s i n the q u a l i t y o f the s t i m u l a n t s , are not coded as v a r i a t i o n s i n the time s t r u c t u r e o f the impulse sequence sent towards the b r a i n . " 44 PRESENT INVESTIGATION The p r e s e n t i n v e s t i g a t i o n was d i v i d e d i n t o three p a r t s : 1) Records o f o l f a c t o r y bulb induced wave a c t i v i t y were made a t a v a r i e t y o f temperatures from a s e l e c t i o n o f p o i k i l o t h e r m s prepared w i t h c h r o n i c a l l y implanted e l e c t r o d e s . The r e l a t i o n s h i p o f induced wave frequency to body temperature was e s t a b l i s h e d so t h a t comparison among p o i k i l o t h e r m i c s p e c i e s and with mammals was p o s s i b l e . A number o f ot h e r experimental procedures were a l s o performed to p r o v i d e f u r t h e r b a s i s f o r comparison with the mammalian s p e c i e s s t u d i e d by oth e r workers. 2) On the b a s i s o f the r e s u l t s o f the f i r s t p a r t o f the i n v e s t i g a t i o n , one s p e c i e s , the r a b b i t , was chosen as the most s u i t a b l e animal f o r more i n t e n s i v e study. A s e r i e s o f acute experiments was performed using r a b b i t s under l i g h t urethane a n a e s t h e s i a . In these the induced wave a c t i v i t y was recorded a t d i f f e r e n t depths through the o l f a c t o r y bulb and the amplitude o f the waves compared with those from a r e f -erence e l e c t r o d e l o c a t e d i n the same bulb. A p r o f i l e was e s t a b l i s h e d o f the induced wave amplitude versus depth below the s u r f a c e o f the bulb. A second s e r i e s o f experiments was peformed using l a r g e m i c r o e l e c t r o d e s i n which the spontaneous f i r i n g o f c e l l s was recorded a t v a r i o u s depths through the o l f a c t o r y bulb. The r e s u l t i n g s i g n a l s were a m p l i f i e d and f e d to a ratemeter (Appendix I I I ) and a p r o f i l e was c o n s t r u c t e d o f c e l l a c t i v i t y versus depth below the s u r f a c e o f the o l f a c t o r y bulb. A comparison was made between the induced wave and 45 the spontaneous c e l l f i r i n g p r o f i l e s . 3) The e x t r a c e l l u l a r e l e c t r i c a l a c t i v i t y o f s i n g l e neurones was recorded from the o l f a c t o r y bulbs o f r a b b i t s under l i g h t urethane a n a e s t h e s i a . The r e c o r d i n g was performed from spontaneously a c t i v e c e l l s and the r e l a t i o n between the induced wave a c t i v i t y and spontaneous f i r i n g was i n v e s t i g a t e d . The r e l a t i o n s h i p o f the c e l l f i r i n g to r e s p i r a t i o n through the i p s i l a t e r a l n o s t r i l was e s t a b l i s h e d f o r a l l c e l l s observed. The r a t e of c e l l f i r i n g was estimated by means o f a "time i n t e r v a l histogram" programme u s i n g a PDP8 computer. EXPERIMENTAL PROCEDURE Chronic Animals ( P o i k i l o t h e r m s ) In a l l cases the animals were a n a e s t h e t i z e d and b i p o l a r e l e c t r o d e s were placed u n i l a t e r a l l y o r b i l a t e r a l l y i n the c e n t r e o f the o l f a c t o r y bulbs under v i s u a l c o n t r o l . The a n a e s t h e t i c used f o r the toads was t r i c a i n e methanesulphonate (MS222, 250 m g / l i t e r of tap water). T h i s gave a good depth of a n a e s t h e s i a which was r a p i d l y r e v e r s e d by removing the animals p o s t - o p e r a t i v e l y to f r e s h tap water. The a n a e s t h e t i c used f o r the iguanas and coach-whip snakes was sodium p e n t o b a r b i t a l (Nembutal, 30 mg/kg). Bu l l s n a k e s were a n a e s t h e t i z e d i n i t i a l l y w ith 4% halothane and then maintained on a 1% mixture. The animals were warmed to about 35° C p r i o r to the a n a e s t h e s i a and were maintained a t t h i s temperature d u r i n g the p o s t - o p e r a t i v e r e c o v e r y p e r i o d to i n c r e a s e t h e i r metabolism. A l l animals were t r e a t e d p o s t - o p e r a t i v e l y with p e n i c i l l i n G (100,000 I.U./Kg). The e l e c t r o d e s c o n s i s t e d o f 2 p a i r s of 0.2 mm d i a . s t a i n l e s s s t e e l wires separated by 1 mm and attached to 2 small s o c k e t s . The wires were then i n s u l a t e d w i t h epoxy cement except f o r { mm a t the t i p s . The s k u l l was exposed and a p a i r o f small holes were d r i l l e d d i r e c t l y over the o l f a c t o r y bulbs and the e l e c t r o d e s p l a c e d i n p o s i t i o n . The sockets were then anchored with a c r y l i c cement to a small screw i n s e r t e d i n the s k u l l . Records o f the e l e c t r i c a l a c t i v i t y o f the o l f a c t o r y bulbs were o b t a i n e d a f t e r a t l e a s t 3 days r e c o v e r y from s u r g e r y , e i t h e r by d i r e c t wire connec-t i o n to the s o c k e t on the animal's head, or by t e l e m e t r y . The s i g n a l s were a m p l i f i e d and r e c o r d e d on an FM tape r e c o r d e r f o r subsequent a n a l y s i s . On playback the s i g n a l s were d i s p l a y e d on a 2 channel o s c i l l o s c o p e w i t h the time base o f f . A moving f i l m camera was used to photograph the s i g n a l s on 35 mm Kodagraph paper or f i l m . A c a l i b r a t i o n frequency was p l a c e d on the magnetic tape a t the time o f r e c o r d i n g the o r i g i n a l r e c o r d s . T h i s c a l i b r a t i o n was used to determine the f r e q u e n c i e s of the induced waves thereby e l i m i n a t i n g the e f f e c t of camera speed v a r i a t i o n s and s h r i n k a g e or s t r e t c h i n g o f the paper or* film.in/'.the photographic p r o c e s s . The f r e q u e n c i e s were read by means of a s p e c i a l p r o p o r t i o n a l r u l e and compared with the c a l i b r a t i o n . Temperatures o f the animals were measured with a c l o a c a l t h e r m i s t o r probe and an e l e c t r o n i c thermometer. The body tempera-t u r e o f the toads was a d j u s t e d by changing the temperature o f the water i n which they were swimming. The temperature o f the r e p t i l e s was changed by p l a c i n g them i n a r e f r i g e r a t o r o r under a heat lamp. Acute Animals ( r a b b i t s ) 43 r a b b i t s weighing between 2.5 and 3 kg were chosen without regard to sex. In the w i n t e r months i t was found necessary to i n j e c t the r a b b i t s with p e n i c i l l i n G (200,000 I.U. i n t r a m u s c u l a r l y ) 3 to 5 days be f o r e the experiment to combat r e s p i r a t o r y c o n g e s t i o n . The animals were anaesthe-t i z e d with 30% urethane ( e t h y l carbamate) admi n i s t e r e d i n t r a v e n o u s l y by means of an i n f u s i o n needle i n s e r t e d i n t o an ear v e i n . Depth o f an a e s t h e s i a was determined by o b s e r v a t i o n and the r e q u i r e d dose was normally between 6 and 12 ml. The animal's head was p l a c e d i n a s t e r o t a x i c apparatus and the e l e c t r o d e s p l a c e d by means o f a micrometer d r i v e . The a n i m a l ' s temperature was maintained by means o f a s p e c i a l l y designed b a t t e r y - o p e r a t e d temperature c o n t r o l u n i t ( d e s c r i b e d i n Appendix I ) . The nasal r e s p i r a t i o n was recorded by means of a r e s p i r a t i o n monitor designed f o r the purpose and d e s c r i b e d i n Appendix I I . The s k i n was removed from the s k u l l over the o l f a c t o r y bulbs and the bone c u t around the o u t s i d e o f the area over the b u l b s . The p i e c e o f bone was then g e n t l y l i f t e d o f f , a v o i d i n g p r e s s u r e to the u n d e r l y i n g t i s s u e . A small hole was made by l i f t i n g the dura and s l i t t i n g i t f o r about 0.5 mm wit h a sharp hypodermic needle. In t h i s way i t was p o s s i b l e to a v o i d damage to the d e l i c a t e blood v e s s e l s on the s u r f a c e o f the o l f a c t o r y bulb. The r e c o r d i n g e l e c t r o d e was l o c a t e d on the s u r f a c e o f the bulb w i t h the a i d o f a d i s s e c t i n g micro-scope. The s u r f a c e o f the bulbs was then covered with a gel o f 2.5% agar i n Ringer's s o l u t i o n . The depth o f the e l e c t r o d e s i n the bulbs was determined by means o f a c a l i b r a t e d micrometer d r i v e and the e l e c t r o d e p o s i t i o n s v e r i f i e d by means o f the h i s t o l o g i c a l s e c t i o n s d e s c r i b e d below. A l l e l e c t r o d e t r a c k s were l o c a t e d as a c c u r a t e l y as p o s s i b l e a t the c e n t r e o f the bulbs both i n the 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 the l a t e r a l d i r e c t i o n . T h i s was done so t h a t the e l e c t r o d e s would pass through the 48 b u l b a r l a y e r s a t r i g h t angles thus maximizing the e l e c t r o d e p o s i t i o n a l accuracy. Macro-electrodes were made by c o a t i n g #00 s t a i n l e s s s t e e l i n s e c t pins with Beldenamel (3 co a t s ) l e a v i n g about 30 jum bare a t the t i p . M i c r o - e l e c t r o d e s were prepared by e t c h i n g #00 s t a i n l e s s s t e e l i n s e c t pins i n a bath o f phosphoric a c i d by pa s s i n g c u r r e n t from a f i l a m e n t t r a n s f o r m e r . The v o l t a g e used was i n i t i a l l y 6 v o l t s with a f i n a l p o l i s h i n g with 3 v o l t s . T h i s i s a m o d i f i c a t i o n o f the method d e s c r i b e d by Green (1958). E l e c t r o d e t i p s i z e s from 1 to 5 Aim were obtained by t h i s method. The e l e c t r o d e s were i n s u l a t e d by c o a t i n g with Beldenamel (3 c o a t s ) to w i t h i n a few jum of the t i p . E l e c t r o d e placements were v e r i f i e d by passing a d i r e c t c u r r e n t o f 10 f o r 30 seconds with the e l e c t r o d e p o l a r i t y p o s i t i v e . At the t e r m i n a t i o n o f experiments the b r a i n was perfused w i t h s a l i n e and then w i t h a 1% s o l u t i o n o f potassium f e r r o c y a n i d e i n 10% f o r m a l i n . The o l f a c t o r y bulbs were then removed and p l a c e d i n 10% f o r m a l i n f o r 24 hours. They were then s e t i n p a r a f f i n wax and c u t i n t o 20 ,um s e c t i o n s . The s e c t i o n s were s e l e c t e d by o b s e r v a t i o n , the e l e c t r o d e marks showing as P r u s s i a n blue d o t s . S e c t i o n s were s t a i n e d w i t h s a f r a n i n a n d mounted on g l a s s s l i d e s . For the induced wave p r o f i l e s where two macro-electrodes were employed the s i g n a l s from the e l e c t r o d e s were a m p l i f i e d (pass band 8 - 8 0 Hz) and d i s p l a y e d on a dual beam o s c i l l o s c o p e . These s i g n a l s were a l s o recorded on a 4 t r a c k FM magnetic tape r e c o r d e r t o g e t h e r with the output from the r e s p i r a t i o n monitor r e c o r d i n g the nasal r e s p i r a t i o n and a v o i c e channel. The three data channels were subsequently d i s p l a y e d on a 4 beam o s c i l l o -scope (one beam not used) with the time base o f f and photographed with a 49 moving f i l m camera. An a l t e r n a t i v e method was employed i n l a t e r e x p e r i -ments. Two i d e n t i c a l narrow band f i l t e r s with a c e n t r e frequency o f 50 Hz were used to e x t r a c t the induced wave frequency from the other components. (Appendix I V ) . One was connected to the a m p l i f i e d s i g n a l from the r e f e r e n c e e l e c t r o d e and the other to the a m p l i f i e d s i g n a l from the e x p l o r i n g e l e c t r o d e . The output o f these f i l t e r s was r e c t i f i e d and smoothed using a r e s i s t i v e c a p a c i t i v e c i r c u i t with a time c o n s t a n t o f 0.1 sec. The r e s u l t i n g "envelope" o f the induced wave a c t i v i t y from each o f the e l e c t r o d e s was d i s p l a y e d on a r e c o r d e r . The induced wave p r o f i l e was produced by p l o t t i n g the r a t i o o f the amplitude o f the s i g n a l from the e x p l o r a t o r y e l e c t r o d e to t h a t from the r e f e r e n c e e l e c t r o d e , a g a i n s t the depth o f the e x p l o r a t o r y e l e c t r o d e . The c e l l f i r i n g p r o f i l e s were obt a i n e d by t a k i n g the s i g n a l from a m a c r o - e l e c t r o d e , a m p l i f y i n g i t (pass band 80 Hz to 10 KHz) and f e e d i n g i t through a f i l t e r (pass band 250 Hz to 2.5 KHz,cut o f f 24 db/octave) to the ratemeter. T h i s e l i m i n a t e d the p o s s i b i l i t y t h a t the induced waves or other base l i n e v a r i a t i o n s c o u l d cause an apparent i n c r e a s e i n the c e l l f i r i n g r a t e . A permanent r e c o r d o f the f i r i n g r a t e a t each depth was obt a i n e d by connecting the ratemeter output to a c h a r t r e c o r d e r . When r e c o r d i n g from s i n g l e neurones using a m i c r o - e l e c t r o d e the s i g n a l was f i r s t f e d to an a m p l i f i e r with high i n p u t impedance and c a p a c i t y compensation. The output from t h i s a m p l i f i e r was high pass f i l t e r e d (low frequency c u t o f f 20 Hz) and d i s p l a y e d on one beam of a dual beam o s c i l l o s c o p e . The s i g n a l was a l s o f i l t e r e d (pass band 250 Hz to 2.5 KHz, c u t o f f 24 db/octave) and f e d to the ot h e r beam of the same 5 0 o s c i l l o s c o p e . These s i g n a l s were a l s o recorded on a 4 t r a c k FM tape r e c o r d e r t o g e t h e r with the output from the r e s p i r a t i o n monitor and a vo i c e channel. Subsequently the three data channels were d i s p l a y e d on a 4 beam o s c i l l o s c o p e and photographed with a moving f i l m camera with the time base o f f . The output from the f i l t e r was a l s o connected to the ratemeter (Appendix I I I ) which provided the i n t e r f a c e necessary to d r i v e a PDP8 computer. The c e l l f i r i n g r a t e s were determined by means o f the PDP8 computer u t i l i z i n g a t i m e - i n t e r v a l histogram programme. This programme produced a d i s p l a y on an o s c i l l o s c o p e showing the number o f occurrences o f c e l l a c t i o n p o t e n t i a l s on the Y a x i s a g a i n s t the time i n t e r v a l values on the X a x i s . The r e s u l t i n g histogram was photographed by means o f a p o l a r o i d camera. The programme a l s o had the c a p a b i l i t y o f being a b l e to produce a d i s p l a y of the i n t e r v a l s between a l t e r n a t e a c t i o n p o t e n t i a l s (second order histogram). Other o r d e r histograms were a v a i l a b l e but were not u t i l i z e d i n t h i s study. 51 R E S U L T S A) INDUCED WAVE ACTIVITY IN POIKILOTHERMS  Toads, Bufo marinus (L.) Two toads were implanted b i l a t e r a l l y and i n both cases the p a i r o f e l e c -trodes i n one bulb gave a b e t t e r r e c o r d o f induced waves than d i d the o t h e r . The a c t i v i t i e s i n the two bulbs were o f approximately the same frequency, and t h e r e f o r e measurements were made from the b e t t e r r e c o r d o n l y . Records were taken a t temperatures from 11° C to 37.9° C and the r e s u l t i n g frequency/temperature dependence i s shown g r a p h i c a l l y i n FIG. l a . I guanas, Iguana iguana (iguana) Three iguanas were implanted with c h r o n i c e l e c t r o d e s i n the o l f a c t o r y b ulbs. One had b i p o l a r e l e c t r o d e s i n one bulb o n l y and the o t h e r s had b i l a t e r a l b i p o l a r e l e c t r o d e s . The s i g n a l s from each bulb were recorded a t a number o f d i f f e r e n t temperatures and the f r e q u e n c i e s c a l c u l a t e d , with the r e l a t i o n s h i p a l s o shown i n FIG. l a . In c a l c u l a t i n g the frequency/temperature dependence no d i s t i n c t i o n was made between the s i g n a l s from the two b u l b s . The e f f e c t o f nasal a i r flow was i n v e s t i g a t e d i n the two iguanas with b i l a t e r a l e l e c t r o d e s . The s i g n a l s from both o l f a c t o r y bulbs were r e -corded with the n o s t r i l s plugged i n t u r n , and with both plugged. No induced waves were seen i n the s i d e with no a i r flow, and plugging both s i d e s e l i m i n a t e d the induced waves b i l a t e r a l l y . In t h i s circumstance 52 the waves c o u l d not be e l i c i t e d by v i s u a l , a u d i t o r y or t a c t i l e s t i m u l i . A t y p i c a l r e s u l t from one iguana i n shown i n FIG. 2. Coachwhip snakes, M a s t i c o p h i s f l a g e l l u m (Shaw) Two coachwhip snakes were implanted with b i p o l a r e l e c t r o d e s i n one o l f a c -t o r y bulb. A r e p r e s e n t a t i v e example o f induced wave a c t i v i t y recorded a t a number o f temperatures i s shown i n FIG. 3. As may be seen i n the r e c o r d s , the frequency w i t h i n each b u r s t was i n i t i a l l y higher and then became constant f o r the remainder of the b u r s t : the l a t t e r p a r t was the one used to o b t a i n the quoted measurements. The r e l a t i o n s h i p between the induced wave frequency and temperature i s i l l u s t r a t e d i n FIG. l a . The induced wave b u r s t s were u s u a l l y o f the form seen i n FIG. 3, but on occ a s i o n s a second harmonic a t h a l f the induced wave frequency was pr e s e n t . T h i s was p a r t i c u l a r l y n o t i c e a b l e when the snakes were warmer and more a l e r t , and s i m i l a r o b s e r v a t i o n s have been r e p o r t e d by o t h e r workers i n ot h e r animals (Ottoson, 1959; Domino and Ueki, 1960). The second harmonic c o u l d appear a t any time d u r i n g the main b u r s t but u s u a l l y not throughout i t s e n t i r e d u r a t i o n . A t y p i c a l example o f t h i s type o f a c t i v i t y i s shown i n FIG. 4. The e f f e c t o f an o l f a c t o r y stimulus was i n v e s t i g a t e d with these snakes and the response was found to be v a r i a b l e . In a n o n - a l e r t snake the e f f e c t o f smoke d e l i v e r e d by a tube to i t s box was to cause a marked i n c r e a s e i n the induced wave (and slow wave) a c t i v i t y o f the bulbs. T h i s e f f e c t i s shown i n FIG. 5a. By c o n t r a s t i n a warmer, a l e r t animal the induced waves were markedly suppressed f o r a p e r i o d o f time (FIG. 5b). 53 In i n t e r m e d i a t e s t a t e s o f a l e r t n e s s , d u r i n g which o l f a c t o r y induced waves were present both before and a f t e r the s t i m u l u s , the frequency of the waves comprising the bu r s t s was unchanged by smoke or by the odour of peppermint (Table I I I ) . B u l l snakes, P i t u o p h i s c a t e n i f e r ( B l a i n v i l l e ) A t o t a l o f 5 b u l l s n a k e s were implanted b i l a t e r a l l y with c h r o n i c b i p o l a r e l e c t r o d e s . The frequency o f the induced wave a c t i v i t y was measured a t a number.of temperatures and the r e s u l t s are shown i n FIG. l a . 54 40.0n 36.0 32.0 H 28.0 24.0 I 20.0 H <u cr <p ,<r 16.0 12.0-1 8.0 H 4.0 0.0 10 15 20 25 30 Cloacal Temperature (°C) —i 35 FIGURE l a Temperature dependence of the frequency of the induced waves recorded from the o l f a c t o r y bulbs of f o u r p o i k i l o t h e r m i c s p e c i e s - L i n e a r t r a n s f o r m a t i o n X Coachwhip snakes ® B u l l s n a k e s S Iguanas A Toads 55 FIGURE l b Temperature dependence o f the frequency of induced waves recorded from the o l f a c t o r y bulbs o f f o u r p o i k i l o t h e r m i c s p e c i e s - Semi l o g a r i t h m i c t r a n s f o r m a t i o n X Coachwhip snakes ® B u l l snakes SI Iguanas A • Toads 56 ROB U ^ u A V y ^ M W t L e f t nostril plugged LOB J ? > J . W V ^ N W ^ ^ a ROB Both nostrils LOB ftt^^Jt*!^^^ ^ ^ M ^ ^ f ^ i u ^ ^ plugged ROB W<ID»III«>" I'*»W»»" i» w>'n»««^»»tw)i>-»)i.ii»t*«<« Rjghj nostril LOB ^ ^ i ^ ^ ^ i ^ ' H plugged sec. FIGURE 2 Si g n a l s recorded from the o l f a c t o r y bulbs o f an iguana a t 26° C. ROB and LOB i n d i c a t e the s i g n a l s from the r i g h t and l e f t o l f a c t o r y bulbs r e s p e c t i v e l y . 57 i i i \ S w * * ^ w » ^ ^ 35.5 ° C i t i j *^tmtf^^^^ 34.0 ° C ^ ^ ^ ^ ^ 32.0 ° C i ! i • I | I 28.0 ° C \t»>t:*\\'immmw>*' 2 0 Hz 2 0 0 juV FIGURE 3 Rep r e s e n t a t i v e examples of induced waves recorded from one o l f a c t o r y bulb o f a coachwhip snake a t v a r i o u s temperatures.. 58 20 Hz 2 0 0 / J V FIGURE 4 An example of the occurrence of a second harmonic i n the induced wave a c t i v i t y recorded from one o l f a c t o r y bulb o f a coachwhip snakg. The f i g u r e shows three c o n s e c u t i v e b u r s t s with the animal a t 29.5 C. 59 T - S m o k e sec. i i i ' ' i i mi |/Slni> •»» W ' 1 1 " " « ~ » S m o k e I sec. FIGURE 5 fctiSit^n^thl^^ st1r3!!s (E1pe s m o k e ) o n t h e 1 n d u c e d a n d s l o w w a v e „ V o l f a c t o r y bulbs o f coachwhip snakes. The records are continuous i n each case. a) n o n - a l e r t snake a t 29° C. b) a l e r t snake a t 34 C. TABLE I I I E f f e c t o f odour on the frequency o f the induced waves i n the o l f a c t o r y bulb o f a coachwhip snake. BEFORE ODOUR AFTER ODOUR ODOUR TEMP MEAN S.D. MEAN S.D. d f P Peppermint 35°C 38.02 +1.33 36.55 +1.57 16 0.1 Smoke 32°C 32.46 +1.20 33.21 +2.53 24 0.5 TABLE IV The r e l a t i o n s h i p s between the frequency of o l f a c t o r y induced waves ( f ) and temperature ( T ) . Li near Semi l o g a r i t h m i c Log/log Toads n = 108 Iguanas n = 189 Coachwhip snakes n = 106 B u l l snakes n = 70 f = 0.33 + 0.36 T r = 0.856 f = 3.10 + 0.44 T r = 0.723 f = -19.39 + 1.63 T r = 0.911 f = 2.48 + 0.66 T r = 0.870 l o g f = 0.46 + 0.020 T l o g f = -0.45 + 1.01 log T r = 0.872 r = 0.889 l o g f = 0.80 + 0.013 T l o g f = 0.15 + 0.71 l o g T r = 0.762 r = 0.756 l o g f = 0.76 + 0.023 T l o g f = -0.92 + 1.61 l o g T r = 0.912 r = 0.908 l o g f = 0.86 + 0.016 T l o g f = 0.032 + 0.89 l o g T r = 0.858 r = 0.893 62 B) INDUCED WAVE AND SPONTANEOUS FIRING PROFILES The amplitude o f the induced wave was recorded from an e l e c t r o d e a t d i f f e r e n t depths i n the o l f a c t o r y bulbs of r a b b i t s using s t a i n l e s s s t e e l e l e c t r o d e s with t i p s i z e s from 7 to 100 jum. The amplitude o f the s i g n a l was compared to t h a t recorded from a f i x e d r e f e r e n c e e l e c t r o d e o f the same t i p s i z e l o c a t e d i n the same o l f a c t o r y bulb. In the f i r s t experiment the r e f e r e n c e e l e c t r o d e was on the s u r f a c e of the bulb where i t y i e l d e d a s m a l l e r l e s s w e l l - d e f i n e d wave. In subsequent experiments the r e f e r e n c e e l e c t r o d e was l o c a t e d a t a depth o f 1000>um where a much l a r g e r induced wave was recorded. Because the amplitude r a t i o was a r e l a t i v e v a l u e , the p o s i t i o n o f the f i x e d r e f e r e n c e e l e c t r o d e was not c r i t i c a l and more a c c u r a t e measurements co u l d be o b t a i n e d w i t h the l a r g e r r e f e r e n c e s i g n a l . The am-p l i t u d e r a t i o was averaged from a s e r i e s o f t h r e e or f o u r measurements a t each depth. P r o f i l e s were a l s o obtained by means o f the matched p a i r of f i l t e r s a l r e a d y d e s c r i b e d and i n a l l cases the r e s u l t s were the same. The induced wave amplitude s t a r t e d to i n c r e a s e r a p i d l y j u s t below the m i t r a l c e l l l a y e r and was l a r g e throughout the granule l a y e r . T h i s was m i r r o r e d i n the lower p a r t of the bulb. A r e p r e s e n t a t i v e p r o f i l e obtained from an e l e c t r o d e t r a c k through the c e n t r e o f the o l f a c t o r y bulb i s shown i n FIG. 6 and one from a t r a c k l a t e r a l to the c e n t r e i n FIG. 7. These p r o f i l e s were obtained from 7 pm e l e c t r o d e s from which the c e l l f i r i n g p r o f i l e was a l s o o b t a i n e d . The spontaneous c e l l u l a r a c t i v i t y was recorded with 7 /am e l e c t r o d e s and the observed s i g n a l c o n s i s t e d o f the e l e c t r i c a l a c t i v i t y from a number o f neurones. This "group" a c t i v i t y was i n t e g r a t e d on a ratemeter, and d i s p l a y e d on a r e c o r d e r . The spontaneous c e l l f i r i n g p r o f i l e was o b t a i n e d by p l o t t i n g the amplitude o f the i n t e g r a t e d s i g n a l a g a i n s t depth from the 63 0 FIGURE 6 Dots - ra t i o of induced wave amplitude from explor ing electrode to that from reference e lect rode. Crosses - c e l l f i r i n g rate from explor ing e lect rode. Both curves plot ted against depth from the surface of the o l fac to ry bulb. I ps i l a te ra l n o s t r i l open, cont ra la tera l nos t r i l plugged, electrode track in centre of bulb. 64 FIGURE 7 Dots - r a t i o o f t h e a m p l i t u d e o f t h e e n v e l o p e o f t h e i n d u c e d wave a c t i v i t y f r o m t h e e x p l o r i n g e l e c t r o d e t o t h a t f r o m t h e r e f e r e n c e e l e c t r o d e . C r o s s e s - c e l l f i r i n g r a t e from e x p l o r i n g e l e c t r o d e . B oth c u r v e s p l o t t e d a g a i n s t d e p t h f r o m t h e s u r f a c e o f t h e o l f a c t o r y b u l b . I p s i l a t e r a l n o s t r i l o pen, c o n t r a l a t e r a l n o s t r i l p l u g g e d , e l e c t r o d e t r a c k l a t e r a l t o mid l i n e o f b u l b . 65 dorsal surface of the olfactory bulb. It was found that the same elec-trode track could be traversed four or five times with negligible change in the profile provided the experimental conditions remained the same. Profiles obtained in this way were always constructed by moving the electrode from the surface downwards through the olfactory bulb, readings were not taken during upward travel of the electrode. Backlash in the electrode drive was eliminated by always returning the electrode to a position slightly above the zero position and then approaching zero in the downward direction. Two representative profiles obtained with the ipsilateral nostril open are shown in figures 6 and 7. Figure 6 represents the profile obtained from an electrode track in the centre of the olfactory bulb and figure 7 shows that from one lateral to the centre. The induced wave and group firing profiles in each figure were obtained from the same electrode by suitable filtering described earlier. The cell firing profile had pronounced peaks of activity centered on the mitral and external plexi-form layers. The cellular activity appeared to be least where the induced wave activity was greatest. This is illustrated in figures 8 and 9 which show the induced waves, the cellular activity and the respiration through the ipsilateral nostril at 200 Aim intervals through the olfactory bulb. The effect of air flow and odour passing over the nasal mucosa on the electrical activity of the olfactory bulb has been the subject of considerable investigation as already discussed. It was therefore of interest to determine the effects of these variables on the cellular activity of the olfactory bulb layers. Group cell activity profiles were constructed in the manner described with the nostrils plugged (respiration through the mouth), the ipsilateral nostril open with the contralateral plugged and finally with the contralateral open and the ipsilateral 66 10 FIGURE 8 FIGURES 8 and 9 Induced wave a c t i v i t y (upper t r a c e ) , c e l l u l a r a c t i v i t y (middle t r a c e ) and r e s p i r a t i o n through i p s i l a t e r a l n o s t r i l (lower t r a c e ) a t 200 urn i n t e r v a l s v e r t i c a l l y through the o l f a c t o r y bulb. The two upper t r a c e s were recorded by s u i t a b l e f i l t e r i n g from the same e l e c t r o d e (7 /jm t i p diameter) and the r e s p i r a t i o n by means o f a nasal t h e r m i s t o r probe. I n s p i r a t i o n i s represented by a downward d e f l e c t i o n . Depth from the s u r f a c e o f the bulb i s represented i n hundreds of microns by the f i g u r e s to the r i g h t o f the r e c o r d s . 46 FIGURE 9 6 8 plugged. A t y p i c a l s e t o f p r o f i l e s o btained by this means i s shown i n f i g u r e 10. I t can be seen t h a t a l a r g e number o f c e l l s i n the e x t e r n a l p l e x i f o r m l a y e r were f i r i n g even with no a i r flow i n t o the nares. Opening the i p s i l a t e r a l n o s t r i l i n c r e a s e d the c e l l u l a r a c t i v i t y w h i l e opening the c o n t r a l a t e r a l n o s t r i l reduced t h i s a c t i v i t y . F r e q u e n t l y the d o r s a l h a l f o f the o l f a c t o r y bulb behaved d i f f e r e n t l y from the v e n t r a l h a l f , the p r o f i l e being r e l a t i v e l y u n a l t e r e d by a i r flow or odour over the o l f a c t o r y mucosa. A t y p i c a l example o f t h i s type o f p r o f i l e i s shown i n f i g u r e 11. This l a c k o f f u n c t i o n a l symmetry w i l l be d i s c u s s e d l a t e r . In o r d e r to e s t a b l i s h the e f f e c t o f the nasal a i r f l o w , the p r o f i l e w ith both n o s t r i l s plugged was used as a c o n t r o l . The f i r i n g r a t e at each depth with the n o s t r i l s plugged was s u b t r a c t e d from the f i r i n g r a t e with the i p s i l a t e r a l n o s t r i l open. The r e s u l t i n g p r o f i l e i n d i c a t e d the change i n c e l l u l a r a c t i v i t y caused by opening the i p s i l a t e r a l n o s t r i l . Such a p r o f i l e i s shown i n f i g u r e 12 which was o b t a i n e d from the r e s u l t s shown i n f i g u r e 10. I t can be seen t h a t a c o n s i d e r a b l e i n c r e a s e i n c e l l a c t i -v i t y i n the m i t r a l and e x t e r n a l granule c e l l l a y e r s was produced by a i r flow through the i p s i l a t e r a l n o s t r i l . No attempt to i n t r o d u c e an odour was made d u r i n g these experiments and normal room a i r was being breathed by the animal. C i g a r e t t e smoking was not p e r m i t t e d i n the r e s e a r c h area a t the time. F i g u r e 13 shows a p r o f i l e o b t a i n e d w i t h the i p s i l a t e r a l n o s t r i l open and from which the c e l l f i r i n g p r o f i l e o b t a i n e d w i t h both n o s t r i l s plugged has been s u b t r a c t e d . In t h i s case the a i r which the r a b b i t was b r e a t h i n g c o n t a i n e d smoke, although t h i s was not d e l i b e r a t e l y blown a t the animal. I t can be seen t h a t the peak of i n c r e a s e d a c t i v i t y i s much broader (shaded area) and now i n c l u d e s the g r a n u l a r l a y e r . T h i s 69 p r o f i l e was obtained from the r e s u l t s shown i n f i g u r e 11. The e f f e c t s i n the lower h a l f o f the bulb, although s m a l l e r , were q u a l i t a t i v e l y s i m i l a r . The e f f e c t o f a i r flow i n the c o n t r a l a t e r a l n o s t r i l can be c a l c u l a t e d i n the same manner. The p r o f i l e o b t a i n e d w i t h both n o s t r i l s plugged was used as a c o n t r o l and was s u b t r a c t e d from the r a t e obtained with the con-t r a l a t e r a l n o s t r i l open. The r a t e change produced r e p r e s e n t s the r e d u c t i o n o f c e l l f i r i n g upon opening the c o n t r a l a t e r a l n o s t r i l . T h i s r e d u c t i o n was c a l c u l a t e d from the p r o f i l e s o f f i g u r e 10 and the r e s u l t s are shown i n f i g u r e 14 (shaded a r e a ) . F i g u r e 15 i l l u s t r a t e s the same phenomenon using the data o f f i g u r e 11. I t w i l l be n o t i c e d t h a t i n the d o r s a l h a l f o f the bulb the l a y e r s showing a r e d u c t i o n o f f i r i n g with the c o n t r a l a t e r a l n o s t r i l open are approximately the same as those t h a t show an i n c r e a s e i n f i r i n g with, the i p s i l a t e r a l n o s t r i l open. Again the e f f e c t s i n the v e n t r a l h a l f o f the bulb were much l e s s marked but t h i s does not n e c e s s a r i l y r e -p r e s e n t a f u n c t i o n a l d i f f e r e n c e as w i l l be d i s c u s s e d l a t e r . Having e s t a b l i s h e d the e f f e c t o f a i r flow through the i p s i l a t e r a l and c o n t r a l a t e r a l n o s t r i l s , i t was necessary to determine the e f f e c t o f a d e l i b e r a t e o l f a c t o r y s t i m u l u s . The most commonly used stimulus employed by previous workers was tobacco smoke blown i n t o the n o s t r i l s o f the animal. Although t h i s was not c o n s i d e r e d to be the best c h o i c e o f s t i m u l u s , i t was the most s u i t a b l e from the p o i n t o f view o f comparison with previous work. Smoke was t h e r e f o r e used as a s t i m u l u s ; however i t was not blown i n t o the n o s t r i l s but a l i g h t c l o u d o f i t was blown i n t o the general area o f the animal's f a c e so t h a t i t was b r e a t h i n g a i r con-taminated with smoke. C e l l f i r i n g p r o f i l e s were measured with the i p s i l a t e r a l n o s t r i l open without smoke pres e n t and again with smoke 70 RATE PULSES/SEC FIGURE 10 Group c e l l a c t i v i t y p r o f i l e o b t a i n e d by i n t e g r a t i o n o f c e l l u l a r a c t i v i t y recorded from a 7 jum e l e c t r o d e . Dots - both n o s t r i l s plugged, c r o s s e s -i p s i l a t e r a l n o s t r i l open, t r i a n g l e s - c o n t r a l a t e r a l n o s t r i l open. A l l curves p l o t t e d a g a i n s t depth from the d o r s a l s u r f a c e o f the o l f a c t o r y bulb. 0 200 400 600 RATE PULSES/SEC FIGURE 11 Group c e l l a c t i v i t y p r o f i l e o b t a i n e d by i n t e g r a t i o n o f c e l l u l a r a c t i v i t y r ecorded from a 7 jum e l e c t r o d e . Dots - both n o s t r i l s plugged, c r o s s e s i p s i l a t e r a l n o s t r i l open, t r i a n g l e s c o n t r a l a t e r a l n o s t r i l open. A l l curves p l o t t e d a g a i n s t depth from the d o r s a l s u r f a c e o f the o l f a c t o r y bulb. R e l a t i v e l y l i t t l e v a r i a t i o n i n the c e l l f i r i n g p r o f i l e i s seen i n the v e n t r a l h a l f o f the o l f a c t o r y bulb. FIGURE 12 P r o f i l e o f i n c r e a s e i n c e l l f i r i n g with the i p s i l a t e r a l n o s t r i l open ( c o n t r a l a t e r a l plugged). Increase i n c e l l f i r i n g r a t e i s p l o t t e d a g a i n s t depth from the d o r s a l s u r f a c e o f the o l f a c t o r y bulb. These r e s u l t s were obtained from the p r o f i l e s shown i n FIG. 10. 7 3 I 1000 Q. Q 2000 3000 4000 - 5000 6000 7000 , — -200 x ' * l x x X X - x I —I 1— 0 + 200 Rate Pulses /sec . 1 + 400 FIGURE 13 P r o f i l e o f i n c r e a s e i n c e l l f i r i n g with the i p s i l a t e r a l n o s t r i l open ( c o n t r a l a t e r a l plugged). Increase i n c e l l f i r i n g r a t e i s p l o t t e d a g a i n s t depth from the d o r s a l s u r f a c e of the o l f a c t o r y bulb (shaded a r e a ) . Decreases i n c e l l f i r i n g a re shown i n the n e g a t i v e d i r e c t i o n by the unshaded areas. These r e s u l t s were obtained from the p r o f i l e s shown i n FIG. 11. 74 2 5. 4000' l> I • » r -200 0 +200 Rate Pulses /sec. FIGURE 14 P r o f i l e o f decrease o f c e l l f i r i n g with the c o n t r a l a t e r a l n o s t r i l open ( i p s i l a t e r a l plugged). Decrease i n c e l l f i r i n g r a t e i s p l o t t e d a g a i n s t depth from the d o r s a l s u r f a c e o f the o l f a c t o r y bulb (shaded a r e a ) . Increases i n c e l l f i r i n g a re shown i n the p o s i t i v e d i r e c t i o n by the unshaded areas. These r e s u l t s were obt a i n e d from the p r o f i l e s shown i n FIG. 10. 75 . 7000, 1 j , -400 -200 0 +200 Rate Pulses / sec. FIGURE 15 P r o f i l e o f decrease i n c e l l f i r i n g with the c o n t r a l a t e r a l n o s t r i l open ( i p s i l a t e r a l plugged). Decrease i n c e l l f i r i n g r a t e i s p l o t t e d a g a i n s t depth from the d o r s a l s u r f a c e o f the o l f a c t o r y bulb (shaded a r e a s ) . Increases i n c e l l f i r i n g a re shown i n the p o s i t i v e d i r e c t i o n by the unshaded ar e a s . These r e s u l t s were obtained from the p r o f i l e s shown i n FIG. 11. present. The f i r i n g r a t e without smoke was s u b t r a c t e d from the f i r i n g r a t e with smoke and a new p r o f i l e produced. A t y p i c a l example i s shown i n f i g u r e 16 f o r the d o r s a l h a l f o f the bulb. A bimodal d i s t r i b u t i o n i s seen with one peak i n the m i t r a l c e l l l a y e r and a second much l a r g e r peak i n the granule c e l l l a y e r . A r e g i o n i n the glomerular l a y e r showed a small decrease i n the c e l l f i r i n g r a t e . In order to determine whether a str o n g s t i m u l u s , which c o u l d be co n s i d e r e d noxious had the same e f f e c t , smoke was blown a t the animal a few seconds before the r a t e was measured. The r e s u l t s o f t h i s experiment are shown i n f i g u r e 17. The method o f o b t a i n i n g the p r o f i l e was the same as t h a t f o r f i g u r e 16. I t can be seen t h a t the r e g i o n o f i n c r e a s e d f i r i n g was l i m i t e d to the granule c e l l l a y e r and the r e g i o n o f reduced f i r i n g became l a r g e r to i n c l u d e the m i t r a l c e l l l a y e r and the e f f e c t was more pronounced. The e f f e c t s o f i n c r e a s i n g c o n c e n t r a t i o n o f smoke i n the a i r being breathed i s summarized i n the f o l l o w i n g t a b l e . The plus s i g n s i n d i c a t e degrees o f c e l l u l a r a c t i v i t y . E x t e r n a l P l e x i f o r m Layer M i t r a l Layer Granule Layer Both n o s t r i l s occluded + + 0 I p s i l a t e r a l n o s t r i l open ( c l e a n room a i r ) +++ +++ 0 (room a i r , t r a c e o f smoke) ++ ++ ++ ( l i g h t smoke) + + +++ (heavy smoke) 0 0 +++ 2000 1 — •400 i -200 0 +200 +400 Rate Pulses /sec + 600 FIGURE 16 The e f f e c t o f an o l f a c t o r y stimulus (smoke) on the c e l l f i r i n g p r o f i l e . The change i n c e l l f i r i n g r a t e with the stimulus p r e s e n t i s - p l o t t e d a g a i n s t depth from the d o r s a l s u r f a c e o f the o l f a c t o r y bulb. Shaded areas r e p r e s e n t an i n c r e a s e i n f i r i n g and unshaded areas a decrease (shown i n the negative d i r e c t i o n ) . 0 i tooo-2 0 0 0 r t i » - 4 0 0 - 2 0 0 0 + 200 +400 R a t e P u l s e s / s e c + 600 FIGURE 17 T h i s p r o f i l e was obtained i n the same way as t h a t shown i n FIG. 16 except t h a t smoke was blown a t the animal a few seconds before the r a t e reading was e s t a b l i s h e d . CD 79 As smoke was blown a t the animal there was a t r a n s i e n t i n c r e a s e i n the f i r i n g r a t e . T h i s i n c r e a s e was c a l c u l a t e d f o r each depth and a p r o f i l e was produced o f the i n c r e a s e i n c e l l f i r i n g upon blowing smoke d i r e c t l y a t the animal. T h i s i s shown i n f i g u r e 18 and can be seen to have i t s peak i n the e x t e r n a l p l e x i f o r m and m i t r a l c e l l l a y e r s . T h i s experiment was repeated m o n i t o r i n g the induced wave s i g n a l as well as the c e l l f i r i n g r a t e . The i n c r e a s e i n each o f these parameters was recorded upon a s t r o n g smoke sti m u l u s and t h i s was repeated every 100/jm to produce a p a i r o f p r o f i l e s . These a r e i l l u s t r a t e d i n f i g u r e 19. The induced wave i n c r e a s e when smoothed by a d i g i t a l smoothing technique (approximate s i n x/x method) g i v e s three peaks, one i n the g l o m e r u l a r l a y e r , one i n the m i t r a l c e l l l a y e r and one i n the g r a n u l e c e l l l a y e r . The i n c r e a s e i n c e l l f i r i n g i n t h i s case shows two peaks, one i n the m i t r a l and e x t e r n a l p l e x i f o r m l a y e r s and another s m a l l e r peak i n the granule c e l l l a y e r . C o n s i d e r a b l e work has been r e p o r t e d by other i n v e s t i g a t o r s on the c e n t r i f u g a l system and the a l e r t n e s s o f the animal. Experiments were performed to determine the e f f e c t s o f a r o u s a l on the c e l l u l a r a c t i v i t y o f the o l f a c t o r y bulb. A p r o f i l e o f c e l l u l a r a c t i v i t y with depth from the s u r f a c e o f the bulb was c o n s t r u c t e d f o r the d o r s a l h a l f o f the bulb with the animal i n a q u i e t environment, t a k i n g care not to touch the animal. T h i s was performed with the i p s i l a t e r a l n o s t r i l plugged. A second p r o f i l e was produced using the same e l e c t r o d e t r a c k t h i s time with the animal d e l i b e r a t e l y a l e r t e d by touching the f u r and by n o i s e . These two p r o f i l e s are shown together i n f i g u r e 20 where i t can be seen t h a t the e f f e c t o f a l e r t i n g the animal i s to i n c r e a s e the c e l l f i r i n g i n the e x t e r n a l p l e x i f o r m and m i t r a l c e l l l a y e r s . No i n c r e a s e i n c e l l f i r i n g was seen i n the granule c e l l l a y e r . The i p s i l a t e r a l n o s t r i l was then 8 0 opened and the p r o f i l e repeated. The r e s u l t was to i n c r e a s e the c e l l f i r i n g i n the. e x t e r n a l p l e x i f o r m and g r a n u l a r l a y e r s . Only a small i n -crease was seen i n the m i t r a l c e l l l a y e r . The p r o f i l e obtained with the i p s i l a t e r a l n o s t r i l plugged i n the a l e r t animal was s u b t r a c t e d from the p r o f i l e o b t a i n e d with the i p s i l a t e r a l open, a l s o i n the a l e r t animal. The r e s u l t i n g p r o f i l e i s shown i n f i g u r e 21 and i n d i c a t e s the i n c r e a s e i n these two r e g i o n s . The s i m i l a r i t y between t h i s p r o f i l e and t h a t of f i g u r e 16 i s obvious and i t may well be t h a t the m i l d smoke stimulus o f f i g u r e 16 caused a l e r t i n g o f the animal to some e x t e n t . E l e c t r o d e t r a c t s were marked by pa s s i n g a c u r r e n t and then by p r u s s i a blue s t a i n i n g as d e s c r i b e d i n the experimental procedure. The e l e c t r o d e p o s i t i o n s were v e r i f i e d h i s t o l o g i c a l l y and a r e p r e s e n t a t i v e photograph of two marks, one a t 800 jum and the other a t 3500 pm i s shown i n f i g u r e 22. Th i s r e p r e s e n t s a t r a c k l a t e r a l to the c e n t r e o f the o l f a c t o r y bulb i n the p o s t e r i o r p a r t o f the bulb. T h i s e l e c t r o d e t r a c k corresponds to one used to produce the p r o f i l e s shown i n f i g u r e s 10, 12, 14, 16, 17, 18, 19 and 20. 81 0 * 1 0 0 0 -C L Q 2 0 0 0 « 0 2 0 0 • 4 0 0 Rate Pulses / s e c . FIGURE 18 The e f f e c t o f blowing smoke d i r e c t l y a t the animal i s shown i n t h i s p r o f i l e . Increase o f c e l l f i r i n g r a t e upon blowing smoke i s p l o t t e d a g a i n s t depth from the d o r s a l s u r f a c e o f the o l f a c t o r y bulb. FIGURE 19 a) Increase i n induced wave amplitude b) Increase i n c e l l f i r i n g r a t e Upon blowing smoke d i r e c t l y a t the animal. Both parameters are p l o t t e d a g a i n s t depth from the d o r s a l s u r f a c e o f the o l f a c t o r y b u l b . Crosses -raw data. Dots smoothed data. RATE PULSES/SEC FIGURE 20 P r o f i l e of c e l l f i r i n g with, i p s i l a t e r a l n o s t r i l plugged. Crosses - n o n - a l e r t animal, dots - a l e r t animal. Both curves p l o t t e d a g a i n s t depth from the d o r s a l s u r f a c e of the o l f a c t o r y bulb. ^ 2000-* • • 0 200 400 RATE P U L S E S / S E C FIGURE 21 P r o f i l e of i n c r e a s e o f c e l l f i r i n g with the i p s i l a t e r a l n o s t r i l open i n a l e r t animal. P l o t t e d a g a i n s t depth from the d o r s a l s u r f a c e o f the o l f a c t o r y bulb. 85 FIGURE 22 H i s t o l o g i c a l s e c t i o n o f r i g h t o l f a c t o r y b u l b o f r a b b i t . E l e c t r o d e t r a c t m a r k e d by p r u s s i a n b l u e r e a c t i o n a t 800 and 3500 iim. C o u n t e r s t a i n e d w i t h s a f r a n i n . 86 C) SINGLE CELL RECORDINGS Recordings were made from s i n g l e neurones i n the o l f a c t o r y bulbs o f r a b b i t s under moderately deep urethane a n a e s t h e s i a using s t a i n l e s s s t e e l m i c r o e l e c t r o d e s with t i p s s i z e s v a r y i n g from 1 to 3 /jm. In some e x p e r i -ments s t i m u l a t i n g e l e c t r o d e s were p l a c e d i n the l a t e r a l o l f a c t o r y t r a c t and the m i t r a l c e l l l a y e r was i d e n t i t i e d by the i n v e r s i o n o f the evoked p o t e n t i a l recorded i n the bulb. F i g u r e 23A shows an example o f such a r e c o r d i n g . The f i g u r e s to the l e f t o f the evoked p o t e n t i a l t r a c e s i n d i c a t e the depth from the d o r s a l s u r f a c e o f the o l f a c t o r y b u l b . The s i g n a l was low pass f i l t e r e d with a c u t o f f at 100 Hz. The n u l l i n the evoked p o t e n t i a l can be seen to appear a t a depth of 3730 jjm, and t h e r e a f t e r the r e c o r d i n g e l e c t r o d e was then p o s i t i o n e d a t t h i s depth. The s i g n a l s shown i n f i g u r e s 23B and C were o b t a i n e d a t t h i s depth. F i g u r e 23B shows the e f f e c t o f a sti m u l u s o f smoke blown a t the r a b b i t w i t h i t s i p s i l a t e r a l n o s t r i l open. L e f t to r i g h t the r e c o r d i n g s a r e : be f o r e smoke, d u r i n g smoke st i m u l u s and a f t e r smoke. The upper t r a c e i n each case i s the s i g n a l from the micro-e l e c t r o d e (low frequency c u t o f f approximately 300 Hz) and the lower t r a c e i s the r e s p i r a t i o n measured by a t h e r m i s t o r probe i n the i p s i l a t e r a l n o s t r i l . I n s p i r a t i o n i s i n d i c a t e d by a downward d e f l e c t i o n . The c e l l s can be seen to be normally q u i e s c e n t and a c t i v a t e d by the smoke s t i m u l u s . A f t e r the smoke st i m u l u s the c e l l s c o n t i n u e d to f i r e , l o c k e d to i n s p i r a t i o n . F i g u r e 23C shows the e f f e c t o f a t a c t i l e s t i m u l u s , no o l f a c t o r y stimulus was employed and the r a b b i t was b r e a t h i n g room a i r . The re c o r d s a t the l e f t were taken before the stimulus and those a t the r i g h t a f t e r the s t i m u l u s . The t a c t i l e s t i m u l u s c o n s i s t e d o f touching the f u r on the r a b b i t ' s back. The c e l l s were q u i e s c e n t before the stimulus but f i r e d l o c k e d to i n s p i r a t i o n a f t e r the s t i m u l u s . The s i m i l a r i t y o f the r e c o r d s 87 before and a f t e r the stimulus i n f i g u r e s 23B and C can e a s i l y be seen. F i g u r e s 24 to 28 r e p r e s e n t c e l l u l a r a c t i v i t y recorded a t d i f f e r e n t depths of a s i n g l e e l e c t r o d e t r a c k . In the records shown, the upper t r a c e i l l u s -t r a t e s the r e s p i r a t i o n recorded by a t h e r m i s t o r probe i n the i p s i l a t e r a l n o s t r i l , i n s p i r a t i o n i s being shown by an upward d e f l e c t i o n . The c e n t r e t r a c e i s the s i g n a l from the m i c r o e l e c t r o d e (low frequency c u t o f f 250 Hz) and the lower t r a c e i s the same s i g n a l with a low frequency c u t o f f o f 8 Hz. The s i g n a l w i t h the 250 Hz low frequency c u t o f f was used to t r i g g e r a ratemeter (Appendix I I I ) which p r o v i d e d a p u l s e s u i t a b l e f o r d r i v i n g a PDP8 computer. The computer was programmed to produce histograms o f the number o f occurrences o f a g i v e n time i n t e r v a l between a c t i o n p o t e n t i a l s ( 1 s t o r d e r ) i n a f i v e second p e r i o d . The programme a l s o had the c a p a b i -l i t y o f producing histograms of the number o f occurrences o f a g i v e n time i n t e r v a l between a l t e r n a t e a c t i o n p o t e n t i a l s (2nd o r d e r ) . The histograms o b t a i n e d before and a f t e r o l f a c t o r y s t i m u l i are shown under the a p p r o p r i a t e r e c o r d i n g s . These are 1st o r d e r histograms u n l e s s marked o t h e r w i s e . The time i n t e r v a l s c a l e has been c a l i b r a t e d i n frequency and a c a l i b r a t i o n u s i ng a s i g n a l generator i s shown a t the bottom l e f t o f f i g u r e 28. F i g u r e 24 g i v i n g an example o f recordsfrom the g r a n u l e c e l l l a y e r , shows a c e l l f i r i n g b e fore the smoke s t i m u l u s . The f i r i n g p a t t e r n o f t h i s c e l l was unusual i n t h a t i t appeared to be frequency modulated with the r a t e o f f i r i n g slowing during i n s p i r a t i o n . The time i n t e r v a l histogram showed a d i f f u s e peak a t the lower frequency end o f the s c a l e . When smoke was blown a t the animal, induced waves were produced and what appeared to be another group of c e l l s s t a r t e d to f i r e . The time i n t e r v a l histogram taken a f t e r the s t i m u l u s i n d i c a t e d a peak o f f i r i n g with a d i s t r i b u t i o n c e n t e r e d on 50 Hz and corresponding to the induced wave frequency. I t i s 88 d i f f i c u l t to t e l l from the o r i g i n a l records what e f f e c t the stimulus had on the c e l l t h a t was a c t i v e before the s t i m u l u s . The i n d i c a t i o n from the time i n t e r v a l histogram was t h a t the c e l l had changed i t s f i r i n g p a t t e r n s i n c e the amplitude o f the histogram a t the low frequency end of the s c a l e was reduced a f t e r the s t i m u l u s . The r e c o r d s shown i n f i g u r e 25 serve to i l l u s t r a t e the e f f e c t s o f d i f f e r e n t o l f a c t o r y s t i m u l i upon c e l l s i n the r e g i o n o f the lower m i t r a l c e l l l a y e r . Before the f i r s t s t imulus (smoke) c e l l s were f i r i n g l o c k e d to i n s p i r a t i o n . Upon p r e s e n t a t i o n o f the smoke st i m u l u s induced waves were produced and the c e l l a c t i v i t y i n c r e a s e d w i t h the f i r i n g s t i l l l o c k e d to i n s p i r a t i o n . A f t e r a w h i l e t h i s a c t i v i t y was s t i l l p r e s e n t but a much l a r g e r a c t i o n p o t e n t i a l was seen to be present i n the second s e t o f t r a c e s which r e p r e s e n t the s i g n a l s approximately 40 seconds a f t e r the smoke s t i -mulus. This type o f a c t i v i t y c ontinued f o r some minutes a t which time a smell o f peppermint was i n t r o d u c e d i n t o the a i r the r a b b i t was b r e a t h i n g . This stimulus caused a s u p p r e s s i o n o f the l a r g e r spikes without i n c r e a s i n g the f i r i n g o f the s m a l l e r s p i k e s . The l a r g e r s p i k e s d i d not r e t u r n spontaneously but c o u l d be evoked again by a second smoke s t i m u l u s , a t which time they continued to be l o c k e d to i n s p i r a t i o n . On i n t r o d u c i n g the smell o f p y r i d i n e the f i r i n g o f the c e l l s was reduced f o r one breath a f t e r which they continued u n a l t e r e d . P y r i d i n e produced no apparent change i n the s m a l l e r amplitude s p i k e s . These complex c e l l u l a r p a t t e r n s were t y p i c a l of the re c o r d s made i n the r e g i o n o f the m i t r a l c e l l s . Other examples o f the c e l l u l a r a c t i v i t y i n the r e g i o n o f the m i t r a l c e l l s are shown i n f i g u r e s 26 and 27 which were recorded 52 urn below the c e l l s i n f i g u r e 25. Again a smoke st i m u l u s e x c i t e d a c e l l with a l a r g e s p i k e and induced waves were seen to be p r e s e n t . A time i n t e r v a l histogram 89 o f t h i s c e l l ' s f i r i n g i n d i c a t e d no r e g u l a r p e r i o d i c i t y ( f i g u r e 26). A f t e r some time the c h a r a c t e r i s t i c f i r i n g l o c k e d to i n s p i r a t i o n appeared ( f i g u r e 27) and the time i n t e r v a l histogram showed a marked p e r i o d i c i t y with the d i s t r i b u t i o n i n d i c a t i n g a predominant frequency of 70 Hz. A second order histogram o f t h i s c e l l u l a r a c t i v i t y showed a more d i f f u s e peak a t 40 - 50 Hz. A smell o f peppermint was i n t r o d u c e d which caused an i n h i b i t i o n o f t h i s c e l l f o r one breath and then the f i r i n g resumed with approximately the same d i s t r i b u t i o n as before the s t i m u l u s . I t i s thought t h a t the l a r g e s p i k e s i n f i g u r e s 25 - 27 r e p r e s e n t the f i r i n g o f m i t r a l c e l l . F i g u r e 28 shows the f i r i n g i n the e x t e r n a l p l e x i f o r m l a y e r . Small s p i k e s were seen l o c k e d to i n s p i r a t i o n b efore the smoke s t i m u l u s . C e l l s e x h i b i t i n g s p i k e s o f l a r g e r amplitude were e x c i t e d by the stimulus and a prolonged induced wave was seen to be p r e s e n t . The computer was t r i g g e r e d o n l y by the a c t i o n p o t e n t i a l s o f l a r g e s t amplitude, and the r e s u l t i n g histogram i s shown. A pronounced peak i n the d i s t r i b u t i o n occurs a t 50 Hz. The s i g n a l s were rec o r d e d on magnetic tape a t the time o f the ex-periment so t h a t i t was p o s s i b l e to a n a l y s e the recorded s i g n a l s by another method l a t e r . The induced wave was used to t r i g g e r the computer ( v i a the ratemeter p u l s e g e n e r a t i n g c i r c u i t ) and the time i n t e r v a l histogram o f t h i s s i g n a l was a l s o computed, and i s shown a t the bottom r i g h t o f f i g u r e 28. I t can be seen t h a t the peaks i n the i n t e r v a l histograms are i n c l o s e agreement. I t should be mentioned a t t h i s p o i n t t h a t the f i l t e r used to separate the s p i k e s from the induced waves had a c u t o f f o f 250 Hz with a 24 db r o l l o f f , thereby e l i m i n a t i n g the p o s s i b i l i t y t h a t an a t t e n u -ated v e r s i o n o f the induced wave frequency c o u l d modulate the s p i k e r e c o r d . The c e l l i n f i g u r e 28 was not i d e n t i f i e d p o s i t i v e l y . I 3650 3 7 3 . ° . M i ^ W - r ^ FIGURE 23 a) Evoked p o t e n t i a l recorded i n the o l f a c t o r y bulb caused by s t i m u l a t i o n o f the l a t e r a l o l f a c t o r y t r a c t . Depth from the do r s a l s u r f a c e of the o l f a c t o r y bulb.shown a t l e f t o f each t r a c e . Time bar 5m sec, v o l t a g e bar 50 juV. Low frequency c u t o f f 100 Hz. b) L e f t to r i g h t : before smoke st i m u l u s , during smoke, a f t e r smoke. c) L e f t to r i g h t : before t a c t i l e s t i m u l u s , a f t e r touching r a b b i t ' s f u r . In B and C the upper t r a c e was the c e l l u l a r a c t i v i t y recorded by the m i c r o e l e c t r o d e and the lower t r a c e was the r e s p i r a t i o n recorded by a the r m i s t o r probe i n the i p s i l a t e r a l n o s t r i l . I n s p i r a t i o n i s downwards. Time bar 1 sec, v o l t a g e bars 50 pV. o 91 I before Smoke after Smoke 6 0 4 0 30 23 Frequency. Depth 2505 jaM FIGURE 24 Upper r e c o r d s are continuous. Top t r a c e , r e s p i r a t i o n recorded by t h e r m i s t o r probe i n i p s i l a t e r a l n o s t r i l , middle t r a c e , s i g n a l from the m i c r o e l e c t r o d e (low frequency cut o f f 300 Hz), bottom t r a c e , same as middle t r a c e but with low frequency c u t o f f of 8 Hz. Time bar, 1/2 second. Graphs are time i n t e r v a l histograms produced by a PDP8 computer. Number o f occurrences o f a gi v e n time i n t e r v a l i s p l o t t e d a g a i n s t the time i n t e r v a l between a c t i o n p o t e n t i a l s ( c a l i b r a t e d i n f r e q u e n c y ) . Smoke Peppermint • Smoke Pyridine I Depth 3602 juM FIGURE 25 Same as FIGURE 24 but without time i n t e r v a l histograms. 93 Smoke after Smoke 1st. Order III ll.j imi,. 2 nd. Order n n J J t 4 d J . . ^ . J j l H , t l 60 40 30 25 Frequency Depth 3654 JDM FIGURE 26 Same as FIGURE 24. 1 s t and second o r d e r histograms are shown a f t e r the s t i m u l u s . The c e l l s were q u i e s c e n t before the st i m u l u s so t h a t no histogram was ob t a i n e d a t t h a t time. Peppermint t before Peppermint after Peppermint 1st. Order 2 nd. Order 60 40 30 25 Frequency Depth 3654 >JM FIGURE 27 Same as FIGURE 24 with both 1st and 2nd order histograms b e f o r e and a f t e r the s t i m u l u s . VO 95 Smoke I before Smoke after Smoke i i I I i..uk&. •, JuiuiL Calibrate induced Waves 11 60 40 30 Zt Frequency Depth 3 8 0 3 / j M FIGURE 28 Same as FIGURE 24 but with a c a l i b r a t i o n , o f the time i n t e r v a l histogram produced by using a s i g n a l generator to d r i v e the computer. The histogram a t the lower r i g h t was o b t a i n e d by using the induced wave s i g n a l to t r i g g e r the computer. 96 DISCUSSION 1. Wave a c t i v i t y A survey of previous work on the e l e c t r i c a l wave a c t i v i t y o f the o l f a c t o r y bulbs has been r e p o r t e d with the f o l l o w i n g f i n d i n g s con-s t i t u t i n g a reasonable b a s i s f o r agreement among the v a r i o u s i n v e s t i -g a t o r s . a) A f l o w o f odourized a i r over the i p s i l a t e r a l o l f a c t o r y mucosa i s necessary f o r the g e n e r a t i o n of induced waves i n the o l f a c t o r y b u l b . b) In the absence of such s t i m u l a t i o n i n t r i n s i c waves are normally p r e s e n t i n the o l f a c t o r y b u l b . c) Odourized a i r f l o w i n g over the o l f a c t o r y mucosa does not produce induced waves i n the c o n t r a l a t e r a l o l f a c t o r y b u l b . d) Blowing odour i n t o the n o s t r i l reduces the amplitude of . n a t u r a l l y o c c u r r i n g induced waves i n the c o n t r a l a t e r a l o l f a c t o r y bulb. e) S e p a r a t i n g the o l f a c t o r y bulb from the remainder of the b r a i n does not a b o l i s h the occurrence o f induced waves i n the i s o l a t e d bulb to an o l f a c t o r y s t i m u l u s . f ) C e n t r i f u g a l c o n t r o l o f the amplitude o f the induced waves has been demonstrated. g) Induced waves have been recorded from many s p e c i e s from lung-f i s h to mammals. 97 The present i n v e s t i g a t i o n has shown induced waves to be present i n the o l f a c t o r y bulbs of two s p e c i e s of snakes and i n iguanas. I t has a l s o confirmed the occurrence o f these waves i n toads. The most s t r i k i n g c h a r a c t e r i s t i c o f the induced waves recorded from the v a r i o u s animals i n t h i s s e r i e s i s the s i m i l a r i t y of t h e i r appearance and o c c u r r e n c e . The obvious q u e s t i o n i s whether the mechanism by which these waves are generated i s common to a l l s p e c i e s . An i n d i c a t i o n i n t h i s d i r e c t i o n can be o b t a i n e d from an examination of the e f f e c t of •temperature on the frequency of the waves. Huggins, Parsons and Pena (1968) with the caiman, Putkonen and S a r a j a s (1968) with hypothermic r a b b i t s and Dupe and Godet (1969) wi t h the l u n g f i s h have a l l r e p o r t e d a l i n e a r dependence of the induced wave frequency with temperature. In the present study the frequency/temperature r e l a t i o n s h i p appeared to be l i n e a r f o r each s p e c i e s , with g r e a t e r s l o p e s f o r the animals having a higher average frequency, as i s i l l u s t r a t e d i n F i g . l a . The narrow range of temperature over which i t i s p o s s i b l e to make the r e c o r d i n g s however precludes a d e c i s i o n whether t h i s simple r e l a t i o n -s h i p i s the c o r r e c t one. F i g . l b i l l u s t r a t e s the l i n e s o b t a i n e d when the l o g (frequency)/temperature r e l a t i o n s h i p s were p l o t t e d , and s i m i l a r l y good l i n e a r f i t s were found when l o g ( f r e q u e n c y ) / l o g (temperature) data were examined. T a b l e IV g i v e s the equation and the c o r r e l a t i o n co-e f f i c i e n t s ( r ) f o r these three s e t s of l i n e s , from which i t appears t h a t on the b a s i s o f the c o r r e l a t i o n c o e f f i c i e n t s i t i s i m p o s s i b l e to determine which t r a n s f o r m a t i o n should be used. However i t can be seen from F i g . l and from the r e g r e s s i o n c o e f f i c i e n t s i n Table IV t h a t there i s c o n s i d e r a b l y 98 l e s s v a r i a t i o n of sl o p e s f o r the l o g (frequency)/temperature method of a n a l y s i s than f o r e i t h e r o f the other two. Although s i m i l a r i t y of s l o p e i s not n e c e s s a r i l y the b a s i s on which to choose the t r a n s f o r m a t i o n , i t i s reasonable to assume t h a t the Q^ Q would be s i m i l a r i f i n f a c t the induced waves i n the d i f f e r e n t s p e c i e s r e s u l t e d from a common mechanism. The Q 1 Q values c a l c u l a t e d from the r e g r e s s i o n c o e f f i c i e n t s o f the s e m i l o g a r i t h m i c t r a n s f o r m a t i o n are given i n Table V. The 0-^ value s , f o r c o nduction v e l o c i t y i n nerve were r e p o r t e d by Maxwell (1907) as 1.78 and Lucas (1908) as 1.79: a l l of the values r e p o r t e d here are lower than these f i g u r e s . By c o n t r a s t the temperature dependence of the s y n a p t i c d e l a y a t the neuromuscular j u n c t i o n o f the f r o g has a of approximately 3, (Katz and M i l e d i , 1965) and the lower values found i n the present study c o u l d be .taken to imply t h a t the mechanism r e g u l a t i n g the frequency w i t h i n each o l f a c t o r y b u r s t may not i n v o l v e a s y n a p t i c p r o c e s s . However i f a r e l a t i v e l y long pathway with a lower Q 1 Q were i n v o l v e d , i t might be expected to obscure the Q^Q e f f e c t o f an i n t e r -vening synapse. The su g g e s t i o n t h a t induced waves have a common genesis i n a l l s p e c i e s i s supported by the f i n d i n g r e p o r t e d here t h a t a i r flow i n t o the nares i s r e q u i r e d f o r the p r o d u c t i o n o f the waves, thus extending the many o b s e r v a t i o n s which have been made upon other animals. The c l a i m of McLennan and Graystone (1965) t h a t the e f f e c t o f a d e l i b e r a t e o l f a c t o r y s timulus to c a t s was to accentuate the induced waves i n a non-a l e r t animal but to depress them i n the a l e r t s t a t e has here been extended to a p o i k i l o t h e r m and once more i n d i c a t e s the common nature o f the o l f a c t o r y p r o c e s s . TABLE V Temperature c o e f f i c i e n t s (QIQ) f o r o l f a c t o r y induced waves. Toads Iguanas Coachwhip snakes B u l l snakes 1.57 1.36 1.71 1.45 100 2. Neuronal A c t i v i t y In the survey of the previous i n v e s t i g a t i o n s of neuronal a c t i v i t y i n the o l f a c t o r y bulb the f o l l o w i n g f i n d i n g s appear to form the b a s i s f o r general agreement or are of importance to the present d i s c u s s i o n . a) M i t r a l and t u f t e d c e l l s show a s p e c i f i c i t y i n t h e i r response to d i f f e r e n t odours and t h i s has been shown to be t r u e a l s o f o r the g l o m e r u l i . There a l s o appears to be a t o p o g r a p h i c a l o r g a n i z a t i o n of these c e l l s w i t h i n the o l f a c t o r y bulb i n r e -l a t i o n to areas of the nasal mucosa. b) A strong stimulus to the nasal mucosa or to the l a t e r a l o l -f a c t o r y t r a c t w i l l i n h i b i t most m i t r a l c e l l s . T h i s may be i n i t i a t e d through axon c o l l a t e r a l s of the m i t r a l c e l l s a c t i n g on granule c e l l s which i n t u r n i n h i b i t the m i t r a l c e l l s , or by way of d e n d r o d e n d r i t i c synapses between the m i t r a l and g r a n u l e c e l l s , or perhaps both mechanisms may p l a y a p a r t i n the p r o c e s s . c) The m a j o r i t y of the c e l l s i n the o l f a c t o r y bulb which are commonly encountered d i s c h a r g e without r e l a t i o n to a i r f l o w or odour. However, those t h a t do^how a g r e a t v a r i e t y o f response. Often c e l l s a few microns a p a r t show q u i t e d i f f e r e n t response c h a r a c t e r i s t i c s to the same o l f a c t o r y s t i m u l u s . d) The f i r i n g p a t t e r n of o l f a c t o r y bulb neurones can be m o d i f i e d by c e n t r a l s t r u c t u r e s . S t i m u l a t i o n o f the mesencephalic r e t i c u l a r f o r m a t i o n changes c e l l u l a r responses i n the o l f a c t o r y bulb to an odour and a l s o has been shown to i n c r e a s e the induced wave amplitude. 101 e) Many workers have searched f o r granule c e l l s a c t i v a t e d e i t h e r by an o l f a c t o r y s timulus or by an e l e c t r i c a l s timulus to the l a t e r a l o l f a c t o r y t r a c t . No d e f i n i t e c o n c l u s i o n s have been reached and some workers have found a c t i v e c e l l s i n the g r a n u l e t l a y e r . However the m a j o r i t y of i n v e s t i g a t o r s have been unable to f i n d s p i k e a c t i v i t y i n granule c e l l s and R a i l e t . a l . (1966) have suggested t h a t these c e l l s may not normally generate an a c t i o n p o t e n t i a l . f ) One group of workers (von Baumgarten e t . a l . (1962a) r e p o r t e d t h a t the f i r i n g of m i t r a l , t u f t e d and granule c e l l s was locked i n phase to the induced waves. They noted the c o r r e l a t i o n was l e s s exact f o r the m i t r a l c e l l s than f o r the other c e l l types and the r e l a t i o n s h i p was much l e s s exact when the induced waves were not of maximum amplitude. 3. The Pathways of the o l f a c t o r y bulb The c u r r e n t knowledge of the o l f a c t o r y pathways i s summarized by the f o l l o w i n g major p o i n t s of i n t e r e s t : a) The main pathway of i n f o r m a t i o n from the o l f a c t o r y bulb i s the l a t e r a l o l f a c t o r y t r a c t c o n t a i n i n g the axons of the m i t r a l c e l l s . b) The axons of the m a j o r i t y o f the t u f t e d c e l l s do not t r a v e l the len g t h o f the l a t e r a l o l f a c t o r y t r a c t though a few may do so. Appa r e n t l y none of these axons t r a v e l i n the a n t e r i o r commissure and the m a j o r i t y synapse i n the a n t e r i o r o l f a c t o r y nucleus. Immediately a f t e r synapsing, c o l l a t e r a l s a t l e a s t o f some o f the axons of the t e r t i a r y neurones^ r e t u r n to the o l f a c t o r y bulb where they presumably..synapse on granule c e l l d e n d r i t e s . The 102 main axons t r a v e l i n the a n t e r i o r limb of the a n t e r i o r commi-ssure where some p r o j e c t to the c o n t r a l a t e r a l a n t e r i o r o l f a c t o r y nucleus and g r a n u l e l a y e r of the c o n t r a l a t e r a l o l f a c t o r y bulb. Others p r o j e c t to a v a r i e t y of c e n t r a l s t r u c t u r e s ( F e r r e r , 1969). c) S t i m u l a t i o n of the c o n t r a l a t e r a l bulb or a n t e r i o r commissure causes an i n h i b i t i o n of m i t r a l c e l l s . The f i b r e s of the a n t e r i o r commissure are thought to e x c i t e interneurones which i n t u r n cause the i n h i b i t i o n o f the m i t r a l c e l l s . d) C e n t r i f u g a l f i b r e s have been shown to run i n the l a t e r a l o l -f a c t o r y t r a c t . The work of Doving and Gemne (1966) i n f i s h has demonstrated t h a t these f i b r e s c a r r y impulses from the i p s i l a t e r a l hemisphere to the b u l b ; a l though s t i m u l a t i o n e i t h e r of the i p s i l a t e r a l or c o n t r a l a t e r a l t r a c t caused an i n c r e a s e of the f i r i n g i n t h i s pathway. Recent work by P r i c e (1969a) has suggested t h a t the c e n t r i f u g a l f i b r e s o r i g i n a t e i n the nucleus of the h o r i z o n t a l limb of the diagonal band i n the basal f o r e -b r a i n . The p r e s e n t i n v e s t i g a t i o n has shown t h a t the m a j o r i t y of neuronal a c t i o n p o t e n t i a l s i n the unstimulated o l f a c t o r y bulb occurs i n the m i t r a l and e x t e r n a l p l e x i f o r m l a y e r s . In these re g i o n s are l o c a t e d c e l l s which are f i r e d by the c e n t r i f u g a l a r o u s a l system, whereas c e l l s i n the g r a n u l a r l a y e r were not. In the e x t e r n a l p l e x i f o r m and m i t r a l c e l l r e g i o n s , a r e a l s o l o c a t e d c e l l s whose spontaneous s p i k e a c t i v i t y i s reduced by room a i r flow over the c o n t r a l a t e r a l mucosa. With the i p s i l a t e r a l n o s t r i l open, the c e l l u l a r a c t i v i t y i n c r e a s e s i n these same r e g i o n s , but o n l y with a strong o l f a c t o r y s t i m u l u s i s s p i k e a c t i v i t y observed i n the g r a n u l e c e l l l a y e r . 103 The d i f f e r e n c e i n the c e l l u l a r p r o f i l e s i n the upper and lower p a r t s of the bulb and the apparent d i f f e r e n c e i n the e f f e c t s o f i p s i -l a t e r a l and c o n t r a l a t e r a l a i r f l o w are not thought to be due to a f u n c t i o n a l d i f f e r e n c e between the two p a r t s o f the bulb. I t seems more l i k e l y t h at the lower h a l f of the bulb has a t o p o g r a p h i c a l r e l a t i o n s h i p to a p a r t of the o l f a c t o r y mucosa which i s more s u s c e p t i b l e to i n f e c t i o n and c o n g e s t i o n so t h a t o l f a c t o r y s t i m u l i may not be as e f f e c t i v e i n these areas. The p r o f i l e o f induced wave amplitude a g a i n s t depth from the s u r f a c e o f the o l f a c t o r y bulb shows t h a t the induced waves have t h e i r maximum amplitude i n the granule l a y e r and l i t t l e evidence f o r a n u l l i n t h i s amplitude d i s t r i b u t i o n was observed. von Baumgarten e t . a l . (1962a) proposed t h a t the induced waves were the r e s u l t o f a generator l o c a t e d i n or near the m i t r a l c e l l l a y e r and t h a t a p o o r l y d e f i n e d n u l l i n the induced waves occurred i n t h i s r e g i o n . I t would seem on the b a s i s o f the present f i n d i n g t h a t the induced waves do not r e p r e s e n t the f i e l d p o t e n t i a l r e s u l t i n g from the s p i k e a c t i v i t y of granule c e l l s i n or near the m i t r a l c e l l l a y e r . R a i l e t . a l . (1966) suggested t h a t the g r a n u l e c e l l s i n v o l v e d i n the proposed d e n d r o d e n d r i t i c synapses with the m i t r a l c e l l s might f u n c t i o n without g e n e r a t i n g a c t i o n p o t e n t i a l s . They d i d not exclude the p o s s i b i l i t y t h a t o c c a s i o n a l f i r i n g o f these c e l l s might occur. They a l s o p r e d i c t e d t h a t these granule c e l l s were s t r a t e g i c a l l y l o c a t e d to enable t h e i r i n -h i b i t o r y a c t i v i t y to pr o v i d e a means f o r the i n t e g r a t i o n o f s e v e r a l i n p u t s to t h e i r deeper l y i n g d e n d r i t e s . The pres e n t r e s u l t s support these pro-p o s a l s i n many ways. I t i s f e l t t h a t even i f the reason f o r the d i f f i c u l t y i n r e c o r d i n g from singl.e.-granule c e l l s was because of t h e i r small s i z e , the present method of r e c o r d i n g would have shown t h e i r group a c t i v i t y to be p r e s e n t . In the case of a strong o l f a c t o r y s t i m u l u s c e l l u l a r s p i k e a c t i v i t y was c l e a r l y seen i n the granule c e l l l a y e r s . I t was never observed however under more p h y s i o l o g i c a l l e v e l s o f s t i m u l a t i o n even when induced waves with l a r g e amplitude were pr e s e n t . F i g u r e s 6, 7 and 12 serve to i l l u s t r a t e t h i s p o i n t and F i g u r e 17 demonstrates the e f f e c t o f a s trong o l f a c t o r y s t i m u l u s . Under t h i s c o n d i t i o n g r a n u l e c e l l f i r i n g has been shown to occur with average r e p e t i t i o n r a t e s approximately the same as the induced wave frequency ( f i g u r e 24). The l o c k i n g of g r a n u l e c e l l f i r i n g r e p o r t e d by von Baumgarten (1962a) was a l s o o n l y obtained with a strong o l f a c t o r y s t i m u l u s . The present f i n d i n g s are t h e r e f o r e i n agreement with the proposal of R a i l e t . a l . (1966) t h a t the granule c e l l s may f u n c t i o n without g e n e r a t i n g an a c t i o n p o t e n t i a l . I t i s a l s o thought t h a t these c e l l s are the s i t e of o r i g i n of the induced waves. I t has been shown t h a t induced wave a c t i v i t y i s s t i l l p r esent i n an o l f a c t o r y bulb i s o l a t e d from the r e s t of the b r a i n but with i t s connections to the mucosa i n t a c t . I t i s obvious t h e r e f o r e t h a t c e n t r -f u g a l c onnections and those from the c o n t r a l a t e r a l o l f a c t o r y b u l b , although they may modify the induced wave a c t i v i t y , are not e s s e n t i a l f o r i t s g e n e r a t i o n . The o n l y other known connections to the granule c e l l s a re from the secondary neurones e i t h e r v i a the proposed d e n d r c - d e n d r i t i c synapses or from the axon c o l l a t e r a l s > a n d induced waves are present with l a r g e amplitude i n response to an o l f a c t o r y s t i m u l u s which i n h i b i t s the m a j o r i t y of these secondary neurones. I t must be remembered t h a t induced waves are not found u n l e s s the o l f a c t o r y mucosa i s s t i m u l a t e d by an odour, though the odour content of room a i r would seem to be s u f f i c i e n t . 105 The i m p l i c a t i o n o f t h i s f i n d i n g i s t h a t i n p u t to the granule c e l l s i s r e q u i r e d f o r the genesis of the induced waves. Westecker (1969, 1970a, 1970b) has presented evidence t h a t the normal pathway of e x c i t a t i o n to the granule c e l l s i s v i a the d e n d r o d e n d r i t i c synapses and not v i a the axon c o l l a t e r a l s . I f t h i s s u g g e s t i o n i s t r u e , then i t would seem t h a t the e s s e n t i a l i n g r e d i e n t f o r the occurrence of induced waves i s the ex-c i t a t i o n of the granule c e l l s a t these d e n d r o d e n d r i t i c synapses. There i s of course always the p o s s i b i l i t y t h a t other c e l l s may be e x c i t a t o r y to the branches of the granule c e l l d e n d r i t e s which extend i n t o the e x t e r n a l p l e x i f o r m l a y e r . I t has been shown i n the present i n v e s t i g a t i o n t h a t the maximum amplitude o f the induced waves occurs deep i n the granule c e l l l a y e r . I f the induced waves o r i g i n a t e a t the d e n d r o d e n d r i t i c synapses and a c t i o n p o t e n t i a l s are not generated by the granule c e l l s , t h i s e l e c t r i c a l a c t i v i t y would have to be propagated to the deeper d e n d r i t e s e l e c t r o n i -c a l l y with an accompanying a t t e n u a t i o n with d i s t a n c e . I t i s not immediately obvious why the maximum amplitude of the induced waves occurs i n the deeper l a y e r s but perhaps the answer l i e s i n the geometry of the d e n d r i t i c branching. I t has been proposed here t h a t e x c i t a t i o n o f the g r a n u l e c e l l s i s r e q u i r e d f o r the genesis of induced wave a c t i v i t y . I t i s a p p r o p r i a t e t h e r e f o r e to d i s c u s s the other connections to the g r a n u l e c e l l s i n t h i s r e g a r d . Yamamoto e t . a l . (1963) proposed t h a t the f i b r e s o f the a n t e r i o r commissure e x c i t e d the g r a n u l e c e l l d e n d r i t e s . S t i m u l a t i o n of the a n t e r i o r commissure or the c o n t r a l a t e r a l o l f a c t o r y bulb causes a r e d u c t i o n of the induced wave amplitude i n the i p s i l a t e r a l bulb. Doving and Gemne (1966) have shown t h a t the c e n t r i f u g a l pathway i n the l a t e r a l o l f a c t o r y 106 t r a c t causes i n h i b i t i o n of the m i t r a l c e l l s and t h a t t h e r e i s a r e d u c t i o n of a c t i v i t y i n these f i b r e s produced by a t a c t i l e s t imulus to the animal. I t i s the present author's i n t e r p r e t a t i o n t h a t these c e n t r i f u g a l f i b r e s a l s o are e x c i t a t o r y to the granule c e l l s and t h a t i n h i b i t i o n of the m i t r a l c e l l s i s mediated v i a the g r a n u l e c e l l s . T a c t i l e s t i m u l i i n other animals have been shown to i n c r e a s e the induced wave a c t i v i t y of the o l -f a c t o r y b u l b . Thus both i n the case o f the f i b r e s of the a n t e r i o r com-missure and the c e n t r i f u g a l f i b r e s , e x c i t a t o r y i n p u t to the deeper den-d r i t e s of the granule c e l l s causes a r e d u c t i o n i n the amplitude o f the induced waves. The f i n d i n g of Do'ving and Gemne (1966) suggests t h a t a r o u s a l causes a r e d u c t i o n o f the e x c i t a t o r y i n p u t to the g r a n u l e c e l l s i f the p r e s e n t i n t e r p r e t a t i o n s are c o r r e c t . I t would seem t h e r e f o r e t h a t an e x c i t a t o r y i n p u t to the deeper d e n d r i t e s of the g r a n u l e c e l l s does not cause an i n c r e a s e i n the induced wave a c t i v i t y but probably a r e d u c t i o n . I t f o l l o w s from t h i s argument t h a t i f two e x c i t a t o r y i n p u t s to the deeper d e n d r i t e s of the granule c e l l s cause a r e d u c t i o n i n the induced wave a c t i v i t y , axon c o l l a t e r a l s o f the m i t r a l c e l l s , which are a l s o proposed as e x c i t a t o r y , would s i m i l a r l y cause a r e d u c t i o n i n the induced wave a c t i v i t y . S i n c e i n f o r m a t i o n probably reaches the g r a n u l e c e l l s by way of the secondary neurones and the induced wave amplitude i s i n c r e a s e d with an odour, then the d e n d r o d e n d r i t i c pathway seems the o n l y p o s s i b i l i t y remaining f o r the t r a n s f e r of t h i s i n f o r m a t i o n . I f t h i s i s the case^then the d e n d r o d e n d r i t i c s y n a p t i c mechanism which i s e x c i t a t o r y to the g r a n u l e c e l l , i n i t i a t e s the induced waves and the s y n a p t i c connections to the deeper d e n d r i t e s , which are a l s o thought to be e x c i t a t o r y , a p p a r e n t l y cause a decrease i n the induced wave amplitude. 107 These a p p a r e n t l y c o n t r a d i c t o r y e f f e c t s can be e x p l a i n e d i n the f o l l o w i n g way: I f the induced waves have t h e i r s i t e of o r i g i n a t the d e n d r o d e n d r i t i c synapses and then propagate e l e c t r o n i c a l l y to the deep d e n d r i t e s , the impedance of the d e n d r i t i c t r e e would appear to be low when viewed from the o r i g i n s i n c e i t i s made up of many p a r a l l e l d e n d r i t i c branches having high impedance. When l o o k i n g from one of these f i n e branches i n the d i r e c t i o n o f the source, a high impedance would be seen. The a c t i o n of an e x c i t a t o r y synapse on the t e r m i n a l branches of the d e n d r i t e s would be to cause a d e p o l a r i z a t i o n of the d e n d r i t e . T h i s would a c t as a low impedance shunt pathway and would swamp the e l e c t r o n i c a l l y propa-gated s i g n a l . I f a l a r g e number of the t e r m i n a l branches of the den-d r i t e s have e x c i t a t o r y s y n a p t i c i n n e r v a t i o n , an e x c i t a t o r y v o l l e y t o these t e r m i n a l s would reduce the amplitude of the observed induced wave s i g n a l recorded from the d e n d r i t i c f i e l d . B I B L I Q G R A P H Y 108 A d r i a n , E.D. and C. Ludwig 1938. Nervous Discharges from the O l f a c t o r y Organs of F i s h . J . P h y s i o l . L o n d . 94: 4 4 1 - 4 6 0 . N A d r i a n , E.D. 1942. 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The Time Course of F a c i l i t a t i o n and I n h i b i t i o n i n the O l f a c t o r y Bulb, I n v e s t i g a t e d with Double Pu l s e S t i m u l a t i o n o f the L a t e r a l O l f a c t o r y T r a c t . B r a i n Res. 16: 527-529. Westecker, Margret E. 1970a. E x c i t a t o r y and I n h i b i t o r y I n t e r a c t i o n s i n the O l f a c t o r y Bulb I n v o l v i n g D e n d r o d e n d r i t i c Synapses between M i t r a l C e l l s and Granular C e l l s . P f l u g e r s A r c h . 317_: 173-186. Westecker, Margret E. 1970b. A l t e r n a t i n g C h a r a c t e r i s t i c s o f the Evoked P o t e n t i a l i n the O l f a c t o r y Bulb i n Response to R e p e t i t i v e S t i m u l a t i o n o f the L a t e r a l O l f a c t o r y T r a c t . B r a i n Res. 17: 142-144. Yamamoto, C. 1961. O l f a c t o r y Bulb P o t e n t i a l s to E l e c t r i c a l S t i m u l a t i o n of the O l f a c t o r y Mucosa. Jap. J . P h y s i o l . 1_1_: 545-554. Yamamoto, C. and K. Iwama. 1961. Arousal R e a c t i o n o f the O l f a c t o r y Bulb. Jap. J . P h y s i o l . T k 335-345. Yamamoto, C. and K. Iwama. 1962. I n t r a c e l l u l a r P o t e n t i a l Recording from O l f a c t o r y Bulb Neurones of the Ra b b i t . Proc. Jap. Acad. 38: 63-67. Yamamoto, C , T. Yamamoto and K. Iwama. 1963. The I n h i b i t o r y Systems i n the O l f a c t o r y Bulb s t u d i e d by the I n t r a c e l l u l a r Recording. J . N e u r o p h y s i o l . 26: 403-415. A P P E N D I X I A SIMPLE BATTERY OPERATED TEMPERATURE CONTROL UNIT FOR SMALL ANIMAL RESEARCH 117 When using small animals f o r acute experimental r e s e a r c h , i t i s necessary to m a i n t a i n the body temperature a t a p h y s i o l o g i c a l l e v e l . In many i n s t a n c e s , p a r t i c u l a r l y i n n e u r o p h y s i o l o g i c a l r e s e a r c h s e n s i -t i v e e l e c t r o n i c equipment i s being used to r e c o r d the parameters under i n v e s t i g a t i o n and the use of a l i n e - o p e r a t e d temperature c o n t r o l u n i t o f t e n i n t r o d u c e s l i n e frequency i n t e r f e r e n c e i n t o the recorded s i g n a l s . I f the c o n t r o l u n i t i s of the discontinuous type c o n t a i n i n g r e l a y s , a d d i t i o n a l a r t i f a c t s a re i n t r o d u c e d by the s w i t c h i n g t r a n s i e n t s . In order to overcome the i n t e r f e r e n c e d i f f i c u l t i e s d e s c r i b e d , a u n i t has been designed which i s powered e n t i r e l y from b a t t e r i e s and r e q u i r e s no l i n e v o l t a g e supply. I t a l s o employs p r o p o r t i o n a l c o n t r o l , thereby e l i m i n a t i n g s w i t c h i n g t r a n s i e n t s . The c i r c u i t o f the temperature c o n t r o l u n i t i s shown i n f i g u r e 1. I t c o n s i s t s o f a Wheatstone b r i d g e , one arm of which i s an e x t e r n a l t h e r m i s t o r probe (Fenwall GB32P2). The "SET TEMP." potentiometer i s c a l i b r a t e d t o read over the range 33~41°C. Adjustment of t h i s c o n t r o l s e l e c t s the chosen n u l l temperature o f the b r i d g e . The "INT CAL" v a r i a b l e r e s i s t o r i s a d j u s t e d to compensate f o r the v a r i a b i l i t y o f t h e r m i s t o r r e s i s t a n c e s . I t i s a d j u s t e d so t h a t the n u l l temperature o f the br i d g e agrees with the c a l i b r a t i o n o f the "SET TEMP" potentiometer a t i t s c e n t e r p o s i t i o n o f 37°C. Because the br i d g e i s balanced a t the n u l l p o s i t i o n , the cen t e r zero meter w i l l read zero a t balance. The balance depends o n l y on the t h e r m i s t o r temperature and the p o s i t i o n o f the "SET TEMP" potentiometer. The "EXT.CAL." v a r i a b l e r e s i s t o r i s a d j u s t e d so t h a t the meter c a l i b r a t i o n i s meaningful. The meter i s c a l i b r a t e d to i n d i c a t e the number of degrees which the animal's temperature i s above or below the s e l e c t e d temperature. 118 Adjustment of the "EXT.CAL." v a r i a b l e r e s i s t o r can be used to c o r r e c t f o r v a r i a t i o n of the b r i d g e supply v o l t a g e ( B l ) as the b a t t e r y v o l t a g e f a l l s with prolonged use. As a l r e a d y d i s c u s s e d , the b r i d g e i s balanced when the t h e r m i s t o r i s a t the temperature s e l e c t e d on the "SET.TEMP." c o n t r o l . I f the animal's temperature i s below t h a t s e l e c t e d , a v o l t a g e w i l l appear acr o s s the meter which w i l l i n d i c a t e the number of degrees which the animal's tempera-t u r e i s below t h a t of the s e l e c t e d temperature. T h i s v o l t a g e i s a m p l i f i e d by means of an o p e r a t i o n a l a m p l i f i e r ( F a i r c h i l d m i n i - d i p ^ 7 4 1 ) and the r e s u l t i n g v o l t a g e used to c o n t r o l the c u r r e n t through a power t r a n s i s t o r (General E l e c t r i c D42C5). T h i s c u r r e n t i s s u p p l i e d from a r e c h a r g e a b l e e x t e r n a l 12 v o l t b a t t e r y (standard l e a d - a c i d automotive b a t t e r y ) . The c o n t r o l i s p r o p o r t i o n a l and the c u r r e n t w i l l s e t t l e a t a v a l u e s u f f i c i e n t to supply the animal's heat l o s s to i t s environment. The best r e s u l t s are obtained i f the heating pad i s c o n s t r u c t e d by sewing a r e s i s t a n c e wire onto a canvas bac k i n g . Two p a r a l l e l s t r a n d s of #26 nichrome wire each 4 f t . long on a 1 f t . square o f m a t e r i a l have been found to be very s a t i s -f a c t o r y . The pad i s then taped around the animal l o o s e l y and the t h e r m i s t o r probe i n s e r t e d r e c t a l l y . I f t h i s i s performed immediately upon anaesthe-t i z i n g the animal and the "SET TEMPT." c o n t r o l i s then a d j u s t e d f o r a meter reading of z e r o ; the animal w i l l be maintained a t i t s o r i g i n a l p h y s i o l o g i c a l temperature. FIGURE 1 C i r c u i t diagram of temperature c o n t r o l u n i t . R e s i s t a n c e s are i n ohms. A P P E N D I X I I A SELF CENTERING RESPIRATION MONITOR FOR SMALL ANIMAL RESEARCH In experiments where a r e c o r d of nasal a i r movement i s r e q u i r e d i n small animals, c o n v e n t i o n a l m o n i t o r i n g equipment such as e n d - t i d a l CO,, monitors have been found to be inadequate. The long time c o n s t a n t o f these u n i t s renders them u n s a t i s f a c t o r y f o r a c c u r a t e i n d i c a t i o n of the temporal r e l a t i o n s h i p s i n the a i r flow p a t t e r n . A r e s p i r a t i o n monitor was designed u t i l i z i n g a very small t h e r m i s t o r as the sensor. T h i s moni-t o r has a time c o n s t a n t of 0.5 second i n moving a i r so t h a t n e g l i g i b l e phase l a g i s in t r o d u c e d i n t o the recorded s i g n a l . The high s e n s i t i v i t y o f the u n i t together with a s e l f c e n t e r i n g f e a t u r e to e l i m i n a t e d r i f t a l l o w s m o n i t o r i n g of nasal a i r flow i n animals with very low t i d a l volumes and r a p i d r e s p i r a t i o n r a t e s . The r e s p i r a t i o n monitor i s b a t t e r y powered to e l i m i n a t e l i n e frequency i n t e r f e r e n c e i n the other s e n s i t i v e r e c o r d i n g equipment. The c i r c u i t o f the r e s p i r a t i o n monitor i s shown i n f i g u r e 1. The thermistor probe, which i s placed i n the nasal a i r flow of the animal, forms p a r t of a p o t e n t i o m e t r i c c h a i n . As the a i r flow v a r i e s the t h e r m i s t o r r e s i s t a n c e changes causing the p o t e n t i a l a t p o i n t X to vary. T h i s poten-t i a l v a r i a t i o n i s t r a n s f e r r e d to the i n p u t of an o p e r a t i o n a l a m p l i f i e r A^. through a r e s i s t a n c e - c a p a c i t a n c e (RC) c i r c u i t with a time c o n s t a n t which i s long with r e s p e c t to the r e s p i r a t o r y p e r i o d . Slow v a r i a t i o n s due to changes i n ambient temperature are e l i m i n a t e d by t h i s RC c i r c u i t . The o p e r a t i o n a l a m p l i f i e r A^ a m p l i f i e s the r e s p i r a t o r y s i g n a l , the s e n s i t i v i t y being s e t by the v a r i a b l e feedback r e s i s t o r l a b e l l e d SWING. The i n i t i a l b a l a n c i n g of the monitor i s achieved by means of the potentiometer l a b e l l e d CENTER. The high l e v e l output of a m p l i f i e r A, i s used to d r i v e a panel 1 2 2 meter to g i v e v i s u a l i n d i c a t i o n o f the r e s p i r a t o r y p a t t e r n . The s i g n a l i s a l s o made a v a i l a b l e on a r e c o r d e r j a c k f o r use where a permanent r e c o r d of r e s p i r a t i o n i s r e q u i r e d . Where an a u d i b l e i n d i c a t i o n of r e s p i r a t i o n i s r e q u i r e d the c i r c u i t shown a t the r i g h t o f f i g u r e 1 may be employed. A second o p e r a t i o n a l a m p l i f i e r Ag i s used to d r i v e a tone generator (SONALERT). At each i n s p i r a t i o n a tone i s sounded and the loudness of t h i s tone may be se t by means of the c o n t r o l l a b e l l e d VOLUME. The tone g e n e r a t o r must be powered from a separate b a t t e r y to e l i m i n a t e p o s i t i v e feedback due to the high g a i n o f the c i r c u i t . o ' i w CM -i CM fO CD e> o c a) R s 2 K A Nominal Rp 3 K J T . Nominal Select to suit Sonalert A | , A 2 Fairchild / J A 7 4 I Mini - Dip + 3 V 3 V Resistor Values in Capacitor Values in pf Switches ganged FIGURE 1 C i r c u i t diagram of s e l f c e n t e r i n g r e s p i r a t i o n monitor to U) A P P E N D I X I I I A RATEMETER FOR RECORDING THE RATE OF FIRING OF NERVE CELLS IN THE BRAINS OF ANIMALS 125 In order to o b t a i n a r e c o r d o f the r a t e o f f i r i n g of nerve c e l l s i n the b r a i n s o f animals a r a p i d l y responding ratemeter i s r e q u i r e d . T h i s i s p a r t i c u l a r l y t r u e when r e c o r d i n g group c e l l a c t i v i t y as opposed to t h a t of s i n g l e c e l l s . In group a c t i v i t y the a c t i o n p o t e n t i a l s may be observed a t r a t e s i n excess o f 200 per second. A ratemeter was t h e r e f o r e designed with a very r a p i d response time and w i t h switched range to accommodate a maximum r a t e o f 800 pulses per second. The ratemeter c i r c u i t was arranged to provide output pulses f o r i n t e n s i t y modulation o f an o s c i l l o s c o p e d i s p l a y so t h a t the f a s t r i s i n g edges of a c t i o n p o t e n t i a l s c o u l d be e a s i l y observed. P r o v i s i o n was a l s o made i n the design o f t h i s u n i t f o r a neg a t i v e - g o i n g p u l s e to be generated a t each a c t i o n p o t e n t i a l occurrence. T h i s p u l s e was used to d r i v e the PDP 8 computer to e s t a b l i s h the time i n t e r v a l histogram of the nerve c e l l f i r i n g p a t t e r n . C i r c u i t D e s c r i p t i o n The a c t i o n p o t e n t i a l recorded from a m i c r o - e l e c t r o d e i s a m p l i f i e d i n the c o n v e n t i o n a l manner and f e d to the in p u t o f the ratemeter. The inp u t requirement o f the ratemeter i s t h a t the s i g n a l has an amplitude o f approximately one v o l t . T h i s s i g n a l i s f u r t h e r a m p l i f i e d and f e d to a Schmitt t r i g g e r c i r c u i t which produces a change i n l e v e l when the i n p u t to i t passes through a t h r e s h o l d v o l t a g e . T h i s t h r e s h o l d can be s e t by means of a potentiometer ( t r i g g e r l e v e l c o n t r o l ) p r o v i d i n g the means to s e l e c t a l e v e l below which s i g n a l v a r i a t i o n s are ign o r e d . The output from the Schmitt t r i g g e r f o r an a c t i o n p o t e n t i a l a t the i n p u t , i s a square wave with f a s t r i s i n g and f a l l i n g edges. T h i s square wave i s d i f f e r e n t i a t e d , and the edge corresponding to the s t a r t of the a c t i o n p o t e n t i a l i s s e l e c t e d by means of a diode . T h i s i s done f o r both complementary outputs of the Schmitt t r i g g e r c i r c u i t , p r o v i d i n g the c h o i c e of a p o s i t i v e or n e g a t i v e pulse f o r i n t e n s i t y modulation and to d r i v e a computer. I f the d i s p l a y o s c i l l o s c o p e has g r i d i n t e n s i t y modulation the p o s i t i v e p u l s e i s used, and the negative p u l s e i f i t has cathode i n t e n s i t y modulation. The p o s i t i v e pulse corresponding to the f a l l i n g edge of the a c t i o n p o t e n t i a l i s s e l e c t e d by means of a diode and used to t r i g g e r a monostable m u l t i v i b r a t o r which produces a f i x e d - w i d t h p u l s e of .05 msec. The f i x e d width pulses corresponding to each a c t i o n p o t e n t i a l are then f e d to an i n t e g r a t o r . T h i s c o n s i s t s of an o p e r a t i o n a l a m p l i f i e r with c a p a c i t i v e feedback . R e s i s t o r s i n p a r a l l e l with t h i s c a p a c i t o r are s e l e c t e d by a switch to g i v e the four ranges of 100, 200, 400 and 800 pulses per second f o r maximum meter d e f l e c t i o n . A s e r i e s r e s i s t o r R 1 i s a d j u s t e d to c a l i b r a t e the instrument. The i n t e g r a t o r output impedance i s low (100 / j) and a low impedance r e c o r d e r may be connected d i r e c t l y to the i n t e g r a t o r output i n order to o b t a i n a permanent r a t e r e c o r d . The c a l i b r a t i o n r e s i s t o r R-| i s a d j u s t e d to g i v e the a p p r o p r i a t e r e c o r d e r c a l i b r a t i o n and then the meter s e r i e s r e s i s t o r R 2 i s a d j u s t e d to r e -c a l i b r a t e the meter. The ratemeter may be operated from any DC supply having both p o s i t i v e and negative output between 6 and 18 v o l t s . I f the i n t e n s i t y modulation f e a t u r e i s r e q u i r e d the u n i t should be s u p p l i e d from plus and minus 18 v o l t s . A block diagram of the ratemeter i s shown i n f i g u r e 1 and the complete c i r c u i t diagram i n f i g u r e 2. 1 Microelectrode Recording Amplifier Trigger Level A V Control \ \ Schmitt / Trigger Amplifier Differentator J l Differentiator! Oscilloscope VerticaL Input e to Grid to Cathode Intensity Modulation to Grid or Cathode of Oscilloscope -I 0.05 Range msec Switch Jl I 1 kl Monostable Integrator '1 Multivibrator Recorder FIGURE 1 Block diagram of ratemeter to + I8V Trigger Level Control o o b Input • — | ( - ^ - T 2 N 5 4 5 7 Note: Resistance in K O Capacities in /jf Transistors 2N5I43 Diodes IN 457 Range Switch 400 Intensity Modulation to Grid Intensity Modulation to Cathode "1 PDP8 Computer Recorder 1.5 V FSD 100 -18 V FIGURE 2 C i r c u i t diagram o f ratemeter. A P P E N D I X I V A TWO CHANNEL BAND PASS FILTER WITH ENVELOPE DETECTION 130 In o r d e r to determine the amplitude d i s t r i b u t i o n of s i g n a l s such as the induced waves of the o l f a c t o r y bulb, i t i s necessary to compare the s i g n a l from the e x p l o r i n g e l e c t r o d e with t h a t from a f i x e d r e f e r e n c e e l e c t r o d e . I t i s of course p o s s i b l e to r e c o r d the two raw s i g n a l s on a s u i t a b l e r e c o r d e r , to compare the amplitudes of the i n d i v i d u a l waves and then to average the r e s u l t f o r each complete b u r s t of induced waves. Th i s i s however a t e d i o u s and time consuming approach. Two i d e n t i c a l band pass f i l t e r s were t h e r e f o r e designed with v a r i a b l e c e n t e r f r e q u e n c i e s and a f i x e d 3 db bandwidth of 10 Hz. The outputs of these f i l t e r s were r e c t i f i e d and smoothed with c i r c u i t s having a d j u s t a b l e time c o n s t a n t s . The r e s u l t i n g envelopes were d i s p l a y e d on a slow speed r e c o r d e r and the r a t i o of the induced wave s i g n a l s i n the two channels obtained by measurement from t h i s r e c o r d . CIRCUIT DESCRIPTION The c i r c u i t diagram of the complete f i l t e r c i r c u i t i s shown i n f i g . 1. The s i g n a l s from the two e l e c t r o d e s were a m p l i f i e d i n the conventional manner (band pass 8 - 8 0 Hz) and f e d to the i n p u t o f the f i l t e r s . The i n p u t requirement of the f i l t e r s was t h a t the s i g n a l should have an amplitude of approximately one v o l t . The output impedance of the a m p l i f i e r was not c r i t i c a l s i n c e the f i r s t stage of each f i l t e r c o n s i s t e d of an impedance matching c i r c u i t ( f i e l d e f f e c t t r a n s i s t o r 2N5457) with an i n p u t impedance of 1 megohm. The output of t h i s f i r s t stage was f e d to the band pass f i l t e r v i a a c o u p l i n g c a p a c i t o r . The output of the f i l t e r was r e c t i f i e d by means of a IN457 diode and f e d to the smoothing c i r c u i t . The low output impedance of the o p e r a t i o n a l ampli-f i e r ( f a i r c h i l d > u A 741) and the diode g i v e a f a s t c h a r g i n g r a t e . The r a t e of d i s c h a r g e was s e t by the r e s i s t a n c e c a p a c i t a n c e network and was v a r i a b l e from 0.1 to 1.1 seconds. The r e c o r d e r had an i n p u t impedance of 1 megohm so t h a t no impedance matching was r e q u i r e d a t the output of the smoothing c i r c u i t . The d e s i g n c h a r a c t e r i s t i c s of the f i l t e r a re as f o l l o w s : where i s the 3 db bandwidth i n Hz A i s the v o l t a g e g a i n f i s the c e n t e r frequency i n Hz C i s the c a p a c i t a n c e i n f a r a d s R.|, R 2, and R^ are the r e s i s t a n c e s i n ohms. The g a i n of the f i l t e r was 5 and the c e n t e r frequency range was 20 to 120 Hz. The induced waves from the o l f a c t o r y bulbs of a r a b b i t a n e a s t h e t i z e d l i g h t l y with urethane have a frequency o f approximately 50 Hz. The f i l t e r s were t h e r e f o r e s e t to t h i s frequency f o r experiments with r a b b i t s . The frequency c h a r a c t e r i s t i c s of the f i l t e r s are shown i n f i g u r e 2. R 2 A T f C 2N5457 + 6V Input Recorder 10 Set Time Constant - 6 V Note : Resistances in K£L Capacitances in jjf FIGURE 1 C i r c u i t diagram o f one channel o f band pass f i l t e r M OJ 0.0 20 30 40 50 60 80 100 Frequency Hz. FIGURE 2 Frequency response c h a r a c t e r i s t i c of band pass f i l t e r . 3db bandwidth 10 Hz, centre frequency 50 Hz. 

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